Bio

Clinical Focus


  • Cancer > Cutaneous (Dermatologic) Oncology
  • Dermatology
  • Autoimmune Blistering Diseases
  • Epidermolysis Bullosa

Academic Appointments


Administrative Appointments


  • Member, Institute for Immunity, Transplantation and Infection (ITI) (2011 - Present)
  • Founding Member/Core Investigator, Program in Epithelial Biology, Stanford University (1999 - Present)
  • Director, Blistering Disease Clinic, Department of Dermatology, Stanford University School of Medicine (1995 - Present)
  • Attending Physician, Dermatology Service, Palo Alto VA Medical Center (1995 - Present)
  • Member, Medical Institutional Review Board 4, Stanford University School of Medicine (2005 - Present)
  • Member, Cancer Center, Stanford University School of Medicine (2004 - Present)

Professional Education


  • Internship:UCSF House Staff Office (1989) CA
  • Medical Education:St Louis University School of Medicine (1988) MO
  • Residency:Oregon Health Science University (1994) OR
  • Board Certification: Dermatology, American Board of Dermatology (1995)
  • Fellowship:Shriner's Hospital - Portland (1990) OR

Research & Scholarship

Current Research and Scholarly Interests


The extracellular matrix of epithelial tissues plays a critical role in many important biological processes such as tissue development and differentiation, wound healing, tumor invasion, cell proliferation and cell migration. A highly organized array of these molecules, termed the basement membrane, lies at the interface of epithelial tissues with surrounding stroma. Cell surface receptors termed integrins transmit the informational cues brought about by changes in the extracellular environment, and transmit them, via intracellular signaling, to effect changes in epithelial gene expression. Laminins and collagens are molecules of the extracellular matrix which play particularly crucial roles in epithelial development.

EXTRACELLULAR MATRIX IN CARCINOMA INVASION
Laminin-5 and its cell surface receptor a6b4 integrin are required for development of squamous cell carcinomas. Lack of either of these molecules results in a lack of tumor growth, whereas overexpression of these molecules correlates with increasing tumor invasiveness and a worsening patient prognosis. We have identified that laminin-5 undergoes proteolytic processing of two of its three chains, via mammalian Tolloid, a metalloprotease of the astacin family. Processing of laminin-5 promotes tumor invasion. We are currently studying the mechanisms whereby these processing events influence tumor cell invasion, migration and metastasis. Type VII collagen appears to play a key role in tumor invasion, and appears to operate through association with laminin-5. We are currently studying the mechanism of this association and its role in tumorigenesis. The laminin-5 receptor a6b4 integrin interacts with laminin-5 at one end and with intracellular protein complexes at the other end, through which it transmits important signaling information to the cell. Disruption of laminin-5 binding or binding to the intracellular protein plectin, through site directed mutagenesis results in a lack of tumor growth, indicating that integrin binding to laminin-5 and integrin binding to plectin are both critical in tumor progression. We are currently studying the mechanisms whereby these binding events promote tumor progression. The molecule collagen XVII is closely associated with laminin-5 and a6b4 integrin and also is required for tumor invasion. The C-terminal extracellular domain of this molecule appears to play a critical role in interaction with extracellular matrix molecules and in organizing cell adhesion structures. It is also a focus of our studies of the role of extracellular matrix in tumor progression.

EXTRACELLULAR MATRIX IN HAIR DEVELOPMENT
Laminin-10 is a widely expressed molecule found in a number of epithelial tissues. Lack of laminin-10 in lama5 -/- mice results in aberrant tissue development. In the skin, there is a complete lack of hair follicle development. Exogenous delivery of laminin-10 rescues hair development in lama5 -/- skin. Laminin-10 appears to act as a potent morphogen, stimulating hair follicle development in the skin of these mice. We are currently examining this system to further understand the mechanisms whereby laminin-10 facilitates hair follicle development and basal cell carcinoma invasion, a developmentally similar process.

EXTRACELLULAR MATRIX IN EPITHELIAL ADHESION
Laminin-5, a6b4 integrin, type VII collagen and type XVII collagen each promote epithelial-mesenchymal cohesion. Defects of these molecule, in the inherited group of diseases known as epidermolysis bullosa, result in profound epithelial adhesion defects, causing extensive skin and mucosal blisters and erosions. As part of a Departmental effort, in association with the Khavari laboratory, our laboratory is participating in the study of new and novel forms of extracellular matrix gene replacement in these adhesion disorders, with the goal of translating these techniques to the clinical setting.

Clinical Trials


  • Characteristics of Adult Patients With Recessive Dystrophic Epidermolysis Bullosa Recruiting

    Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited blistering disease caused by the absence of type VII collagen. Patients with RDEB develop large, severely painful blisters and open wounds from minor trauma to their skin. We are screening RDEB subjects to determine additional characteristics of patients who survive to adulthood.

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  • Grafting of Epidermolysis Bullosa Wounds Using Cultured Revertant Autologous Keratinocytes Not Recruiting

    The term epidermolysis bullosa (EB) is used to describe a group of genetic skin diseases associated with skin weakness, blisters, and chronic wounds. "Revertant mosaicism" means that there are two genetically different populations of cells due to spontaneous mutations. Some EB patients have normal, non-fragile skin patches which may be areas of revertant mosaicism. In the revertant areas, the proteins function normally, like non-EB skin. In this study, we plan to culture cells from the revertant areas and graft them on to the wounded areas.

    Stanford is currently not accepting patients for this trial. For more information, please contact Emily S Gorell, MS, 650-721-7166.

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  • Gene Transfer for Recessive Dystrophic Epidermolysis Bullosa Recruiting

    Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited blistering skin disease caused by absence of a protein known as type VII collagen. Patients with RDEB develop large, severely painful blisters and open wounds from minor trauma to their skin. This trial will create a graft, which the investigators call "LEAES," of the patient's own skin that has been genetically engineered in the investigators lab to express this missing protein. The purpose of this study is to achieve proof-of-concept for this general approach to cell-based gene therapy in humans and to set the stage for further therapeutic extension in RDEB. The investigators will basically take a subject's own cells, correct them in culture, and then transplant the corrected cells back onto them.

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  • Characteristics of Patients With Dystrophic Epidermolysis Bullosa Recruiting

    Dystrophic epidermolysis bullosa (DEB) is a group of diseases caused by genetic mutations in the gene for type VII collagen. DEB can be severe or mild with the recessive disease usually being more severe. Patients with DEB develop large, severely painful blisters and open wounds from minor trauma to their skin. We are screening subjects with DEB to evaluate characteristics of the subjects and their cells in order to develop new strategies of therapy.

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Teaching

2013-14 Courses


Publications

Journal Articles


  • Laminin-511 is an epithelial message promoting dermal papilla development and function during early hair morphogenesis GENES & DEVELOPMENT Gao, J., DeRouen, M. C., Chen, C., Nguyen, M., Nguyen, N. T., Ido, H., Harada, K., Sekiguchi, K., Morgan, B. A., Miner, J. H., Oro, A. E., Marinkovich, M. P. 2008; 22 (15): 2111-2124

    Abstract

    Hair morphogenesis takes place through reciprocal epithelial and mesenchymal signaling; however, the mechanisms controlling signal exchange are poorly understood. Laminins are extracellular proteins that play critical roles in adhesion and signaling. Here we demonstrate the mechanism of how laminin-511 controls hair morphogenesis. Dermal papilla (DP) from laminin-511 mutants showed developmental defects by E16.5, including a failure to maintain expression of the key morphogen noggin. This maintenance was critical as exogenous introduction of noggin or sonic hedgehog (Shh) produced downstream from noggin was sufficient to restore hair follicle development in lama5(-/-) (laminin-511-null) skin. Hair development required the beta1 integrin binding but not the heparin binding domain of laminin-511. Previous studies demonstrated that Shh signaling requires primary cilia, microtubule-based signaling organelles. Laminin-511 mutant DP showed decreased length and structure of primary cilia in vitro and in vivo. Laminin-511, but not laminin-111, restored primary cilia formation in lama5(-/-) mesenchyme and triggered noggin expression in an Shh- and PDGF-dependent manner. Inhibition of laminin-511 receptor beta1 integrin disrupted DP primary cilia formation as well as hair development. These studies show that epithelial-derived laminin-511 is a critical early signal that directs ciliary function and DP maintenance as a requirement for hair follicle downgrowth.

    View details for DOI 10.1101/gad.1689908

    View details for Web of Science ID 000258117500012

    View details for PubMedID 18676816

  • Targeting a tumor-specific laminin domain critical for human carcinogenesis CANCER RESEARCH Tran, M., Rousselle, P., Nokelainen, P., Tallapragada, S., Nguyen, N. T., Fincher, E. F., Marinkovich, M. P. 2008; 68 (8): 2885-2894

    Abstract

    Laminin-332 is critical for squamous cell carcinoma (SCC) tumorigenesis, but targeting it for cancer therapy has been unachievable due to key role of laminin-332 in promoting tissue integrity. Here, we show that a portion of laminin-332, termed G45, which is proteolytically removed and absent in normal tissues, is prominently expressed in most human SCC tumors and plays an important role in human SCC tumorigenesis. Primary human keratinocytes lacking G45 (DeltaG45) showed alterations of basal receptor organization, impaired matrix deposition, and increased migration. After SCC transformation, the absence of G45 domain in DeltaG45 cells was associated with deficient extracellular signal-regulated kinase and phosphotidylinositol 3-kinase (PI3K) pathway activation, impaired invasion, deficient metalloproteinase activity, and absent tumorgenicity in vivo. Expression of G45 or activated PI3K subunit in DeltaG45 cells reversed these abnormalities. G45 antibody treatment induced SCC tumor apoptosis, decreased SCC tumor proliferation, and markedly impaired human SCC tumorigenesis in vivo without affecting normal tissue adhesion. These results show a remarkable selectivity of expression and function for laminin-332 G45 in human SCC tumorigenesis and implicate it as a specific target for anticancer therapy.

    View details for DOI 10.1158/0008-5472.CAN-07-6160

    View details for Web of Science ID 000255100500041

    View details for PubMedID 18413757

  • A laminin-collagen complex drives human epidermal carcinogenesis through phosphoinositol-3-kinase activation CANCER RESEARCH Waterman, E. A., Sakai, N., Nguyen, N. T., Horst, B. A., Veitch, D. P., Dey, C. N., Ortiz-Urda, S., Khavari, P. A., Marinkovich, M. P. 2007; 67 (9): 4264-4270

    Abstract

    Laminin-332 (formerly laminin-5) and collagen VII are basement membrane proteins expressed at the invasive front of human squamous cell carcinoma (SCC) tumors. These proteins have protumorigenic properties, but whether laminin-332 and collagen VII promote SCC tumors by providing adhesion or other nonadhesive extracellular cues, or whether laminin-332 and collagen VII interact together in this process remains unknown. In this study, we examined the role of these molecules by a structural approach using an in vivo model of human SCC tumorigenesis. Here, we show that individual domains (VI and V-III) on the laminin-332 beta3 chain provide distinct and highly divergent cell adhesion and tumor-promoting functions. We found that laminin beta3 domain VI provided a critical role in the assembly of stable adhesion complexes, but this domain was not required in SCC tumors. Instead, we found that laminin beta3 domain V-III played an essential role in SCC carcinogenesis/invasion through binding to collagen VII, which in turn, led to phosphoinositol-3-kinase activation and protection from apoptosis. Overexpression of constitutively active p110 phosphoinositol-3-kinase subunit was sufficient to restore invasion and tumorigenesis in transformed cells lacking laminin-332/collagen VII interaction in a manner independent of cellular adhesion. These studies show distinctive adhesive and signaling functions in individual domains of laminin-332, one which is required for normal epithelial adhesion and one which is required for SCC tumorigenesis. This uncoupling of stable adhesion from tumor progression in our studies suggests that laminin-332/collagen VII interaction promotes epidermal carcinogenesis through signaling rather than adhesion.

    View details for DOI 10.1158/0008-5472.CAN-06-4141

    View details for Web of Science ID 000246330300034

    View details for PubMedID 17483338

  • Tumour microenvironment: laminin 332 in squamous-cell carcinoma. Nature reviews. Cancer Marinkovich, M. P. 2007; 7 (5): 370-380

    Abstract

    Basement membranes can be a barrier to tumour growth, but basement membrane molecules, including laminins, are also important autocrine factors produced by cancers to promote tumorigenesis. Many studies have shown the importance of laminin 332 (previously known as laminin 5) in this process, especially in squamous cell carcinoma. Through interactions with several cell-surface receptors (including alpha6beta4 and alpha3beta1 integrins, epidermal growth factor receptor and syndecan 1) and other basement membrane components (including type VII collagen), laminin 332 drives tumorigenesis through phosphatidylinositol-3 kinase (PI3K) and RAC1 activation, promoting tumour invasion and cell survival. The extracellular interactions of laminin 332 appear amenable to antibody-mediated therapies.

