Bio

Clinical Focus


  • Gastroenterology/Nutrition/Hepatology, Pediatric
  • Pediatric Gastroenterology

Academic Appointments


Administrative Appointments


  • Assistant Dean for Academic Advising, Stanford University School of Medicine (2012 - Present)
  • Senator, School of Medicine Faculty Senate (2009 - Present)
  • Associate Director (Research), Stanford Pediatric Gastroenterology Training Program (2004 - Present)
  • Chair/Vice-Chair, Growth, Development and Aging Section, American Gastroenterology Association (2007 - 2011)
  • Editor-In-Chief, Journal of Pediatric Gastroenterology and Nutrition (2006 - 2010)
  • Member, Clinical and Integrative Gastrointestinal Pathobiology Study Section, NIH (2004 - 2007)
  • Admissions Committee, Stanford University School of Medicine (1998 - 2001)

Honors & Awards


  • Member, American Clinical and Climatological Association (2010-present)
  • Council, elected, American Gastroenterology Association (2007-2011)
  • Council, elected, Society for Pediatric Research (2005-2008)
  • Named Investigator Award, Stanford Digestive Disease Center (2002-2003)
  • Young Faculty Investigator Award, North American Society for Pediatric Gastroenterology and Nutrition (1998)

Professional Education


  • Undergraduate Education, Harvard College, A.B., Biochemical Sciences (1982)
  • Medical Education:Johns Hopkins University School of Medicine (1990) MD
  • Graduate Education, The Johns Hopkins University, Ph.D., Biological Chemistry (1990)
  • Internship:Harbor-UCLA Medical Center (1991) CA
  • Residency:Harbor-UCLA Medical Center (1993) CA
  • Fellowship:Stanford University School of Medicine (1996) CA
  • Board Certification: Pediatric Gastroenterology, American Board of Pediatrics (1997)

Research & Scholarship

Current Research and Scholarly Interests


Transcriptional Regulation of Intestine-Specific Gene Expression during Gut Development and Maturation.

The mammalian gastrointestinal tract matures from a primitive tube into morphologically and functionally distinct regions during development. The mature small intestine functions in the digestion and absorption of ingested nutrients. Expression of several nutrient hydrolases is spatially restricted to distinct segments along the cephalo-caudal axis of the small intestine and is temporally regulated during postnatal maturation. Intestinal lactase, the hydrolase responsible for the digestion of lactose in milk, is expressed at highest levels in the jejunal segment of the small intestine shortly after birth and then declines dramatically just prior to weaning in most mammals.

Our research is directed towards defining the mechanisms regulating the spatial and temporal restriction of lactase gene expression during intestinal development. The normal maturational decline in lactase enzymatic activity is correlated with a decline in lactase messenger RNA levels and is transcriptionally regulated. We are currently identifying maturation-specific lactase gene cis elements and characterizing the nuclear proteins interacting with those elements in cell culture and transgenic animals. We seek to define the interactions of the lactase gene elements and nuclear factors involved in mediating transcriptional control. The overall goal is to relate these lactase control mechanisms to the broader pathways specifying acquisition of a small intestinal phenotype. We are also investigating gene transfer methods to deliver specific genes to the intestine.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Intestinal Pdx1 mediates nutrient metabolism gene networks and maternal expression is essential for perinatal growth in mice BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Chen, C., Leavitt, T., Sibley, E. 2012; 424 (3): 549-553

    Abstract

    The homeodomain transcription factor Pdx1 is essential for pancreas formation and functions in pancreatic islets cells to regulate genes involved in maintenance of glucose homeostasis. In order to investigate a role for Pdx1 in intestinal cells, we analyzed the functions and networks associated with genes differentially expressed by Pdx1 overexpression in human Caco-2 cells. In agreement with previous results for intestine isolated from mice with Pdx1 inactivation, functional analysis of genes differentially expressed with Pdx1 overexpression revealed functions significantly associated with nutrient metabolism. Similarly, network analysis examining the interactions among the differentially expressed genes revealed gene networks involved in lipid metabolism. Consistent with defects in maternal nutrient metabolism, mouse pups born to dams with intestine-specific Pdx1 inactivation are underweight and fail to thrive in the neonatal period compared to pups born to control dams. We conclude that Pdx1 mediates lipid metabolism gene networks in intestinal cells and that maternal expression is essential for perinatal growth in mice.