    View details for PubMedID 17457303

  • Type VII collagen is required for Ras-driven human epidermal tumorigenesis SCIENCE Ortiz-Urda, S., Garcia, J., Green, C. L., Chen, L., Lin, Q., Veitch, D. P., Sakai, L. Y., LEE, H., Marinkovich, M. P., Khavari, P. A. 2005; 307 (5716): 1773-1776

    Abstract

    Type VII collagen defects cause recessive dystrophic epidermolysis bullosa (RDEB), a blistering skin disorder often accompanied by epidermal cancers. To study the role of collagen VII in these cancers, we examined Ras-driven tumorigenesis in RDEB keratinocytes. Cells devoid of collagen VII did not form tumors in mice, whereas those retaining a specific collagen VII fragment (the amino-terminal noncollagenous domain NC1) were tumorigenic. Forced NC1 expression restored tumorigenicity to collagen VII-null epidermis in a non-cell-autonomous fashion. Fibronectin-like sequences within NC1 (FNC1) promoted tumor cell invasion in a laminin 5-dependent manner and were required for tumorigenesis. Tumor-stroma interactions mediated by collagen VII thus promote neoplasia, and retention of NC1 sequences in a subset of RDEB patients may contribute to their increased susceptibility to squamous cell carcinoma.

    View details for DOI 10.1126/science.1106209

    View details for Web of Science ID 000227883900044

    View details for PubMedID 15774758

  • A critical reappraisal of the current data on drug-induced linear immunoglobulin A bullous dermatosis: A real and separate nosological entity? JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY Fortuna, G., Cesar Salas-Alanis, J., Guidetti, E., Marinkovich, M. P. 2012; 66 (6): 988-994

    Abstract

    Linear IgA disease (LAD) has been associated with a variety of drugs over the past 30 years.To review current literature on all available cases of drug-induced LAD, in order to ascertain whether a close relationship is justified, so that it constitutes a real and separate nosological entity.The PubMed database was searched for all articles written in English related to drug-induced LAD published between January 1980 and December 2010.The literature review shows that at least 84 articles were published, describing a total of 103 patients. Of these articles, only 46, from 13 countries, were included in this analysis, with a total of 52 patients: 24 (46.2%) were believed to be induced by vancomycin and 28 (53.8%) by drugs other than vancomycin. Challenge-dechallenge-rechallenge testing protocol was performed on only 6 (11.5%) of 52 patients, of which only 5 showed a positive result, while the Naranjo algorithm was performed on only 2 of them (0.3%).The evidence of this review analysis is based only on case reports. No study on large samples of drug-induced LAD is currently available.The literature analysis reveals no strong scientific evidence to support the notion that some drugs have induced LAD; therefore in many reviewed cases, we must question whether drug-induced LAD is really the underlying entity. Further and thorough investigations using one of the available algorithms for adverse drug reaction are warranted.

    View details for DOI 10.1016/j.jaad.2011.09.018

    View details for Web of Science ID 000303979900033

    View details for PubMedID 22169257

  • Linear immunoglobulin A bullous dermatosis CLINICS IN DERMATOLOGY Fortuna, G., Marinkovich, M. P. 2012; 30 (1): 38-50

    Abstract

    Linear immunoglobulin A (IgA) bullous dermatosis, also known as linear IgA disease, is an autoimmune mucocutaneous disorder characterized by subepithelial bullae, with IgA autoantibodies directed against several different antigens in the basement membrane zone. Its immunopathologic characteristic resides in the presence of a continuous linear IgA deposit along the basement membrane zone, which is clearly visible on direct immunofluorescence. This disorder shows different clinical features and distribution when adult-onset of linear IgA disease is compared with childhood-onset. Diagnosis is achieved via clinical, histopathologic, and immunopathologic examinations. Two common therapies are dapsone and sulfapyridine, which reduce the inflammatory response and achieve disease remission in a variable period of time.

    View details for DOI 10.1016/j.clindermatol.2011.03.008

    View details for Web of Science ID 000298212600005

    View details for PubMedID 22137225

  • Molecular organization of the basement membrane zone CLINICS IN DERMATOLOGY Hashmi, S., Marinkovich, M. P. 2011; 29 (4): 398-411

    Abstract

    The dermal-epidermal basement membrane is a complex assembly of proteins that provide adhesion and regulate many important processes such as development, wound healing, and cancer progression. This contribution focuses on the structure and function of individual components of the basement membrane, how they assemble together, and how they participate in human tissues and diseases, with an emphasis on skin involvement. Understanding the composition and structure of the basement membrane provides insight into the pathophysiology of inherited blistering disorders, such as epidermolysis bullosa, and acquired bullous diseases, such as the pemphigoid group of autoimmune diseases and epidermolysis bullosa acquisita.

    View details for DOI 10.1016/j.clindermatol.2011.01.009

    View details for Web of Science ID 000292353800006

    View details for PubMedID 21679867

  • Laminin-511 and integrin beta-1 in hair follicle development and basal cell carcinoma formation BMC DEVELOPMENTAL BIOLOGY DeRouen, M. C., Zhen, H., Tan, S. H., Williams, S., Marinkovich, M. P., Oro, A. E. 2010; 10

    Abstract

    Initiation of the hair follicle placode and its subsequent growth, maturation and cycling in post-natal skin requires signaling interactions between epithelial cells and adjacent dermal cells and involves Shh signaling via the primary cilium. Previous reports have implicated laminins in hair follicle epithelial invagination.Here we use a human BCC model system and mouse mutants to re-evaluate the role of laminin-511 in epithelial invagination in the skin. Blocking laminin 511 and 332 in BCCs maintains primary cilia and Shh signalling, but prevents invagination. Similarly, in laminin-511 and dermal beta-1 integrin mutants, dermal papilla development and primary cilia formation are normal. Dermal beta-1 integrin mutants have normal hair follicle development.Our data provides support for a primary role of laminin-511 promoting hair follicle epithelial downgrowth without affecting dermal primary cilia and Shh target gene induction.

    View details for DOI 10.1186/1471-213X-10-112

    View details for Web of Science ID 000284878300001

    View details for PubMedID 21067603

  • Observations of Skin Grafts Derived from Keratinocytes Expressing Selectively Engineered Mutant Laminin-332 Molecules JOURNAL OF INVESTIGATIVE DERMATOLOGY Sakai, N., Waterman, E. A., Nguyen, N. T., Keene, D. R., Marinkovich, M. P. 2010; 130 (8): 2147-2150

    View details for DOI 10.1038/jid.2010.85

    View details for Web of Science ID 000279984000029

    View details for PubMedID 20393483

  • Role of Dermal-Epidermal Basement Membrane Zone in Skin, Cancer, and Developmental Disorders DERMATOLOGIC CLINICS Ko, M. S., Marinkovich, M. P. 2010; 28 (1): 1-?

    Abstract

    The dermal-epidermal basement membrane zone is an important epithelial and stromal interface, consisting of an intricately organized collection of intracellular, transmembrane, and extracellular matrix proteins. The basement membrane zone has several main functions including acting as a permeability barrier, forming an adhesive interface between epithelial cells and the underlying matrix, and controlling cellular organization and differentiation. This article identifies key molecular players of the dermal-epidermal membrane zone, and highlights recent research studies that have identified structural and functional roles of these components in the context of various blistering, neoplastic, and developmental syndromes.

    View details for DOI 10.1016/j.det.2009.10.001

    View details for Web of Science ID 000278970000002

    View details for PubMedID 19945611

  • Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies JOURNAL OF INVESTIGATIVE DERMATOLOGY Nguyen, B., Dusek, R. L., Beaudry, V. G., Marinkovich, M. P., Attardi, L. D. 2009; 129 (7): 1710-1718

    Abstract

    Pemphigus vulgaris (PV) is an autoimmune bullous disease in which autoantibodies against proteins of the desmosomal adhesion complex perturb desmosomal function, leading to intercellular adhesion defects in the oral mucosa and skin. Previous studies have demonstrated a central role for downregulation of the desmosomal cadherin desmoglein 3 (DSG3) in the pathogenesis of PV. However, the effects of non-cadherin desmosomal proteins in modulating the cellular manifestations of PV remain poorly understood. Here, we characterize the expression and functional importance of Perp, a newly discovered tetraspan desmosomal protein, in PV. Our data demonstrate that PV autoantibodies disrupt Perp expression at the membrane and trigger its internalization along with DSG3 into the endosomal pathway, where it is ultimately targeted to the lysosome for degradation. We further show that Perp deficiency exacerbates the pathogenic effects of PV autoantibodies on keratinocytes by enhancing both the depletion of desmosomal DSG3 and intercellular adhesion defects. Together, our findings highlight the importance of non-cadherin desmosomal proteins in modulating PV phenotypes and provide new insight into Perp's role in the desmosome.

    View details for DOI 10.1038/jid.2008.419

    View details for Web of Science ID 000267270300019

    View details for PubMedID 19158843

  • Bridging structure with function: Structural, regulatory, and developmental role of laminins INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY Tzu, J., Marinkovich, M. P. 2008; 40 (2): 199-214

    Abstract

    The basement membrane is a highly intricate and organized portion of the extracellular matrix that interfaces with a variety of cell types including epithelial, endothelial, muscle, nerve, and fat cells. The laminin family of glycoproteins is a major constituent of the basement membrane. The 16 known laminin isoforms are formed from combinations of alpha, beta, and gamma chains, with each chain containing specific domains capable of interacting with cellular receptors such as integrins and other extracellular ligands. In addition to its role in the assembly and architectural integrity of the basement membrane, laminins interact with cells to influence proliferation, differentiation, adhesion, and migration, processes activated in normal and pathologic states. In vitro these functions are regulated by the post-translational modifications of the individual laminin chains. In vivo laminin knockout mouse studies have been particularly instructive in defining the function of specific laminins in mammalian development and have also highlighted its role as a key component of the basement membrane. In this review, we will define how laminin structure complements function and explore its role in both normal and pathologic processes.

    View details for DOI 10.1016/j.biocel.2007.07.015

    View details for Web of Science ID 000252061800006

    View details for PubMedID 17855154

  • Discovery of basement membrane components. journal of investigative dermatology Marinkovich, M. P. 2008; 128 (E2): E3-4

    View details for DOI 10.1038/skinbio.6250012

    View details for PubMedID 21233818

  • What's new in blistering disorders? CURRENT ALLERGY AND ASTHMA REPORTS Chaudbari, P., Marinkovich, M. P. 2007; 7 (4): 255-263

    Abstract

    From the characterization of new animal models for the study of disease pathogenesis, to the demonstration of new therapeutic modalities, many developments have revolutionized the field of autoimmune bullous diseases in the past several years. This review highlights many of the significant advances that have taken place in the diagnosis, pathogenesis, and treatment options for pemphigus, pemphigoid, epidermolysis bullosa acquisita, and immunoglobulin (Ig) A-mediated bullous disorders.

    View details for Web of Science ID 000247296400004

    View details for PubMedID 17547846

  • Integrin beta 4 regulates migratory behavior of keratinocytes by determining laminin-332 organization JOURNAL OF BIOLOGICAL CHEMISTRY Sehgal, B. U., DeBiase, P. J., Matzno, S., Chew, T., Claiborne, J. N., Hopkinson, S. B., Russell, A., Marinkovich, M. P., Jones, J. C. 2006; 281 (46): 35487-35498

    Abstract

    Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.

    View details for DOI 10.1074/jbc.M606317200

    View details for Web of Science ID 000241933700082

    View details for PubMedID 16973601

  • beta 4 integrin and epidermal growth factor coordinately regulate electric field-mediated directional migration via Rac1 MOLECULAR BIOLOGY OF THE CELL Pullar, C. E., Baier, B. S., Kariya, Y., Russell, A. J., Horst, B. A., Marinkovich, M. P., Isseroff, R. R. 2006; 17 (11): 4925-4935

    Abstract

    Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (beta)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), beta4 integrin-null human keratinocytes (beta4-), or those in which beta4 integrin was reexpressed (beta4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of beta4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding-defective beta4 (beta4+AD) or beta4 with a truncated cytoplasmic tail (beta4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the beta4 integrin ligand-binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus.

    View details for DOI 10.1091/mbc.E06-05-0433

    View details for Web of Science ID 000241993500030

    View details for PubMedID 16914518

  • Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells JOURNAL OF VIROLOGY Culp, T. D., Budgeon, L. R., Marinkovich, M. P., Meneguzzi, G., Christensen, N. D. 2006; 80 (18): 8940-8950

    Abstract

    Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing alpha6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking alpha6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing alpha6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.

    View details for DOI 10.1128/JVI.00724-06

    View details for Web of Science ID 000240384800010

    View details for PubMedID 16940506

  • Overexpression of laminin-8 in human dermal microvascular endothelial cells promotes angiogenesis-related functions JOURNAL OF INVESTIGATIVE DERMATOLOGY Li, J., Zhou, L., Tran, H. T., Chen, Y., Nguyen, N. E., Karasek, M. A., Marinkovich, M. P. 2006; 126 (2): 432-440

    Abstract

    This study examined the effects of endogenous overexpression of laminin-8 on angiogenesis and wound healing in primary human dermal microvascular endothelial cells (HDMECs). HDMECs expressed laminin-8 and laminin-10, but no other laminins, as determined by radioimmunoprecipitation assay using a panel of antibodies to individual laminin chains. To study laminin-8 function, full-length human laminin alpha4 cDNA was retrovirally transferred to HDMEC, and specific overexpression of laminin-8 was verified by Western blot. Laminin-8 overexpression promoted endothelial cell spreading and migration in scratch assays and accelerated angiogenic tubule formation in collagen gel overlay assays. Strong inhibitory effect of beta1 integrin and weak inhibition by alphavbeta3 integrin antibodies were observed in laminin-8-stimulated cell migration, but only beta1 integrin antibodies affected tubule formation. These studies suggest that laminin-8 overexpression may prove to be a useful method to engineer HDMECs to promote angiogenesis and wound repair.

    View details for DOI 10.1038/sj.jid.5700089

    View details for Web of Science ID 000238968400028

    View details for PubMedID 16374451

  • A simplified laminin nomenclature MATRIX BIOLOGY Aumailley, M., Bruckner-Tuderman, L., Carter, W. G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., ENGVALL, E., Hohenester, E., Jones, J. C., Kleinman, H. K., Marinkovich, M. P., Martin, G. R., Mayer, U., Meneguzzi, G., Miner, J. H., Miyazaki, K., PATARROYO, M., Paulsson, M., Quaranta, V., Sanes, J. R., Sasaki, T., Sekiguchi, K., Sorokin, L. M., Talts, J. F., Tryggvason, K., UITTO, J., VIRTANEN, I., von der Mark, K., Wewer, U. M., Yamada, Y., Yurchenco, P. D. 2005; 24 (5): 326-332

    Abstract

    A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

    View details for DOI 10.1016/j.matbio.2005.05.006

    View details for Web of Science ID 000231205300002

    View details for PubMedID 15979864

  • Involvement of p53 and p16 tumor suppressor genes in recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma JOURNAL OF INVESTIGATIVE DERMATOLOGY Arbiser, J. L., Fan, C. Y., Su, X. B., van Emburgh, B. O., Cerimele, F., Miller, M. S., Harvell, J., Marinkovich, M. P. 2004; 123 (4): 788-790

    Abstract

    Recessive dystrophic epidermolysis bullosa (RDEB) is an autosomal recessive disorder characterized by the loss of collagen type VII, an intrinsic component of the anchoring fibrils, which attach the epidermis to the dermis. Of the genetic blistering disorders, RDEB has the highest rate of morbidity and mortality, with morbidity arising from fusion of digits in a mitten-glove deformity and growth retardation associated with anemia. The leading cause of death in RDEB is cutaneous squamous cell carcinoma, which causes death through invasion and metastasis. In order to better understand the pathogenesis of these rare but aggressive squamous cell carcinoma (SCC), we analyzed them for mutations in p53 and loss of p16ink4a. Three tumors demonstrated mutations in the p53 tumor suppressor gene. We also analyzed SCC from patients with RDEB for the presence of p16ink4a hypermethylation, and found two tumors that have loss of p16ink4a through hypermethylation. This is the first description of specific abnormalities in tumor suppressor genes in RDEB associated SCC, and demonstrates that alterations in both p53 and p16ink4a can contribute to RDEB associated SCC.

    View details for DOI 10.1111/j.0022-202X.2004.23418.x

    View details for Web of Science ID 000223982900031

    View details for PubMedID 15373786

  • Kinetics and specificity of Fas ligand induction in toxic epidermal necrolysis ARCHIVES OF DERMATOLOGY Chang, H. Y., Cooper, Z. A., Swetter, S. A., Marinkovich, M. P. 2004; 140 (2): 242-244

    View details for Web of Science ID 000188877100024

    View details for PubMedID 14967808

  • Mature human thymocytes migrate on laminin-5 with activation of metalloproteinase-14 and cleavage of CD44 JOURNAL OF IMMUNOLOGY Vivinus-Nebot, M., Rousselle, P., Breittmayer, J. P., Cenciarini, C., Berrih-Aknin, S., Spong, S., Nokelainen, P., Cottrez, F., Marinkovich, M. P., Bernard, A. 2004; 172 (3): 1397-1406

    Abstract

    We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 gamma(2) chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.

    View details for Web of Science ID 000188378700009

    View details for PubMedID 14734715

  • Autocrine laminin-5 ligates alpha 6 beta 4 integrin and activates RAC and NF kappa B to mediate anchorage-independent survival of mammary tumors JOURNAL OF CELL BIOLOGY Zahir, N., Lakins, J. N., Russell, A., Ming, W. Y., Chatterjee, C., Rozenberg, G. I., Marinkovich, M. P., Weaver, V. M. 2003; 163 (6): 1397-1407

    Abstract

    Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling.

    View details for DOI 10.1083/jcb.200302023

    View details for Web of Science ID 000187583500021

    View details for PubMedID 14691145

  • alpha 6 beta 4 integrin regulates keratinocyte chemotaxis through differential GTPase activation and antagonism of alpha 3 beta 1 integrin JOURNAL OF CELL SCIENCE Russell, A. J., Fincher, E. F., Millman, L., Smith, R., Vela, V., Waterman, E. A., Dey, C. N., Guide, S., Weaver, V. M., Marinkovich, M. P. 2003; 116 (17): 3543-3556

    Abstract

    Growth factor-induced cell migration and proliferation are essential for epithelial wound repair. Cell migration during wound repair also depends upon expression of laminin-5, a ligand for alpha 6 beta 4 integrin. We investigated the role of alpha 6 beta 4 integrin in laminin-5-dependent keratinocyte migration by re-expressing normal or attachment-defective beta 4 integrin in beta 4 integrin null keratinocytes. We found that expression of beta 4 integrin in either a ligand bound or ligand unbound state was necessary and sufficient for EGF-induced cell migration. In a ligand bound state, beta 4 integrin supported EGF-induced cell migration though sustained activation of Rac1. In the absence of alpha 6 beta 4 integrin ligation, Rac1 activation became tempered and EGF chemotaxis proceeded through an alternate mechanism that depended upon alpha 3 beta 1 integrin and was characterized by cell scattering. alpha 3 beta 1 integrin also relocalated from cell-cell contacts to sites of basal clustering where it displayed increased conformational activation. The aberrant distribution and activation of alpha 3 beta 1 integrin in attachment-defective beta 4 cells could be reversed by the activation of Rac1. Conversely, in WT beta 4 cells the normal cell-cell localization of alpha 3 beta 1 integrin became aberrant after the inhibition of Rac1. These studies indicate that the extracellular domain of beta 4 integrin, through its ability to bind ligand, functions to integrate the divergent effects of growth factors on the cytoskeleton and adhesion receptors so that coordinated keratinocyte migration can be achieved.

    View details for DOI 10.1242/jcs.00663

    View details for Web of Science ID 000187394900010

    View details for PubMedID 12865436

  • Topical tacrolimus is a useful adjunctive therapy for bullous pemphigoid ARCHIVES OF DERMATOLOGY Chu, J., Bradley, M., Marinkovich, M. P. 2003; 139 (6): 813-815

    View details for Web of Science ID 000183478000027

    View details for PubMedID 12810523

  • Laminin-10 is crucial for hair morphogenesis EMBO JOURNAL Li, J., Tzu, J., Chen, Y., Zhang, Y. P., Nguyen, N. T., Gao, J., Bradley, M., Keene, D. R., Oro, A. E., Miner, J. H., Marinkovich, M. P. 2003; 22 (10): 2400-2410

    Abstract

    The role of the extracellular matrix in cutaneous morphogenesis is poorly understood. Here, we describe the essential role of laminin-10 (alpha5beta1gamma1) in hair follicle development. Laminin-10 was present in the basement membrane of elongating hair germs, when other laminins were downregulated, suggesting a role for laminin-10 in hair development. Treatment of human scalp xenografts with antibodies to laminin-10, or its receptor beta1 integrin, produced alopecia. E16.5 Lama5 -/- mouse skin, lacking laminin-10, contained fewer hair germs compared with controls, and after transplantation, Lama5 -/- skin showed a failure of hair germ elongation followed by complete hair follicle regression. Lama5 -/- skin showed defective basement membrane assembly, without measurable increases in anoikis. Instead, Lama5 -/- skin showed decreased expression of early hair markers including sonic hedgehog and Gli1, implicating laminin-10 in developmental signaling. Intriguingly, treatment of Lama5 -/- skin with purified laminin-10 corrected basement membrane defects and restored hair follicle development. We conclude that laminin-10 is required for hair follicle development and report the first use of exogenous protein to correct a cutaneous developmental defect.

    View details for Web of Science ID 000182957100010

    View details for PubMedID 12743034

  • Mammalian tolloid metalloproteinase, and not matrix metalloprotease 2 or membrane type 1 metalloprotease, processes laminin-5 in keratinocytes and skin JOURNAL OF BIOLOGICAL CHEMISTRY Veitch, D. P., Nokelainen, P., McGowan, K. A., Nguyen, T. T., Nguyen, N. E., Stephenson, R., Pappano, W. N., Keene, D. R., Spong, S. M., Greenspan, D. S., Findell, P. R., Marinkovich, P. M. 2003; 278 (18): 15661-15668

    Abstract

    Laminin-5, a major adhesive ligand for epithelial cells, undergoes processing of its gamma2 and alpha3 chains. This study investigated the mechanism of laminin-5 processing by keratinocytes. BI-1 (BMP-1 isoenzyme inhibitor-1), a selective inhibitor of a small group of astacin-like metalloproteinases, which includes bone morphogenetic protein 1 (BMP-1), mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mammalian Tolloid-like 2 (mTLL-2), inhibited the processing of laminin-5 gamma2 and alpha3 chains in keratinocyte cultures in a dose-dependent manner. In a proteinase survey, all BMP-1 isoenzymes processed human laminin-5 gamma2 and alpha3 chains to 105- and 165-kDa fragments, respectively. In contrast, MT1-MMP and MMP-2 did not cleave the gamma2 chain of human laminin-5 but processed the rat laminin gamma2 chain to an 80-kDa fragment. An immunoblot and quantitative PCR survey of the BMP-1 isoenzymes revealed expression of mTLD in primary keratinocyte cultures but little or no expression of BMP-1, mTLL-1, or mTLL-2. mTLD was shown to cleave the gamma2 chain at the same site as the previously identified BMP-1 cleavage site. In addition, mTLD/BMP-1 null mice were shown to have deficient laminin-5 processing. Together, these data identify laminin-5 as a substrate for mTLD, suggesting a role for laminin-5 processing by mTLD in the skin.

    View details for DOI 10.1074/jbc.M210588200

    View details for Web of Science ID 000182680000027

    View details for PubMedID 12473650

  • NF-kappa B blockade and oncogenic Ras trigger invasive human epidermal neoplasia NATURE Dajee, M., Lazarov, M., Zhang, J. Y., Cai, T., Green, C. L., Russell, A. J., Marinkovich, M. P., Tao, S. Y., Lin, Q., Kubo, Y., Khavari, P. A. 2003; 421 (6923): 639-643

    Abstract

    The nuclear factor NF-kappaB and oncogenic Ras can alter proliferation in epidermis, the most common site of human cancer. These proteins are implicated in epidermal squamous cell carcinoma in mice, however, the potential effects of altering their function are uncertain. Whereas inhibition of NF-kappaB enhances apoptosis in certain tumours, blockade of NF-kappaB predisposes murine skin to squamous cell carcinoma. Because therapeutics inhibiting Ras and NF-kappaB pathways are being developed to treat human cancer, it is essential to assess the effects of altering these regulators. The medical relevance of murine studies is limited, however, by differences between mouse and human skin, and by the greater ease of transforming murine cells. Here we show that in normal human epidermal cells both NF-kappaB and oncogenic Ras trigger cell-cycle arrest. Growth arrest triggered by oncogenic Ras can be bypassed by IkappaBalpha-mediated blockade of NF-kappaB, generating malignant human epidermal tissue resembling squamous cell carcinoma. Human cell tumorigenesis is dependent on laminin 5 and alpha6beta4 integrin. Thus, IkappaBalpha circumvents restraints on growth promotion induced by oncogenic Ras and can act with Ras to induce invasive human tissue neoplasia.