    View details for DOI 10.1016/j.bbrc.2012.06.153

    View details for Web of Science ID 000307618800032

    View details for PubMedID 22771330

  • The human lactase persistence-associated SNP -13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression HUMAN GENETICS Fang, L., Ahn, J. K., Wodziak, D., Sibley, E. 2012; 131 (7): 1153-1159

    Abstract

    Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype.

    View details for DOI 10.1007/s00439-012-1140-z

    View details for Web of Science ID 000305195400014

    View details for PubMedID 22258180

  • PDX1 regulation of FABP1 and novel target genes in human intestinal epithelial Caco-2 cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Chen, C., Fang, R., Chou, L., Lowe, A. W., Sibley, E. 2012; 423 (1): 183-187

    Abstract

    The transcription factor pancreatic and duodenal homeobox 1 (PDX1) plays an essential role in pancreatic development and in maintaining proper islet function via target gene regulation. Few intestinal PDX1 targets, however, have been described. We sought to define novel PDX1-regulated intestinal genes. Caco-2 human intestinal epithelial cells were engineered to overexpress PDX1 and gene expression profiles relative to control cells were assessed. Expression of 80 genes significantly increased while that of 49 genes significantly decreased more than 4-fold following PDX1 overexpression in differentiated Caco-2 cells. Analysis of the differentially regulated genes with known functional annotations revealed genes encoding transcription factors, growth factors, kinases, digestive glycosidases, nutrient transporters, nutrient binding proteins, and structural components. The gene for fatty acid binding protein 1, liver, FABP1, is repressed by PDX1 in Caco-2 cells. PDX1 overexpression in Caco-2 cells also results in repression of promoter activity driven by the 0.6kb FABP1 promoter. PDX1 regulation of promoter activity is consistent with the decrease in FABP1 RNA abundance resulting from PDX1 overexpression and identifies FABP1 as a candidate PDX1 target. PDX1 repression of FABP1, LCT, and SI suggests a role for PDX1 in patterning anterior intestinal development.

    View details for DOI 10.1016/j.bbrc.2012.05.113

    View details for Web of Science ID 000307087700031

    View details for PubMedID 22640736

  • Expression profiling identifies novel gene targets and functions for Pdx1 in the duodenum of mature mice AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Chen, C., Sibley, E. 2012; 302 (4): G407-G419

    Abstract

    Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, the functions of Pdx1 in mature small intestine are less known. We aimed to investigate the intestinal role of Pdx1 by profiling the expression of genes differentially regulated in response to inactivation of Pdx1 specifically in the intestinal epithelium. Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1(flox/flox);VilCre mice. Total RNA was isolated from the first 5 cm of the small intestine from mature Pdx1(flox/flox);VilCre and littermate control mice. Microarray analysis identified 86 probe sets representing 68 genes significantly upregulated or downregulated 1.5-fold or greater in Pdx(flox/flox);VilCre mice maintained under standard conditions. Ingenuity Pathway Analysis revealed that functions of the differentially expressed genes are significantly associated with metabolism of nutrients including lipids and iron. Network analysis examining the interactions among the differentially expressed genes further supports the notion that Pdx1 may modulate metabolism of lipids and iron from mature intestinal epithelium. Following forced oil feeding, Pdx1(flox/flox);VilCre mice showed diminished lipid staining in the duodenal epithelium and decreased serum triglyceride levels, indicating reduced lipid absorption compared with control duodenal epithelium. Blood samples from Pdx1(flox/flox);VilCre mice have significantly lower mean values for mean corpuscular volume and mean corpuscular hemoglobin, consistent with iron deficiency. The absence of nonheme iron in the villous epithelium and lamina propria of Pdx1(flox/flox);VilCre duodenum indicates that the duodenal epithelium lacking Pdx1 may have defects in importing iron through enterocytes, resulting in iron deficiency in Pdx1(flox/flox);VilCre mice.