    View details for DOI 10.1038/nature01283

    View details for Web of Science ID 000180803200044

    View details for PubMedID 12571598

  • Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue JOURNAL OF CLINICAL INVESTIGATION Ortiz-Urda, S., Lin, Q., Green, C. L., Keene, D. R., Marinkovich, M. P., Khavari, P. A. 2003; 111 (2): 251-255

    Abstract

    Current therapeutic strategies for genetic skin disorders rely on the complex process of grafting genetically engineered tissue to recipient wound beds. Because fibroblasts synthesize and secrete extracellular matrix, we explored their utility in recessive dystrophic epidermolysis bullosa (RDEB), a blistering disease due to defective extracellular type VII collagen. Intradermal injection of RDEB fibroblasts overexpressing type VII collagen into intact RDEB skin stably restored correctly localized type VII collagen expression in vivo and normalized hallmark RDEB disease features, including subepidermal blistering and anchoring fibril defects. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

    View details for DOI 10.1172/JCI.200317193

    View details for Web of Science ID 000180672900016

    View details for PubMedID 12531881

  • The first international consensus on mucous membrane pemphigoid - Definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators ARCHIVES OF DERMATOLOGY Chan, L. S., Ahmed, A. R., ANHALT, G. J., Bernauer, W., Cooper, K. D., Elder, M. J., Fine, J. D., Foster, S., Ghohestani, R., Hashimoto, T., Hoang-Xuan, T., Kirtschig, G., Korman, N. J., Lightman, S., Lozado-Nur, F., Marinkovich, M. P., Mondino, B. J., Prost-Squarcioni, C., Rogers, R. S., Setterfield, J. F., West, D. P., Wojnarowska, F., Woodley, D. T., Yancey, K. B., Zillikens, D., Zone, J. J. 2002; 138 (3): 370-379

    Abstract

    We aimed to develop consensus-based recommendations for streamlining medical communication among various health care professionals, to improve accuracy of diagnosis and treatment, and to facilitate future investigations for mucous membrane pemphigoid.Because of the highly specific nature of this group of diseases, the 26 invited participants included either international scholars in the field of mucous membrane pemphigoid or experts in cutaneous pharmacology representing the 3 medical disciplines ophthalmology, oral medicine, and dermatology.The first author (L.S.C.) conducted a literature search. Based on the information obtained, international experts who had contributed to the literature in the clinical care, diagnosis, and laboratory investigation for mucous membrane pemphigoid were invited to participate in a consensus meeting aimed at developing a consensus statement.A consensus meeting was convened and conducted on May 10, 1999, in Chicago, Ill, to discuss the relevant issues. The first author drafted the statement based on the consensus developed at the meeting and the participants' written comments. The draft was submitted to all participants for 3 separate rounds of review, and disagreements were reconciled based on literature evidence. The third and final statement incorporated all relevant evidence obtained in the literature search and the consensus developed by the participants. The final statement was approved and endorsed by all 26 participants.Specific consensus-based recommendations were made regarding the definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators for mucous membrane pemphigoid. A system of standard reporting for these patients was proposed to facilitate a uniform data collection.

    View details for Web of Science ID 000174367300010

    View details for PubMedID 11902988

  • Epidermolysis bullosa: new and emerging trends. American journal of clinical dermatology Pai, S., Marinkovich, M. P. 2002; 3 (6): 371-380

    Abstract

    Epidermolysis bullosa is a family of inherited blistering skin disorders characterized by blister formation in response to mechanical trauma. Major types of epidermolysis bullosa include epidermolysis bullosa simplex, hemidesmosomal epidermolysis bullosa, junctional epidermolysis bullosa, and dystrophic epidermolysis bullosa. Current treatment for epidermolysis bullosa consists of supportive care for skin and other organ systems and entails a combination of wound management, infection support for chronic wounds, surgical management as needed, nutritional support, and preventative screening for squamous cell carcinoma in recessive dystrophic epidermolysis bullosa. The regimen must be tailored specifically to the severity and extent of skin and systemic involvement in each case. Recent studies have identified specific protein and genetic abnormalities for most epidermolysis bullosa subtypes. These new advancements in the understanding of molecular pathophysiology have provided much of the basis for current efforts to develop effective gene and protein therapy for epidermolysis bullosa.

    View details for PubMedID 12113646

  • Linear IgA bullous dermatosis CLINICS IN DERMATOLOGY Guide, S. V., Marinkovich, M. P. 2001; 19 (6): 719-727

    View details for Web of Science ID 000172273000009

    View details for PubMedID 11705681

  • A spontaneous canine model of mucous membrane (cicatricial) pemphigoid, an autoimmune blistering disease affecting mucosae and mucocutaneous junctions JOURNAL OF AUTOIMMUNITY Olivry, T., Dunston, S. M., Schachter, M., Xu, L. T., Nguyen, N., Marinkovich, M. P., Chan, L. S. 2001; 16 (4): 411-421

    Abstract

    Mucous membrane pemphigoid (MMP) is a rare autoimmune blistering dermatosis of humans that was previously known as cicatricial pemphigoid. It is characterized by vesicles, ulcers and scarring that affect predominantly mucosae and mucocutaneous junctions. Circulating autoantibodies recognize epitopes on basement membrane proteins such as collagen XVII or laminin-5/6. Herein, we describe the clinico-pathological and immunological characteristics of 17 dogs afflicted with a dermatosis homologous to MMP of humans. Patients exhibited vesicles and erosions predominantly on mucous membranes or mucocutaneous junctions of the mouth, nose, eyes, genitalia or anus. Histopathology revealed subepithelial vesicles with variable dermal inflammation. Direct immunofluorescence demonstrated IgG or complement at the dermoepithelial junction. Indirect immunofluorescence using salt-split epithelia permitted the detection of circulating basement membrane-specific IgG autoantibodies in 15 cases. In 11 patients, autoantibodies recognized the NC16A segment of collagen XVII, as determined by salt-split indirect immunofluorescence, immunoblotting using canine keratinocytes and ELISA with synthetic canine peptides. In one dog, autoantiodies bound to the dermal side of salt-split epithelia and recognized epitopes within the 30 kDa carboxy-terminal segment of human collagen XVII. Canine MMP, like its human counterpart, exhibits distinctive clinical signs and histopathological lesions, yet circulating autoantibodies target different antigenic epitopes. This spontaneous canine model of MMP could prove useful for studies on the pathogenesis or therapy of this human disease.

    View details for DOI 10.1006/jaut.2001.0510

    View details for Web of Science ID 000169515500005

    View details for PubMedID 11437489

  • IgG anti-LABD97 antibodies in bullous pemphigoid patients' sera react with the mid-portion of the BPAg2 ectodomain JOURNAL OF INVESTIGATIVE DERMATOLOGY Egan, C. A., Reddy, E. D., Nie, Z. X., Taylor, T. B., Schmidt, L. A., Meyer, L. J., Petersen, M. J., Hashimoto, T., Marinkovich, M. P., Zone, J. J. 2001; 116 (2): 348-350

    View details for Web of Science ID 000167170700021

    View details for PubMedID 11180014

  • Properties of the collagen type XVII ectodomain - Evidence for N- to C-terminal triple helix folding JOURNAL OF BIOLOGICAL CHEMISTRY Areida, S. K., Reinhardt, D. P., Muller, P. K., FIETZEK, P. P., Kowitz, J., Marinkovich, M. P., Notbohm, H. 2001; 276 (2): 1594-1601

    Abstract

    Collagen XVII is a transmembrane component of hemidesmosomal cells with important functions in epithelial-basement membrane interactions. Here we report on properties of the extracellular ectodomain of collagen XVII, which harbors multiple collagenous stretches. We have recombinantly produced subdomains of the collagen XVII ectodomain in a mammalian expression system. rColXVII-A spans the entire ectodomain from deltaNC16a to NC1, rColXVII-B is similar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains deltaNC16a to C15, and a small C-terminal polypeptide rColXVII-D comprises domains NC6 to NC1. Amino acid analysis of rColXVII-A and -C demonstrated prolyl and lysyl hydroxylation with ratios for hydroxyproline/proline of 0.4 and for hydroxylysine/lysine of 0.5. A small proportion of the hydroxylysyl residues in rColXVII-C ( approximately 3.3%) was glycosylated. Limited pepsin and trypsin degradation assays and analyses of circular dichroism spectra clearly demonstrated a triple-helical conformation for rColXVII-A, -B, and -C, whereas the C-terminal rColXVII-D did not adopt a triple-helical fold. These results were further substantiated by electron microscope analyses, which revealed extended molecules for rColXVII-A and -C, whereas rColXVII-D appeared globular. Thermal denaturation experiments revealed melting temperatures of 41 degrees C (rColXVII-A), 39 degrees C (rColXVII-B), and 35 degrees C (rColXVII-C). In summary, our data suggest that triple helix formation in the ectodomain of ColXVII occurs with an N- to C-terminal directionality.

    View details for Web of Science ID 000166430900098

    View details for PubMedID 11042218

  • Laminins and human disease MICROSCOPY RESEARCH AND TECHNIQUE McGowan, K. A., Marinkovich, M. P. 2000; 51 (3): 262-279

    Abstract

    The laminin protein family has diverse tissue expression patterns and is involved in the pathology of a number of organs, including skin, muscle, and nerve. In the skin, laminins 5 and 6 contribute to dermal-epidermal cohesion, and mutations in the constituent chains result in the blistering phenotype observed in patients with junctional epidermolysis bullosa (JEB). Allelic heterogeneity is observed in patients with JEB: mutations that results in premature stop codons produce a more severe phenotype than do missense mutations. Gene therapy approaches are currently being studied in the treatment of this disease. A blistering phenotype is also observed in patients with acquired cicatricial pemphigoid (CP). Autoantibodies targeted against laminins 5 and 6 destabilize epithelial adhesion and are pathogenic. In muscle cells, laminin alpha 2 is a component of the bridge that links the actin cytoskeleton to the extracellular matrix. In patients with laminin alpha 2 mutations, the bridge is disrupted and mature muscle cells apoptose. Congenital muscular dystrophy (CMD) results. The role of laminin in diseases of the nervous system is less well defined, but the extracellular protein has been shown to serve an important role in peripheral nerve regeneration. The adhesive molecule influences neurite outgrowth, neural differentiation, and synapse formation. The broad spatial distribution of laminin gene products suggests that laminin may be involved in a number of diseases for which pathogenic mechanisms are still being unraveled.

    View details for Web of Science ID 000089953000006

    View details for PubMedID 11054876

  • Autoantibodies to BP180 associated with bullous pemphigoid release interleukin-6 and interleukin-8 from cultured human keratinocytes JOURNAL OF INVESTIGATIVE DERMATOLOGY Schmidt, E., Reimer, S., Kruse, N., Jainta, S., Brocker, E. B., Marinkovich, M. P., Giudice, G. J., Zillikens, D. 2000; 115 (5): 842-848

    Abstract

    Bullous pemphigoid is an inflammatory subepidermal blistering disease that is associated with auto- antibodies to the keratinocyte surface protein, BP180. In addition to the binding of autoantibodies, the infiltration of inflammatory cells is necessary for blister formation. Cytokines, including interleukin-6 and interleukin-8, have been implicated in the disease process of both human and experimental murine bullous pemphigoid. This study was aimed at testing the hypothesis that the binding of anti-BP180 antibodies to their target antigen triggers a signal transduction event that results in the secretion of these pro-inflammatory cytokines. Consistent with this hypothesis, treatment of cultured normal human epidermal keratinocytes with bullous pemphigoid IgG, but not control IgG, led to increased levels of interleukin-6 and interleukin-8, but not interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, interleukin-10, or monocyte chemoattractant protein-1, in the culture medium. This effect was concentration- and time-dependent and was abolished by depleting the bullous pemphigoid IgG of reactivity to two distinct epitopes on the BP180 NC16A domain. Upregulation of interleukin-6 and interleukin-8 was found at both protein and mRNA levels. In addition, bullous pemphigoid IgG did not induce the release of interleukin-6 and interleukin-8 from BP180-deficient keratinocytes obtained from a patient with generalized atrophic benign epidermolysis bullosa. These data indicate that bullous pemphigoid-associated autoantibodies to the human BP180 ectodomain trigger a signal transducing event that leads to expression and secretion of interleukin-6 and interleukin-8 from human keratinocytes.

    View details for Web of Science ID 000165219000010

    View details for PubMedID 11069622

  • Autoantibodies against the processed ectodomain of collagen XVII (BPAG2, BP180) define a canine homologue of linear IgA disease of humans VETERINARY PATHOLOGY Olivry, T., Dunston, S. M., Fahey, M., Nguyen, N., Marinkovich, M. P. 2000; 37 (4): 302-309

    Abstract

    Linear IgA disease (LAD) is an acquired autoimmune subepidermal blistering dermatosis that affects human children and adults. In contrast to bullous pemphigoid, in which autoantibodies recognize transmembrane type XVII collagen (BP180, BPAG2), LAD is associated with skin-fixed and circulating IgA autoantibodies that target LAD-1, the processed extracellular form of type XVII collagen. An immunologic homologue of LAD in humans was identified in two dogs according to the following criteria: 1) erosive, ulcerative, and crusted lesions seen on the face, in the oral cavity, and on the extremities, 2) dermoepidermal clefting present in the basement membrane lamina lucida without inflammation or with mild neutrophilic infiltration, 3) basement membrane-fixed IgG and/or IgA antibodies, and 4) circulating IgA and IgG autoantibodies that target the 120-kd soluble protein LAD-1. The present study establishes unequivocally the existence of a naturally occurring canine model of LAD of humans.

    View details for Web of Science ID 000087996900003

    View details for PubMedID 10896391

  • Subepidermal blistering disease with autoantibodies against a novel dermal 200-kDa antigen JOURNAL OF DERMATOLOGICAL SCIENCE Kawahara, Y., Zillikens, D., Yancey, K. B., Marinkovich, M. P., Nie, Z., Hashimoto, T., Nishikawa, T. 2000; 23 (2): 93-102

    Abstract

    A number of autoimmune subepidermal blistering diseases are characterized by the distinct autoantigens of the cutaneous basement membrane zone. Recently, a few cases with autoantibodies against a novel 200-kDa dermal protein have been reported. We collected nine cases of subepidermal blistering disease with IgG antibodies against this 200-kDa antigen. In this report, we describe the clinical and immunological appearances in these cases. Five cases showed bullous pemphigoid-like features, one case resembled dermatitis herpetiformis, and another case showed mixed features of bullous pemphigoid and linear IgA bullous dermatosis. It was interesting to note that psoriasis coexisted in four cases. By indirect immunofluorescence on 1 M NaCl split skin, IgG antibodies from all sera reacted with the dermal side of the split. By immunoblot analysis, IgG antibodies recognized a 200-kDa protein of dermal extract. IgG affinity-purified antibodies on the 200-kDa immunoblot membrane stained the dermal side of 1 M NaCl split skin. Various examinations suggested that the 200-kDa antigen is not identical to any chains of laminins-1, -5 or -6. This autoimmune subepidermal blistering disease against the dermal 200-kDa protein may form a new distinct entity, which often associates with psoriasis.

    View details for Web of Science ID 000087425100003

    View details for PubMedID 10808126

  • Compound heterozygosity for novel splice site mutations in the BPAG2/COL17A1 gene underlies generalized atrophic benign epidermolysis bullosa JOURNAL OF INVESTIGATIVE DERMATOLOGY Pulkkinen, L., Marinkovich, M. P., Tran, H. T., Lin, L., Herron, G. S., Uitto, J. 1999; 113 (6): 1114-1118

    Abstract

    Generalized atrophic benign epidermolysis bullosa, GABEB (OMIM# 226650), is a nonlethal variant of epidermolysis bullosa with autosomal recessive inheritance pattern. The pathogenesis of this disorder can be caused by mutations affecting two different gene/protein systems. Most of the mutations have been identified in the BPAG2/COL17A1 gene encoding a hemidesmosomal transmembrane protein, the 180 kDa bullous pemphigoid antigen (BP180), also known as type XVII collagen. The minority of the mutations are localized in the LAMB3 gene encoding the beta3 polypeptide of laminin 5. In In this study we describe a GABEB patient who showed absent expression of BP180 in the cultured keratinocytes as well as in the skin. The patient was a compound heterozygote for two different splice site mutations, 3053-1G-->C and 3871+1G-->C, affecting the extra-cellular domain of the protein. These mutations resulted in multiple aberrant splice variants, three of them causing premature termination codons for translation. This case, dealing with out-of-frame splice site mutations in BPAG2/COL17A1, attests to the molecular heterogeneity of GABEB.

    View details for Web of Science ID 000084436600038

    View details for PubMedID 10636730

  • Reduced anchoring fibril formation and collagen VII immunoreactivity in feline dystrophic epidermolysis bullosa VETERINARY PATHOLOGY Olivry, T., Dunston, S. M., Marinkovich, M. P. 1999; 36 (6): 616-618

    Abstract

    Dystrophic epidermolysis bullosa was diagnosed in a cat with juvenile-onset epithelial sloughing of the oral mucosa, footpads, and haired skin. Dermoepidermal separation occurred in the absence of inflammation or cytolysis of basal epidermal cells. Collagen IV-specific immunostaining corroborated the fact that clefting took place below the epidermal basement membrane. Ultrastructural examination revealed that the proband's anchoring fibrils exhibited a filamentous morphology and were decreased in number compared with those in a normal cat. Finally, the attenuated immunoreactivity for collagen VII in our patient led us to suspect that its encoding gene, COL7A1, could be mutated in this case of feline dystrophic epidermolysis bullosa.

    View details for Web of Science ID 000085281300014

    View details for PubMedID 10568446

  • Update on inherited bullous dermatoses DERMATOLOGIC CLINICS Marinkovich, M. P. 1999; 17 (3): 473-?

    Abstract

    Bullous diseases are becoming increasingly better understood owing to the active research which has taken place in this field over the past decade. Advances in understanding of bullous disease pathophysiology is translating into clinical applications for diagnosis and therapy that will greatly enhance the quality of care bullous disease patients may receive now and in the future. This review focuses on the progress which has been achieved in inherited bullous dermatoses.

    View details for Web of Science ID 000081318700003

    View details for PubMedID 10410853

  • Novel feline autoimmune blistering disease resembling bullous pemphigoid in humans: IgG autoantibodies target the NC16A ectodomain of type XVII collagen (BP180/BPAG2) VETERINARY PATHOLOGY Olivry, T., Chan, L. S., Xu, L., Chace, P., Dunston, S. M., Fahey, M., Marinkovich, M. P. 1999; 36 (4): 328-335

    Abstract

    In humans and dogs, bullous pemphigoid (BP) is an autoimmune blistering disease associated with the production of basement membrane autoantibodies that target the 180-kd type XVII collagen (BP180, BPAG2) and/or the 230-kd plakin epidermal isoform BPAG1e (BP230). In two adult cats, an acquired dermatosis and stomatitis was diagnosed as BP subsequent to the fulfillment of the following criteria: 1) presence of cutaneous vesicles, erosions, and ulcers; 2) histologic demonstration of subepidermal vesiculation with inflammatory cells, including eosinophils; 3) in vivo deposition of IgG autoantibodies at the epidermal basement membrane zone; and 4) serum IgG autoantibodies targeting a 180-kd epidermal protein identified as type XVII collagen. In both cats, the antigenic epitopes targeted by IgG autoantibodies were shown to be situated in the NC16A ectodomain of type XVII collagen, a situation similar to that of humans and dogs with BP. Feline BP therefore can be considered a clinical, histopathologic, and immunologic homologue of BP in humans and dogs.

    View details for Web of Science ID 000083807900008

    View details for PubMedID 10421100

  • Melanocytes adhere to and synthesize laminin-5 in vitro EXPERIMENTAL DERMATOLOGY Scott, G. A., Cassidy, L., Tran, H., Rao, S. K., Marinkovich, M. P. 1999; 8 (3): 212-221

    Abstract

    Melanocytes arise from the neural crest, migrate to the skin, and can be detected in the basal layer of the epidermis in skin biopsies of human fetuses as early as 11 weeks gestational age. During post-natal life, melanocytes reside at the basal layer of the epidermis, but the ligands to which they attach are unknown. Laminin-5 is a component of anchoring filaments of the lamina lucida of the epidermal basement membrane. In this report we show that human melanocytes adhere to purified laminin-5 to a level comparable with normal human keratinocytes. Blocking antibodies to the 165 kDa subunit of laminin-5 significantly inhibited fetal and neonatal melanocyte attachment to the surface of salt-split skin, which exposes laminin-5 on its surface, suggesting that laminin-5 is a ligand for melanocyte attachment to the basement membrane in vivo. Western blotting of concentrated culture supernatant of fetal and neonatal melanocytes with anti-laminin-5 antibodies demonstrated a single immunoreactive band of the expected size of laminin-5. In contrast, 3 human metastatic melanoma cell lines did not produce laminin-5. Immunofluorescence microscopy with antibodies to each of the three chains of laminin-5 confirmed the presence of laminin-5 in a peri-cellular distribution around melanocytes, but not melanoma cells. Our results suggest that laminin-5 may be a ligand for normal human melanocytes in the basement membrane, and that loss of laminin-5 production by melanoma cells may be a marker for malignant transformation.

    View details for Web of Science ID 000080694800007

    View details for PubMedID 10389639

  • NC1 domain of type VII collagen binds to the beta 3 chain of laminin 5 via a unique subdomain within the fibronectin-like repeats JOURNAL OF INVESTIGATIVE DERMATOLOGY Chen, M., Marinkovich, M. P., Jones, J. C., O'Toole, E. A., Li, Y. Y., Woodley, D. T. 1999; 112 (2): 177-183

    Abstract

    Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, globular domains, NC1 and NC2. Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC1 binds to various extracellular matrix components including a complex of laminin 5 and laminin 6 (Chen et al, 1997a). In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments). Both authentic and purified recombinant NC1 bound to human and rat laminin 5 as measured by enzyme-linked immunosorbant assay and by binding of 125I-radiolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. NC1 bound predominantly to the beta3 chain of laminin 5, but also to the gamma2 chain when examined by a protein overlay assay. The binding of 125I-NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-glycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclonal antibody to the beta3 chain. In contrast, the NC1-laminin 5 interaction was not affected by a monoclonal antibody to the alpha3 chain. Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760-1045) subdomain of NC1 was identified as the binding site for laminin 5. IgG from an epidermolysis bullosa acquisita serum containing autoantibodies to epitopes within NC1 that colocalized with the laminin 5 binding site inhibited the binding of NC1 to laminin 5. Thus, perturbation of the NC1-laminin 5 interaction may contribute to the pathogenesis of epidermolysis bullosa acquisita.

    View details for Web of Science ID 000078287000007

    View details for PubMedID 9989793

  • Gene therapy for a lethal genetic blistering disease: a status report. Transactions of the American Clinical and Climatological Association BAUER, E. A., Herron, G. S., Marinkovich, M. P., Khavari, P. A., Lane, A. T. 1999; 110: 86-92

    View details for PubMedID 10344009

  • BP180 gene delivery in junctional epidermolysis bullosa GENE THERAPY Seitz, C. S., Giudice, G. J., Balding, S. D., Marinkovich, M. P., Khavari, P. A. 1999; 6 (1): 42-47

    Abstract

    Epidermolysis bullosa (EB) comprises a family of inherited blistering skin diseases for which current therapy is only palliative. Junctional EB (JEB) involves dissociation of the dermal-epidermal junction and results from mutations in a number of genes that encode vital structural proteins, including BP180 (type XVII collagen/BPAG2). In order to develop a model of corrective gene delivery for JEB, we produced a retroviral expression vector for wild-type human BP180 and used it to restore BP180 protein expression to primary keratinocytes from BP180-negative patients with generalized atrophic JEB. Restoration of full-length BP180 protein expression was associated with adhesion parameter normalization of primary JEB keratinocytes in vitro. These cells were then used to regenerate human skin on immune-deficient mice. BP180 gene-transduced tissue demonstrated restoration of BP180 gene expression at the dermal-epidermal junction in vivo while untransduced regenerated JEB skin entirely lacked BP180 expression. These findings provide a basis for future efforts to achieve gene delivery in human EB skin tissue.

    View details for Web of Science ID 000078169300007

    View details for PubMedID 10341874

  • Self-assembly of laminin isoforms JOURNAL OF BIOLOGICAL CHEMISTRY Cheng, Y. S., Champliaud, M. F., Burgeson, R. E., Marinkovich, M. P., Yurchenco, P. D. 1997; 272 (50): 31525-31532

    Abstract

    The alpha, beta, and gamma subunits of basement membrane laminins can combine into different heterotrimeric molecules with either three full short arms (e.g. laminins-1-4), or molecules containing one (laminins-6-9) or more (laminin-5) short arm truncations. Laminin-1 (alpha1beta1gamma1), self-assembles through a calcium-dependent thermal gelation requiring binding interactions between N-terminal short arm domains, forming a meshwork polymer thought to contribute to basement membrane architecture (Yurchenco, P. D., and Cheng, Y. S. (1993) J. Biol. Chem. 268, 17286-17299). However, it has been unclear whether other isoforms share this property, and if so, which ones. To begin to address this, we evaluated laminin-2 (alpha2beta1gamma1), laminin-4 (alpha2beta2gamma1), laminin-5 (alpha3Abeta3gamma2), and laminin-6 (alpha3Abeta1gamma1). The first two isoforms were found to self-aggregate in a concentration- and temperature-dependent manner and a close self-assembly relationship among laminins-1, -2, and -4 were demonstrated by: (a) polymerization of all three proteins was inhibited by EDTA and laminin-1 short arm fragments, (b) polymerization of laminin-1 was inhibited by fragments of laminins-2 and -4, (c) laminin-2 and, to a lesser degree, laminin-4, even well below their own critical concentration, co-aggregated with laminin-1, evidence for co-polymerization. Laminin-5, on the other hand, neither polymerized nor co-polymerized with laminin-1. Laminin-6 failed to co-aggregate with laminin-1 at all concentrations evaluated, evidence for a lack of a related self-assembly activity. The data support the hypothesis that all three short arms are required for self-assembly and suggest that the short arm domain structure of laminin isoforms affect their architecture-forming properties in basement membranes.