    View details for DOI 10.1152/ajpgi.00314.2011

    View details for Web of Science ID 000300318000001

    View details for PubMedID 22135308

  • Theodore E. Woodward Award: lactase persistence SNPs in African populations regulate promoter activity in intestinal cell culture. Transactions of the American Clinical and Climatological Association Sibley, E., Ahn, J. K. 2011; 122: 155-165

    Abstract

    Lactase-phlorizin hydrolase, lactase, is the intestinal enzyme responsible for the digestion of the milk sugar lactose. The majority of the world's human population experiences a decline in expression of the lactase gene by late childhood (lactase non-persistence). Individuals with lactase persistence, however, continue to express high levels of the lactase gene throughout adulthood. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ?14 kb upstream of the lactase gene in different ethnic populations: -13910*T in Europeans and -13907*G, -13915*G, and -14010*C in several African populations. The coincidence of the four SNPs clustering within 100 bp strongly suggests that this region mediates the lactase non-persistence/persistence phenotype. Having previously characterized the European SNP, we aimed to determine whether the African SNPs similarly mediate a functional role in regulating the lactase promoter. Human intestinal Caco-2 cells were transfected with lactase SNP/promoter-reporter constructs and assayed for promoter activity. The -13907*G and -13915*G SNPs result in a significant enhancement of lactase promoter activity relative to the ancestral lactase non-persistence genotype. Such differential regulation by the SNPs is consistent with a causative role in the mechanism specifying the lactase persistence phenotype.

    View details for PubMedID 21686221

  • -13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1 HUMAN GENETICS Olds, L. C., Ahn, J. K., Sibley, E. 2011; 129 (1): 111-113

    Abstract

    Lactase gene expression declines with aging (lactase non-persistence) in the majority of humans worldwide. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ~14-kb upstream (-13907, -13910 and -13915) of the lactase gene in different ethnic populations. In contrast to the -13907*G and -13910*T SNPs, the -13915*G SNP was previously believed not to interact with Oct-1. In the present study, however, Oct-1 is shown to interact with the -13915*G SNP region DNA sequence by EMSAs and gel supershift. In addition, Oct-1 is capable of enhancing promoter activity of a lactase promoter-reporter construct harboring the 13915*G SNP sequence in cell culture. Oct-1 binding to the -13907 to -13915 SNP region therefore remains a candidate interaction involved in lactase persistence.

    View details for DOI 10.1007/s00439-010-0898-0

    View details for Web of Science ID 000286396200012

    View details for PubMedID 20960210

  • Increased HLA-DR Expression on Tissue Eosinophils in Eosinophilic Esophagitis JOURNAL OF PEDIATRIC GASTROENTEROLOGY AND NUTRITION Patel, A. J., Fuentebella, J., Gernez, Y., Nguyen, T., Bass, D., Berquist, W., Cox, K., Sibley, E., Kerner, J., Nadeau, K. 2010; 51 (3): 290-294

    Abstract

    The aim of the study was to investigate whether eosinophils have increased human leukocyte antigen (HLA)-DR expression in subjects with eosinophilic esophagitis (EoE) compared with controls.Patients who were undergoing an upper endoscopy with biopsies for suspected gastroesophageal reflux disease (GERD) or EoE at Lucile Packard Children's Hospital were enrolled. In total, the blood and tissue samples of 10 healthy controls (HC), 11 subjects with GERD, and 10 with EoE were studied. Multiple tissue staining to identify eosinophils (via eosinophil cationic protein-clone EG2) and major histocompatibility complex class II cell surface receptors (via HLA-DR) was performed via immunohistochemistry. The peripheral blood was analyzed using flow cytometry to detect eosinophil HLA-DR expression among these subjects.In the tissue, a greater proportion of eosinophils expressed HLA-DR among the subjects with EoE (mean 0.83 +/- 0.14, n = 9) relative to those with GERD (mean 0.18 +/- 0.19, n = 8, P < 0.01) and HC (mean 0.18 +/- 0.13, n = 6, P < 0.01). In total, 6 participants (4 HC subjects and 2 subjects with GERD) did not have any eosinophils identified on tissue staining and were unable to be included in the present statistical analysis. In the blood, there was no statistically significant difference in eosinophil HLA-DR expression among HC subjects (mean 415 +/- 217, n = 6), subjects with GERD (mean 507 +/- 429, n = 2), and those with EoE (mean 334 +/- 181, n = 6).These data demonstrate that the eosinophils from the esophagus of subjects with EoE have increased HLA-DR expression within this tissue.