    View details for Web of Science ID A1997YL41900048

    View details for PubMedID 9395489

  • Hypoxia increases human keratinocyte motility on connective tissue JOURNAL OF CLINICAL INVESTIGATION O'Toole, E. A., Marinkovich, M. P., Peavey, C. L., Amieva, M. R., FURTHMAYR, H., Mustoe, T. A., Woodley, D. T. 1997; 100 (11): 2881-2891

    Abstract

    Re-epithelialization of skin wounds depends upon the migration of keratinocytes from the cut margins of the wound and is enhanced when human keratinocytes are covered with occlusive dressings that induce hypoxia. In this study, two independent migration assays were used to compare cellular motility on connective tissue components under normoxic or hypoxic conditions. Human keratinocytes apposed to collagens or fibronectin exhibited increased motility when subjected to hypoxic (0.2 or 2% oxygen) conditions compared with normoxic (9 or 20% oxygen) conditions. When compared with normoxic cells, hypoxic keratinocytes exhibited increased expression and redistribution of the lamellipodia-associated proteins (ezrin, radixin, and moesin). Furthermore, hypoxic keratinocytes demonstrated decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility. Hypoxia did not alter the number of integrin receptors on the cell surface, but did induce enhanced secretion of the 92-kD type IV collagenase. These data demonstrate that hypoxia promotes human keratinocyte motility on connective tissue. Hypoxia-driven motility is associated with increased expression of lamellipodia proteins, increased expression of collagenase and decreased expression of laminin-5, the locomotion brake for keratinocytes.

    View details for Web of Science ID 000071007300031

    View details for PubMedID 9389755

  • Immunodissection of the connective tissue matrix in human skin MICROSCOPY RESEARCH AND TECHNIQUE Keene, D. R., Marinkovich, M. P., Sakai, L. Y. 1997; 38 (4): 394-406

    Abstract

    Much of what has been learned of the components and structure of human skin over the past few years has been accomplished with the aid of antibody technology. Antibodies are used in techniques such as affinity chromatography to isolate individual molecules and by immunofluorescence and immunoelectron microscopy to identify each of those molecules as components of specific macromolecular assemblies present within the dermis. This manuscript is meant not as a review of technique but instead as a summary of recent progress made in the understanding of dermal matrix architecture.

    View details for Web of Science ID A1997XU93000007

    View details for PubMedID 9297689

  • Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components - A potential role in epidermal-dermal adherence in human skin JOURNAL OF BIOLOGICAL CHEMISTRY Chen, M., Marinkovich, M. P., Veis, A., Cai, X. Y., Rao, C. N., OToole, E. A., Woodley, D. T. 1997; 272 (23): 14516-14522

    Abstract

    Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC1 domain contains multiple submodules with homology to known adhesive molecules including fibronectin type III-like repeats and the A domain of von Willebrand factor. In this study, we produced the entire NC1 domain of human type VII collagen in the stably transfected human kidney 293 cell clones and purified large quantities of the recombinant NC1 protein from serum-free culture media. The recombinant NC1 formed interchain disulfide-bonded dimers and trimers and was N-linked glycosylated. Tunicamycin inhibited the cellular secretion of NC1, suggesting that N-linked glycosylation may play a role in NC1 secretion. The recombinant NC1 was indistinguishable from the authentic NC1 obtained from human amnions or WISH cells with respect to N-linked sugar content, electrophoretic mobility, rotary shadow imaging, and binding affinity to type IV collagen. Purified recombinant NC1, like authentic NC1, also bound specifically to fibronectin, collagen type I, and a laminin 5/6 complex. Both monomeric and trimeric forms of NC1 exhibited equal affinity for these extracellular matrix components, suggesting that the individual arms of NC1 can function independently. The multiple interactions of NC1 with other extracellular matrix components may support epidermal-dermal adhesion.

    View details for Web of Science ID A1997XC32700005

    View details for PubMedID 9169408

  • A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone. journal of investigative dermatology Zillikens, D., Kawahara, Y., Ishiko, A., Shimizu, H., Mayer, J., Rank, C. V., Liu, Z., Giudice, G. J., Tran, H. H., Marinkovich, M. P., Brocker, E. B., Hashimoto, T. 1996; 106 (6): 1333-1338

    Abstract

    Several components of the basement membrane zone (BMZ) have been identified as antigenic targets in autoimmune bullous diseases. We report a novel disease with autoantibodies to a BMZ antigen that is different from the targets described so far. The patient suffering from this disorder showed tense bullae and severe mucous membrane involvement rapidly responding to oral tetracyclines and colchicine. Histopathologic findings resembled those of dermatitis herpetiformis. Direct immunofluorescence microscopy showed linear deposits of IgG and C3 at the BMZ. By indirect immunofluorescence studies on split human skin, using both 1 M NaCl and suction blistering for dermal-epidermal separation, IgG antibodies localized exclusively to the dermal side of the split. The antibodies were mainly of the IgG4 subclass. By Western blot analysis of epidermal and dermal extracts, the patient's serum unequivocally reacted with a dermal antigen of 200 kDa. It did not recognize bullous pemphigoid antigens, the autoantigen of epidermolysis bullosa acquisita, purified preparations of laminin-1 and laminin-5, or the recently described 105-kDa BMZ antigen. By immunoblotting of concentrated conditioned SCC-25 medium, the patient's antibodies reacted with a band of 200 kDa and several hands of lower molecular weight. No reactivity was seen with extracts of cultured human fibroblasts. By indirect immunogold electron microscopy, immunoreactants localized to the lower lamina lucida. After clearance of skin lesions, both indirect immunofluorescence and Western blot analysis became negative. This patient suffers from a novel autoimmune bullous disease with autoantibodies to a 200-kDa antigen of the BMZ.

    View details for PubMedID 8752680

  • LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kDa anchoring filament protein synthesized by epidermal cells JOURNAL OF INVESTIGATIVE DERMATOLOGY Marinkovich, M. P., Taylor, T. B., Keene, D. R., Burgeson, R. E., Zone, J. J. 1996; 106 (4): 734-738

    Abstract

    This study characterizes a novel basement membrane component that is the target of autoantibodies in patients with linear IgA bullous dermatosis. Tissue surveys showed that this protein localized to the epidermal side of 1 M NaCl split skin and to basement membranes in cornea, oral mucosa, esophagus, intestine, kidney collecting ducts, ureter, bladder, urethra, and thymus, but was absent in lung, blood vessels, skeletal muscle, and nerve. Monoclonal antibody 123, which recognizes this protein, induced dermal-epidermal separation of human skin in situ, and this protein was found, by immunoelectron microscopy, to localize exclusively to anchoring filaments. This protein was secreted as as a 120-kDa peptide from primary cultures of keratinocytes as determined by radioimmunoprecipitation. Monoclonal antibody 123 recognized this protein as a 120-kDa band from conditioned cell culture medium and a 97-kDa band from human skin extracts as shown by immunoblot. Serum from five patients with the autoimmune blistering disorder linear IgA bullous dermatosis specifically recognized bands of 120 and 97 kDa from culture medium and skin extracts, respectively, that were of identical electrophoretic migration to the bands recognized by monoclonal antibody 123. In summary, this study characterizes a novel anchoring filament protein that is the target of linear IgA bullous dermatosis autoantibodies. Because monoclonal antibody 123 induces blistering of human skin, we hypothesize that this protein functions to maintain dermal-epidermal cohesion and that autoantibodies in this disease are themselves pathogenic. We propose LAD-1 as the name for this protein.

    View details for Web of Science ID A1996UC78700025

    View details for PubMedID 8618013

  • A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone JOURNAL OF INVESTIGATIVE DERMATOLOGY Zillikens, D., Kawahara, Y., Ishiko, A., Shimizu, H., Mayer, J., Rank, C. V., Liu, Z., Giudice, G. J., Tran, H. H., Marinkovich, M. P., Brocker, E. B., Hashimoto, T. 1996; 106 (3): 465-470

    Abstract

    Several components of the basement membrane zone (BMZ) have been identified as antigenic targets in autoimmune bullous diseases. We report a novel disease with autoantibodies to a BMZ antigen that is different from the targets described so far. The patient suffering from this disorder showed tense bullae and severe mucous membrane involvement rapidly responding to oral tetracyclines and colchicine. Histopathologic findings resembled those of dermatitis herpetiformis. Direct immunofluorescence microscopy showed linear deposits of IgG and C3 at the BMZ. By indirect immunofluorescence studies on split human skin, using both 1 M NaCl and suction blistering for dermal-epidermal separation, IgG antibodies localized exclusively to the dermal side of the split. The antibodies were mainly of the IgG4 sub-class. By Western blot analysis of epidermal and dermal extracts, the patient's serum unequivocally reacted with a dermal antigen of 200 kDa. It did not recognize bullous pemphigoid antigens (the autoantigen of epidermolysis bullosa acquisita), purified preparations of laminin-1 and laminin-5, or the recently described 105-kDa BMZ antigen. By immunoblotting of concentrated conditioned SCC-25 medium, the patient's antibodies reacted with a band of 200 kDa and several bands of lower molecular weight. No reactivity was seen with extracts of cultured human fibroblasts. By indirect immunogold electron microscopy, immunoreactants localized to the lower lamina lucida. After clearance of skin lesions, both indirect immunofluorescence and Western blot analysis became negative. This patient suffers from a novel autoimmune bullous disease with autoantibodies to a 200-kDa antigen of the BMZ.

    View details for Web of Science ID A1996UB96800014

    View details for PubMedID 8648178

  • PRENATAL-DIAGNOSIS OF HERLITZ JUNCTIONAL EPIDERMOLYSIS-BULLOSA BY AMNIOCENTESIS PRENATAL DIAGNOSIS Marinkovich, M. P., Meneguzzi, G., Burgeson, R. E., BLANCHETBARDON, C., Holbrook, K. A., Smith, L. T., Christiano, A. M., Ortonne, J. P. 1995; 15 (11): 1027-1034

    Abstract

    Herlitz junctional epidermolysis bullosa (HJEB) is a severe blistering disorder which usually results in death during infancy. We have previously shown that the anchoring filament protein laminin-5 (kalinin/nicein), which mediates keratinocyte attachment and dermal-epidermal cohesion, is abnormally expressed in individuals with HJEB. Laminin-5 was detected by Western blot analysis in amniotic fluid from 44 consecutive normal second-trimester control pregnancies, but was undetectable in second-trimester amniotic fluid from four pregnancies with fetuses affected by HJEB. In one case of severe non-Herlitz JEB, laminin-5 was detected in both amniotic fluid and skin. In human amniotic fluid, the laminin-5 a3 subunit was processed to a major 165 kD species and a minor 145 kD species and the beta 2 subunit was partially processed to 105 kD. Although laminin-5 was covalently associated with laminin-6 (K-laminin) in amniotic membrane, no covalent interaction was detected in amniotic fluid. Laminin-5 from amniotic fluid strongly supported keratinocyte attachment. These results suggest that Western blot analysis of second-trimester amniotic fluid is useful in determining the prenatal diagnosis of HJEB and that laminin-5 may serve a physiologically important function in amniotic fluid.

    View details for Web of Science ID A1995TG44000005

    View details for PubMedID 8606881

  • ANTIBASEMENT MEMBRANE AUTOANTIBODIES IN PATIENTS WITH ANTI-EPILIGRIN CICATRICIAL PEMPHIGOID BIND THE ALPHA-SUBUNIT OF LAMININ-5 JOURNAL OF INVESTIGATIVE DERMATOLOGY Kirtschig, G., Marinkovich, M. P., Burgeson, R. E., Yancey, K. B. 1995; 105 (4): 543-548

    Abstract

    Recent studies have identified a group of cicatricial pemphigoid patients who have IgG anti-basement membrane autoantibodies that recognize epiligrin, a set of disulfide-linked polypeptides closely related if not identical to laminin 5 (formerly called kalinin, nicein, or BM600). To further understand the pathophysiology of blister formation in these patients, we have sought to identify the specific polypeptide(s) targeted by their autoantibodies. Comparative studies show that sera from these patients (nine of nine), P1E1 monoclonal anti-epiligrin antibody, and polyclonal as well as monoclonal anti-laminin 5 antibodies immunoprecipitate the same set of disulfide-linked polypeptides from media of biosynthetically radiolabeled human keratinocytes. Moreover, sera from eight of nine patients with anti-epiligrin cicatricial pemphigoid immunoblot the alpha subunit of laminin 5 but show no reactivity to its beta or gamma subunits. In addition, circulating IgG from a representative patient was affinity-purified against the alpha subunit of laminin 5 and shown to bind the dermal side of 1 M NaC1 split skin in the same manner as autoantibodies from all patients with anti-epiligrin cicatricial pemphigoid. Sera from patients with bullous pemphigoid (n = 5), other forms of cicatricial pemphigoid (n = 5), epidermolysis bullosa acquisita (n = 4), or bullous systemic lupus erythematosus (n = 1) show no reactivity against any subunit of this laminin isoform in immunoprecipitation or immunoblot experiments. These findings correlate with prior reports showing that a monoclonal antibody directed against the alpha subunit of laminin 5 (i.e., laminin subunit alpha 3) induces detachment of human keratinocytes from extracellular matrix in vitro as well as epidermis from human skin in situ. Together, these studies suggest that laminin subunit alpha 3 mediates attachment of basal keratinocytes to epidermal basement membrane and that autoantibodies directed against it may be pathogenic.

    View details for Web of Science ID A1995RY59800004

    View details for PubMedID 7561156

  • A NEWLY IDENTIFIED 105-KD LOWER LAMINA-LUCIDA AUTOANTIGEN IS AN ACIDIC PROTEIN DISTINCT FROM THE 105-KD GAMMA-2 CHAIN OF LAMININ-5 JOURNAL OF INVESTIGATIVE DERMATOLOGY Chan, L. S., Wang, X. S., Lapiere, J. C., Marinkovich, M. P., Jones, J. C., Woodley, D. T. 1995; 105 (1): 75-79

    Abstract

    A 105-kD lower lamina lucida antigen (p105) has been detected by autoantibodies (anti-p105) from patients with a novel immunobullous disease. To distinguish p105 from other known lamina lucida components, we performed comparative immunoblotting on purified human amniotic laminin-5 (kalinin), 804G matrix (enriched in laminin-5), and keratinocyte and fibroblast proteins using anti-804G matrix antibody (J-18) and anti-p105. J-18 labeled the truncated laminin-5 gamma 2 chain in amniotic laminin-5, 804G matrix, and keratinocyte conditioned medium, but did not label fibroblast cytosol. Conversely, anti-p105 did not label amniotic laminin-5 or 804G matrix, but did label p105 in both keratinocyte conditioned medium and fibroblast cytosol. J-18 labeled the 105-kD laminin-5 gamma 2 chain in reduced keratinocyte proteins and a 400-kD laminin-5 complex under non-reducing conditions. In contrast, anti-p105 labeled p105 under both reducing and non-reducing conditions but did not label a 400-kD protein complex. Similarly, comparative immunoblotting on keratinocyte proteins using anti-p105 and anti-laminin-1 revealed no commonly labeled protein bands. Electrophoretic fractionations by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of these fractions revealed that the peak fractions of keratinocyte proteins reactive with anti-p105 are different from those reactive with J-18. Furthermore, keratinocyte proteins fractionated by Mono Q anion-exchange chromatography revealed fractions immunoreactive with anti-p105, whereas J-18 showed no reactivity with these fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-p105 revealed p105 to be an acidic protein with isoelectric points between 5.7 and 6.3, distinct from the isoelectric points of laminin-5 gamma 2 chain. We conclude that p105 is an acidic protein located in the lamina lucida and distinct from the truncated laminin-5 gamma 2 chain and the laminin-1 family.

    View details for Web of Science ID A1995RJ63200014

    View details for PubMedID 7615980

  • NECROLYTIC MIGRATORY ERYTHEMA WITHOUT GLUCAGONOMA IN PATIENTS WITH LIVER-DISEASE JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY Marinkovich, M. P., Botella, R., DATLOFF, J., Sangueza, O. P. 1995; 32 (4): 604-609

    Abstract

    Necrolytic migratory erythema (NME) is an uncommon inflammatory dermatosis with a distinct clinical and histologic appearance. NME is usually associated with glucagonoma. Only a few cases of NME in the absence of glucagonoma have been previously reported.We sought to understand further the pathogenesis of NME by analyzing data from three patients.Three patients were examined both clinically and histopathologically.Each patient had an extensive erythematous scaling eruption in intertriginous, perioral, and acral areas, and a markedly red, smooth tongue. Skin biopsy specimens showed confluent parakeratosis, epidermal pallor, papillary edema, and a lymphohistiocytic infiltrate. Two patients had alcoholic liver disease and one had liver dysfunction as a result of hemochromatosis. Serum albumin level was depressed, and liver enzyme values were increased in all three patients. Glucagonoma was undetectable in these patients.In the absence of glucagonoma, hepatocellular dysfunction and hypoalbuminemia appear to be the most common factors associated with NME.

    View details for Web of Science ID A1995QP04800009

    View details for PubMedID 7896950

  • THE MOLECULAR-GENETICS OF BASEMENT-MEMBRANE DISEASES ARCHIVES OF DERMATOLOGY Marinkovich, M. P. 1993; 129 (12): 1557-1565

    Abstract

    Keratin filaments, hemidesmosomes, anchoring filaments, the lamina densa, and anchoring fibrils each function to maintain different levels of basement membrane cohesion.Keratin 5 or 14 mutations are present in epidemiolysis bullosa simplex. Herlitz junctional epidermolysis bullosa is characterized by defects of the anchoring filament protein kalinin (alternatively known as nicein). Mutations of the type VII collagen gene appear to be the primary cause of dominant and recessive dystrophic epidermolysis bullosa. Two hemidesmosomal components are the bullous pemphigoid (BP) antigens: BP230 shows homology to desmoplakin, a desmosomal component; BP180 contains extracellular collagen domains. The autoantigens in cicatricial pemphigiod and IgA-mediated autoimmune diseases are less well understood. Type IV collagen chains are affected in Alport's and Goodpasture's syndromes.New diagnostic and therapeutic techniques based on these genetic/biochemical advances are currently being developed.

    View details for Web of Science ID A1993MW82600002

    View details for PubMedID 8250577

  • BASEMENT-MEMBRANE PROTEINS KALININ AND NICEIN ARE STRUCTURALLY AND IMMUNOLOGICALLY IDENTICAL LABORATORY INVESTIGATION Marinkovich, M. P., Verrando, P., Keene, D. R., Meneguzzi, G., Lunstrum, G. P., Ortonne, J. P., Burgeson, R. E. 1993; 69 (3): 295-299

    Abstract

    The purpose of this investigation was 2-fold: (a) to compare two recently described proteins, the anchoring filament protein, kalinin and the hemidesmosome-associated protein, nicein (formerly called BM-600) which are both absent in junctional epidermolysis bullosa (JEB) Herlitz's disease; (b) to further define the structural defect in JEB Herlitz's disease.Cultured keratinocytes were analyzed with monoclonal antibodies (mAbs) against kalinin and nicein by indirect immunofluorescence. These mAbs were also used to immunoprecipitate radiolabeled proteins from keratinocyte cultures and to immunoaffinity purify proteins from keratinocyte conditioned culture medium. The precipitated or purified products were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, partial V8 protease digestion, and rotary shadowing.Kalinin and nicein mAbs show identical immunofluorescent staining patterns on cultured keratinocytes. Kalinin and nicein mAbs immunoprecipitate peptides from radiolabeled normal human keratinocyte cell and medium fractions that are electrophoretically identical. Partial V8 protease digestion patterns of the reduced 140 kilodalton peptides precipitated by nicein and kalinin mAbs are identical. Kalinin (like nicein) is absent from JEB Herlitz keratinocyte conditioned medium although K-laminin, another anchoring filament component, is present in these cultures. Kalinin, purified from conditioned keratinocyte medium by antibody affinity chromatography with K140-Sepharose (mAb against kalinin) and nicein purified from conditioned keratinocyte medium with GB3-Sepharose (mAb against nicein) are electrophoretically identical by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunologically identical by immunoblotting using kalinin and nicein mAbs. Rotary shadowed images of kalinin and nicein molecules are identical.We demonstrate that kalinin and nicein are identical by biochemical and immunologic analysis. We also verify that kalinin, like nicein, is absent in the conditioned medium of cultured JEB Herlitz keratinocytes, although another anchoring filament protein, K-laminin, is secreted by these cultures. These results correlate with previous immunofluorescent findings that show that while kalinin or nicein is absent in basement membranes of individuals with JEB Herlitz's disease, K-laminin appears to be present.

    View details for Web of Science ID A1993LZ43000005

    View details for PubMedID 8377472

  • CELLULAR-ORIGIN OF THE DERMAL-EPIDERMAL BASEMENT-MEMBRANE DEVELOPMENTAL DYNAMICS Marinkovich, M. P., Keene, D. R., RIMBERG, C. S., Burgeson, R. E. 1993; 197 (4): 255-267

    Abstract

    The basement membrane underlying epithelium of skin is generally believed to be of epithelial origin, but a mesenchymal contribution to the basement membrane has not been directly examined. The purpose of this study was to directly evaluate both epithelial and mesenchymal contributions to the basement membrane. Fetal bovine keratinocytes cultured on the surface of collagen gels in the absence of fibroblasts did not produce an ultrastructurally recognizable basement membrane; however, when these cells were cultured in the presence of dermal fibroblasts a basement membrane at the keratinocyte-fibroblast interface was produced after 1 week which was very similar in biochemical composition and ultrastructural appearance to dermal-epidermal basement membrane in human skin. When dual species cultures of bovine keratinocytes and human fibroblasts were analyzed by indirect immunofluorescent microscopy (IF)1 with human specific antibodies against basement membrane components, dermal fibroblasts were shown to synthesize and deposit type IV collagen, type VII collagen, and laminin in a linear manner into the basement membrane zone. Fetal bovine keratinocytes cultured in the presence or absence of fibroblasts synthesized and deposited type IV collagen, type VII collagen, laminin, K-laminin, kalinin, and basement membrane associated heparan sulfate proteoglycan (HSPG) into the underlying basement membrane zone. In organ culture, a subpopulation of fibroblasts initially migrating from human foreskin explants was found to stain strongly for types VII and IV collagen and laminin by IF whereas after subculture all cells showed a uniform low staining. Based on these observations we propose that differentiated fibroblasts exist adjacent to epithelial tissues in vivo which produce basement membrane components and assist in basement membrane assembly.

    View details for Web of Science ID A1993ME56500003

    View details for PubMedID 8292823

  • Kalinin is abnormally expressed in epithelial basement membranes of Herlitz's junctional epidermolysis bullosa patients. Experimental dermatology Meneguzzi, G., Marinkovich, M. P., Aberdam, D., Pisani, A., Burgeson, R., Ortonne, J. P. 1992; 1 (5): 221-229

    Abstract

    Kalinin is an extracellular adhesion molecule specific to epithelial basement membranes (BM) identified as a component of anchoring filaments of hemidesmosomes. This heterotrimeric protein is synthesized by cultured normal human keratinocytes and is involved in cell attachment. In indirect immunofluorescence studies, the epidermal BM of patients with junctional epidermolysis bullosa (JEB) of Herlitz's type were found not to be reactive with the anti-kalinin monoclonal antibodies ka146 and K140 and displayed a decreased immunoreactivity to two anti-kalinin antibodies cross-reacting with K-laminin, an anchoring filament component recently described. The intrinsic defect of JEB keratinocytes in the synthesis of kalinin was further documented by indirect immunofluorescence on in vitro cultures of these cells. In non-Herlitz JEB patients, staining of BM was constantly detected. Impairment of expression of kalinin correlated with the lack of reactivity to the monoclonal antibody GB3, which detects the BM component nicein/BM600. These results clearly demonstrate a defect of kalinin expression in epithelial basement membranes of Herlitz JEB patients and suggest that kalinin may play a role in the pathogenesis of the disease. Further studies are in progress to define possible relationships between kalinin and nicein.

    View details for PubMedID 1365323

  • THE DERMAL EPIDERMAL JUNCTION OF HUMAN SKIN CONTAINS A NOVEL LAMININ VARIANT JOURNAL OF CELL BIOLOGY Marinkovich, M. P., Lunstrum, G. P., Keene, D. R., Burgeson, R. E. 1992; 119 (3): 695-703

    Abstract

    We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant.

    View details for Web of Science ID A1992JV26900019

    View details for PubMedID 1383241

  • IDENTIFICATION AND PARTIAL-PURIFICATION OF A LARGE, VARIANT FORM OF TYPE-XII COLLAGEN JOURNAL OF BIOLOGICAL CHEMISTRY Lunstrum, G. P., McDonough, A. M., Marinkovich, M. P., Keene, D. R., Morris, N. P., Burgeson, R. E. 1992; 267 (28): 20087-20092

    Abstract

    A large, alternate form of type XII collagen has been identified in cultures of the human epidermoid cell line WISH. This form, designated XIIA, is comprised of alpha chains that are approximately 90 kDa larger than the 220-kDa alpha chain previously characterized in extracts of fetal chicken and bovine tissues. Results from both collagenase digestion and rotary shadow analysis of partially purified material show that the increase is due to a larger NC3 domain. While both the large (XIIA) and the small (XIIB) forms of type XII collagen are identified in pulse-chase radiolabeling of fetal bovine skin explant culture, they are not related in a precursor-product fashion. Inhibition studies with alpha, alpha'-dipyridyl indicate that proper folding of the collagen helix is required for complete assembly and secretion of type XIIA in WISH cell culture. The 310-kDa alpha 1A chain is likely to represent the bovine equivalent of a second translation product, estimated to be 340 kDa, predicted from analysis of one complete chick cDNA sequence. Additionally, the amino-terminal amino acid sequence of the 220-kDa bovine alpha 1B chain was determined. This sequence is very near a potential alternate splice site predicted from analysis of chicken type XII cDNA.