    View details for DOI 10.1097/MPG.0b013e3181e083e7

    View details for Web of Science ID 000281453500008

    View details for PubMedID 20639774

  • Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution JOURNAL OF BIOMEDICAL OPTICS Fu, Y., Lu, C., Lin, C., Juang, J., Enikolopov, G., Sibley, E., Chiang, A., Tang, S. 2010; 15 (4)

    Abstract

    Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.

    View details for DOI 10.1117/1.3470241

    View details for Web of Science ID 000281335400053

    View details for PubMedID 20799820

  • Eotaxin and FGF enhance signaling through an extracellular signal-related kinase (ERK)-dependent pathway in the pathogenesis of Eosinophilic esophagitis. Allergy, asthma, and clinical immunology : official journal of the Canadian Society of Allergy and Clinical Immunology Huang, J. J., Joh, J. W., Fuentebella, J., Patel, A., Nguyen, T., Seki, S., Hoyte, L., Reshamwala, N., Nguyen, C., Quiros, A., Bass, D., Sibley, E., Berquist, W., Cox, K., Kerner, J., Nadeau, K. C. 2010; 6 (1): 25-?

    Abstract

    Eosinophilic esophagitis (EoE) is characterized by the inflammation of the esophagus and the infiltration of eosinophils into the esophagus, leading to symptoms such as dysphagia and stricture formation. Systemic immune indicators like eotaxin and fibroblast growth factor were evaluated for possible synergistic pathological effects. Moreover, blood cells, local tissue, and plasma from EoE and control subjects were studied to determine if the localized disease was associated with a systemic effect that correlated with presence of EoE disease.Real-time polymerase chain reaction from peripheral blood mononuclear cells (PBMC), immunohistochemistry from local esophageal biopsies, fluid assays on plasma, and fluorescence-activated cell sorting on peripheral blood cells from subjects were used to study the systemic immune indicators in newly diagnosed EoE (n = 35), treated EoE (n = 9), Gastroesophageal reflux disease (GERD) (n = 8), ulcerative colitis (n = 5), Crohn's disease (n = 5), and healthy controls (n = 8).Of the transcripts tested for possible immune indicators, we found extracellular signal-regulated kinase (ERK), Bcl-2, bFGF (basic fibroblast growth factor), and eotaxin levels were highly upregulated in PBMC and associated with disease presence of EoE. Increased FGF detected by immunohistochemistry in esophageal tissues and in PBMC was correlated with low levels of pro-apoptotic factors (Fas, Caspase 8) in PBMC from EoE subjects. Plasma-derived bFGF was shown to be the most elevated and most specific in EoE subjects in comparison to healthy controls and disease control subjects.We describe for the first time a possible mechanism by which increased FGF is associated with inhibiting apoptosis in local esophageal tissues of EoE subjects as compared to controls. Eotaxin and FGF signaling pathways share activation through the ERK pathway; together, they could act to increase eosinophil activation and prolong the half-life of eosinophils in local tissues of the esophagus in EoE subjects.

    View details for DOI 10.1186/1710-1492-6-25

    View details for PubMedID 20815913

  • Pdx1 inactivation restricted to the intestinal epithelium in mice alters duodenal gene expression in enterocytes and enteroendocrine cells AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Chen, C., Fang, R., Davis, C., Maravelias, C., Sibley, E. 2009; 297 (6): G1126-G1137

    Abstract

    Null mutant mice lacking the transcription factor pancreatic and duodenal homeobox 1 (Pdx1) are apancreatic and survive only a few days after birth. The role of Pdx1 in regulating intestinal gene expression has therefore yet to be determined in viable mice with normal pancreatic development. We hypothesized that conditional inactivation of Pdx1 restricted to the intestinal epithelium would alter intestinal gene expression and cell differentiation. Pdx1(flox/flox);VilCre mice with intestine-specific Pdx1 inactivation were generated by crossing a transgenic mouse strain expressing Cre recombinase, driven by a mouse villin 1 gene promoter fragment, with a mutant mouse strain homozygous for loxP site-flanked Pdx1. Pdx1 protein is undetectable in all epithelial cells in the intestinal epithelium of Pdx1(flox/flox);VilCre mice. Goblet cell number and mRNA abundance for mucin 3 and mucin 13 genes in the proximal small intestine are comparable between Pdx1(flox/flox);VilCre and control mice. Similarly, Paneth cell number and expression of Paneth cell-related genes Defa1, Defcr-rs1, and Mmp7 in the proximal small intestine remain statistically unchanged by Pdx1 inactivation. Although the number of enteroendocrine cells expressing chromogranin A/B, gastric inhibitory polypeptide (Gip), or somatostatin (Sst) is unaffected in the Pdx1(flox/flox);VilCre mice, mRNA abundance for Gip and Sst is significantly reduced in the proximal small intestine. Conditional Pdx1 inactivation attenuates intestinal alkaline phosphatase (IAP) activity in the duodenal epithelium, consistent with an average 91% decrease in expression of the mouse enterocyte IAP gene, alkaline phosphatase 3 (a novel Pdx1 target candidate), in the proximal small intestine following Pdx1 inactivation. We conclude that Pdx1 is necessary for patterning appropriate gene expression in enterocytes and enteroendocrine cells of the proximal small intestine.

    View details for DOI 10.1152/ajpgi.90586.2008

    View details for Web of Science ID 000272151900010

    View details for PubMedID 19808654

  • Microtome-Free 3-Dimensional Confocal Imaging Method for Visualization of Mouse Intestine With Subcellular-Level Resolution GASTROENTEROLOGY Fu, Y., Lin, C., Enikolopov, G., Sibley, E., Chiang, A., Tang, S. 2009; 137 (2): 453-465

    Abstract

    The intrinsic opacity of mouse intestinal tissue prevents its evaluation by high-resolution, in-depth optical microscopy. Instead, intestinal tissue is usually sectioned to expose the interior domains of the mucosa and submucosa for microscopic examination. However, microtome sectioning can cause distortions and artifacts that prevent acquisition of an accurate view of the sample. We therefore attempted to develop a microtome-free 3-dimensional (3D) confocal imaging method for characterization of mouse intestine.We applied an optical-clearing solution, FocusClear, to permeate and reduce the opacity of mouse colon and ileum. Tissues were labeled with fluorescent probes and examined by confocal microscopy with efficient fluorescence excitation and emission in the FocusClear solution. The voxel-based confocal micrographs were processed with Amira software for 3D visualization and analysis.Treatment of tissues with the optical-clearing solution improved photon penetration, resulting in the acquisition of images with subcellular-level resolution across the mucosa, submucosa, and muscle layers. Collectively, the acquired image stacks were processed by projection algorithms for 3D analysis of the spatial relations in villi, crypts, and connective tissues. These imaging technologies allowed for identification of spatiotemporal changes in crypt morphology of colon tissues from mice with dextran sulfate sodium-induced colitis as well as detection of transgenic fluorescent proteins expressed in the colon and ileum.This new optical method for penetrative imaging of mouse intestine does not require tissue sectioning and provides a useful tool for 3D presentation and analysis of diseased and transgenic intestine in an integrated fashion.