    View details for Web of Science ID A1992JR85800055

    View details for PubMedID 1400326

  • THE ANCHORING FILAMENT PROTEIN KALININ IS SYNTHESIZED AND SECRETED AS A HIGH-MOLECULAR-WEIGHT PRECURSOR JOURNAL OF BIOLOGICAL CHEMISTRY Marinkovich, M. P., Lunstrum, G. P., Burgeson, R. E. 1992; 267 (25): 17900-17906

    Abstract

    Kalinin, a recently characterized novel protein component of anchoring filaments, has been shown to be involved in keratinocyte attachment to culture substrates and to dermis in vivo, and to exist in keratinocyte-conditioned culture medium in two heterotrimeric forms of 440 and 400 kDa (Rousselle, P., Lunstrum, G.P., Keene, D.R., and Burgeson, R.E. (1991) J. Cell Biol. 114, 567-576). This study demonstrates that kalinin is initially synthesized in a cell-associated form estimated to be 460 kDa. By second dimension reduced electrophoresis, V8 protease digestion, and immunoblot analysis, we demonstrate that the cell form contains nonidentical subunits of 200, 155, and 140 kDa. The 440-kDa medium form is derived from the cell form by extracellular processing of the 200-kDa subunit to 165 kDa, a step which also occurs in skin organ culture. The 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155 kDa-subunit to 105 kDa. The cell form is secreted by keratinocytes, deposited onto culture substratum, and is the form which facilitates attachment and adhesion of growing and spreading keratinocytes. It is also the form initially synthesized in skin organ culture. Kalinin purified from tissue, which appears to facilitate epithelial-mesenchymal cohesion in vivo, is closely related to the 400-kDa medium form purified from culture.

    View details for Web of Science ID A1992JM22300064

    View details for PubMedID 1517226

  • SYNTHESIS OF CALELECTRINS AND CALPACTIN-I DURING CYTOCHALASIN MEDIATED CELL SPREADING INHIBITION CELL CALCIUM Hom, Y. K., Marinkovich, M. P., Lozano, J. J., Rocha, V. 1989; 10 (3): 135-144

    Abstract

    Mammary epithelial cell spreading on collagen gels has previously been shown to be correlated with the synthesis of a group of calcium-binding proteins (CBPs) which we have identified as the calcium-binding proteins termed calelectrins and calpactin I monomer/p36. To determine whether cell spreading per se is required for CBP synthesis, we examined the effect of cytochalasin D on these two events. Concentrations of cytochalasin D that did not reduce total protein synthesis, caused inhibition of cell spreading in a dose-dependent manner, but did not cause inhibition of CBP synthesis. Synthesis of collagen also continued during cytochalasin inhibition of cell spreading. Removal of the inhibitor from the cultures initiated cell spreading and CBP synthesis continued. Membrane-cytoskeleton complexes from control and CD treated cells were identical in regard to binding CBPs in a calcium-dependent manner. Colchicine, which inhibited cell spreading, was shown to be toxic to general protein synthesis at 75 nM. The data clearly indicate that mere inhibition of epithelial cell spreading does not automatically suppress CBP synthesis.

    View details for Web of Science ID A1989T948700002

    View details for PubMedID 2524259

  • BASAL LAMINA INHIBITION SUPPRESSES SYNTHESIS OF CALCIUM-DEPENDENT PROTEINS ASSOCIATED WITH MAMMARY EPITHELIAL-CELL SPREADING EXPERIMENTAL CELL RESEARCH Rocha, V., Hom, Y. K., Marinkovich, M. P. 1986; 165 (2): 450-460

    Abstract

    Spreading of mouse mammary epithelial cells on collagen gels is closely correlated with the synthesis of a group of putative calcium-binding proteins (CBP) (Braslau et al., Exp cell res 155 (1984) 213). Collagen synthesis was shown to occur during cell spreading, while omission of serum prevented cell spreading and the synthesis of collagen. The proline analogues cis-hydroxyproline and L-azetidine-2-carboxylic acid were shown to inhibit epithelial cell spreading and to suppress the collagen synthesis that occurs during serum-supported cell spreading. Inhibition of collagen synthesis resulted in the inhibition of CBP synthesis associated with cell spreading. In contrast, the collagen cross-linking inhibitor B-aminopropionitrile did not inhibit cell spreading nor did it suppress collagen synthesis; CBP synthesis was also normal during treatment with this inhibitor. Thus, mammary epithelial cell spreading on collagen gels and CBP synthesis can both be suppressed by inhibition of collagen synthesis indicating that they may be integrated in some manner. It is suggested that inhibition of cell spreading during inhibition of collagen synthesis results from failure to assemble a normal basal lamina; this may in turn signal suppression of CBP synthesis.

    View details for Web of Science ID A1986D238000016

    View details for PubMedID 3720859

  • COLLAGEN-SYNTHESIS AND DEPOSITION DURING MAMMARY EPITHELIAL-CELL SPREADING ON COLLAGEN GELS JOURNAL OF CELLULAR PHYSIOLOGY Marinkovich, M. P., Rocha, V. 1986; 128 (1): 61-70

    Abstract

    Mouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dose-dependent manner. Cytochalasin D inhibition of F-actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreading.

    View details for Web of Science ID A1986D155700010

    View details for PubMedID 3722273

Conference Proceedings


  • Bullous systemic lupus erythematosus with autoantibodies recognizing multiple skin basement membrane components, bullous pemphigoid antigen 1, laminin-5, laminin-6, and type VII collagen Chan, L. S., Lapiere, J. C., Chen, M., Traczyk, T., Mancini, A. J., Paller, A. S., Woodley, D. T., Marinkovich, M. P. AMER MEDICAL ASSOC. 1999: 569-573

    Abstract

    Bullous systemic lupus erythematosus is a generalized subepidermal blistering skin eruption in patients suffering from systemic lupus erythematosus. Type VII collagen was initially identified as the target antigen.We studied an unusual patient who had bullous systemic lupus crythematosus. The patient fulfilled the criteria of systemic lupus with an antinuclear antibody titer of 1:5120. Immunopathological testing revealed in vivo deposition of all IgG subclasses, secretory IgA1, and both light chains at the patient's skin basement membrane. The in vivo-bound IgG and IgA were localized at the hemidesmosomes and lamina densa. The patient's IgG and IgA circulating autoantibodies labeled both the epidermal roof and the dermal floor of salt-split skin and recognized the hemidesmosomal protein BP230 as well as the full-length native form and the recombinant noncollagenous domain 1 of type VII collagen (anchoring fibril). In addition, the patient's IgG autoantibodies recognized the anchoring filament proteins laminin-5 and laminin-6 (alpha3 chain and gamma2 chain).We conclude that patients with bullous systemic lupus erythematosus may have autoantibodies to multiple basement membrane components critical for epidermal-dermal junctional adhesion. Possible pathogenic mechanisms in this patient's clinical diseases include provocation of organ-specific disease (bullous disease) by systemic autoimmunity (lupus) and the "epitope spreading" immune phenomenon.

    View details for Web of Science ID 000080167000012

    View details for PubMedID 10328198

  • LAD-1 is absent in a subset of junctional epidermolysis bullosa patients Marinkovich, M. P., Tran, H. H., Rao, S. K., Giudice, G. J., Balding, S., Jonkman, M. F., Pas, H. H., McGuire, J. S., Herron, G. S., BRUCKNERTUDERMAN, L. NATURE PUBLISHING GROUP. 1997: 356-359

    Abstract

    The anchoring filament protein LAD-1 has been recently identified as the target of autoantibodies in the acquired blistering disorder linear IgA bullous dermatosis. Because this protein appears to be involved in the process of dermal-epidermal cohesion, this study sought to determine the involvement of LAD-1 in the pathology of junctional epidermolysis bullosa (JEB). To this end, 44 patients with a variety of subtypes of JEB were analyzed by indirect immunofluorescence microscopy with antibodies to LAD-1, BP180, and laminin-5. We found that only patients with generalized atrophic benign epidermolysis bullosa (GABEB) contained LAD-1 defects. Of the 16 GABEB patients studied, 13 showed absent or greatly reduced expression of LAD-1 (including 2 patients with a peculiar interrupted staining pattern) and 3 patients showed defects of laminin-5 expression with normal LAD-1 expression. Patients who showed LAD-1 defects also showed abnormal expression of BP180. Keratinocytes were cultured from the skin of two GABEB patients and analyzed by indirect immunofluorescent microscopy. One culture demonstrated defects of BP180 and LAD-1 expression (which was also verified by radioimmunoprecipitation assay), and one culture showed decreased laminin-5 expression but normal BP180 and LAD-1 expression. Thus, these studies demonstrate that: (i) LAD-1 and BP180 are normally expressed in all subtypes of JEB except GABEB, (ii) the majority of GABEB patients show absent or near absent expression of both LAD-1 and BP180 but normal expression of laminin-5, and (iii) a smaller subset of GABEB patients show normal LAD-1 and BP180 expression but express persistent but reduced levels of laminin-5.

    View details for Web of Science ID A1997XT10300016

    View details for PubMedID 9284104

  • Laminin-5 inhibits human keratinocyte migration OToole, E. A., Marinkovich, M. P., Hoeffler, W. K., FURTHMAYR, H., Woodley, D. T. ELSEVIER INC. 1997: 330-339

    Abstract

    Laminin-5 (previously known as kalinin, epiligrin, and nicein) is an adhesive protein localized to the anchoring filaments within the lamina lucida space of the basement membrane zone lying between the epidermis and dermis of human skin. Anchoring filaments are structures within the lamina lucida and lie immediately beneath the hemidesmosomes of the overlying basal keratinocytes apposed to the basement membrane zone. Human keratinocytes synthesize and deposit laminin-5. Laminin-5 is present at the wound edge during reepithelialization. In this study, we demonstrate that laminin-5, a powerful matrix attachment factor for keratinocytes, inhibits human keratinocyte migration. We found that the inhibitory effect of laminin-5 on keratinocyte motility can be reversed by blocking the alpha3 integrin receptor. Laminin-5 inhibits keratinocyte motility driven by a collagen matrix in a concentration-dependent fashion. Using antisense oligonucleotides to the alpha3 chain of laminin-5 and an antibody that inhibits the cell binding function of secreted laminin-5, we demonstrated that the endogenous laminin-5 secreted by the keratinocyte also inhibits the keratinocyte's own migration on matrix. These findings explain the hypermotility that characterizes keratinocytes from patients who have forms of junctional epidermolysis bullosa associated with defects in one of the genes encoding for laminin-5 chains, resulting in low expression and/or functional inadequacy of laminin-5 in these patients. These studies also suggest that during reepithelialization of human skin wounds, the secreted laminin-5 stabilizes the migrating keratinocyte to establish the new basement membrane zone.

    View details for Web of Science ID A1997XE31700010

    View details for PubMedID 9194495

  • Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid Chan, L. S., MAJMUDAR, A. A., Tran, H. H., Meier, F., SCHAUMBURGLEVER, G., Chen, M., Anhalt, G., Woodley, D. T., Marinkovich, M. P. NATURE PUBLISHING GROUP. 1997: 848-853

    Abstract

    We characterized basement membrane zone (BMZ) autoantigens targeted by autoantibodies (AAb) from patients with cicatricial pemphigoid. Serum from a patient with severe oral cicatricial pemphigoid contained IgG anti-BMZ AAb. The AAb labeled a lower BMZ component on salt-split skin and localized to the lower lamina lucida/lamina densa by direct and indirect immunoelectron microscopy (IEM) but did not label blood vessels. The AAb did not react with EHS laminin-1 and type IV collagen, pepsinized human type IV collagen, recombinant entactin, or NC1 domain of type VII collagen by dot blotting and western blotting. We focused our studies on the laminin family, as laminin-5 was identified as an autoantigen in cicatricial pemphigoid. Culture-conditioned media from normal keratinocytes (containing laminin-6 and laminin-5) and JEB keratinocytes (containing laminin-6 but not laminin-5) were studied by western blotting. Under nonreducing conditions, the patient's AAb recognized a 600-kDa protein (laminin-6) intensely and a 400-kDa protein (laminin-5) weakly in normal keratinocyte medium even though abundant laminin-5 was present. InJEB keratinocyte medium, however, the 600-kDa protein (laminin-6) alone was recognized by the patient's AAb. The AAb also immunolabeled BMZ of JEB skin that lacked laminin-5. The AAb from this patient and two other patients with anti-laminin-5 cicatricial pemphigoid immunoprecipitated both laminin-6 and laminin-5. Taken together, the results of IEM, non-reducing western blotting, immunoprecipitation, and JEB skin BMZ immunolabeling indicate that laminin-6, as well as laminin-5, is identified by the AAb from a subset of cicatricial pemphigoid patients. We propose the name "anti-laminin cicatricial pemphigoid" for this subset.

    View details for Web of Science ID A1997XB83600003

    View details for PubMedID 9182809

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