    View details for DOI 10.1053/j.gastro.2009.05.008

    View details for Web of Science ID 000268551000019

    View details for PubMedID 19447107

  • Transient cytochalasin-D treatment induces apically administered rAAV2 across tight junctions for transduction of enterocytes JOURNAL OF GENERAL VIROLOGY Fu, Y., Sibley, E., Tang, S. 2008; 89: 3004-3008

    Abstract

    Enteropathogens are known to disrupt apical actin filaments and/or tight-junction barriers of intestinal epithelial cells to promote infection. In this study, we show that a controlled, cytochalasin-D (Cyto-D)-mediated disruption of actin filaments and tight junctions enhanced the apical delivery of the gene-therapy vector recombinant adeno-associated virus serotype 2 (rAAV2). This increase in transduction efficiency can be attributed to the enhanced delivery of rAAV2 across the Cyto-D disrupted tight junctions, allowing basolateral entry of rAAV2. Previously, we have shown that MG101 and doxorubicin are capable of overcoming proteasome-mediated transduction barriers of rAAV2 in enterocytes. In this study, when Cyto-D was combined with MG101 and doxorubicin in apical delivery of rAAV2 to transduce the differentiated Caco-2 enterocytes, a synergistic >2300-fold increase in transgene expression was achieved. We conclude that Cyto-D is capable of permeating the polarized enterocytes for rAAV2 transduction, which may potentially be a useful device to facilitate intestinal gene transfer via the gut lumen.

    View details for DOI 10.1099/vir.0.2008/001446-0

    View details for Web of Science ID 000261527800010

    View details for PubMedID 19008386

  • Application of doxorubicin-induced rAAV2-p53 gene delivery in combined chemotherapy and gene therapy for hepatocellular carcinoma CANCER BIOLOGY & THERAPY Chen, C., Lo, C., Lin, B., Sibley, E., Tang, S. 2008; 7 (2): 303-U3

    Abstract

    p53 gene transfer has been proposed as a potential therapeutic option for treatment of hepatocellular carcinoma (HCC). Compared to other commonly used gene transfer vectors such as adenovirus and retrovirus, recombinant adeno-associated virus serotype 2 (rAAV2) has shown promising results in human clinical trials. Significant enhancement in the gene transfer efficiency is needed, however, for HCC applications. In the present study, we applied chemotherapy drug Doxorubicin (DOX) to induce rAAV2 transduction of hepatomas. Using reporter assays, we showed that the DOX-treated hepatomas became more susceptible to rAAV2 infection in comparison to untreated controls: the permissiveness increased >350-fold and >120-fold for HepG2 (p53 wild-type) and Hep3B (p53 null) hepatomas, respectively. Using the induced permissiveness, we applied rAAV2-p53 transduction to restore p53 expression in the p53-null Hep3B hepatomas. Compared to rAAV2-p53 transduction alone, rAAV2-p53 transduction with DOX resulted in a >16-fold induction of p53 expression. The transduced Hep3B expressed as much as 380% more immunoreactive p53 in comparison to the wild-type p53 expression in the HepG2 hepatomas. Significantly, when Hep3B cells were treated with 0.5 muM of DOX and rAAV2-p53 (MOI = 10) for twelve hours, the cell viability dropped to 66% four days after the administration. This decrease in cell viability was similar to that of treatment with 1 microM of DOX alone in the absence of rAAV2. The 50% reduction in DOX administration--from 1 microM to 0.5 microM--revealed the antitumor property of the rAAV2-p53 transduction as well as the joint cytotoxicity of DOX and rAAV2-p53 against the p53-null hepatomas. We conclude that DOX mediates the enhancement effect on rAAV2 transduction of human hepatomas. Combined DOX and rAAV2-p53 administration may facilitate more efficient treatment for the HCC caused by p53 mutations.

    View details for Web of Science ID 000256658500025

    View details for PubMedID 18059187

  • Genetic modification of somatic gut mucosa: An adeno-associated virus approach JOURNAL OF PEDIATRIC GASTROENTEROLOGY AND NUTRITION Tang, S., Sibley, E. 2006; 43 (2): 158-159

    View details for Web of Science ID 000239542200003

    View details for PubMedID 16877978

  • Spatio-temporal patterns of intestine-specific transcription factor expression during postnatal mouse gut development GENE EXPRESSION PATTERNS Fang, R. X., Olds, L. C., Sibley, E. 2006; 6 (4): 426-432

    Abstract

    The small intestine matures from a primitive tube into morphologically and functionally distinct regions during gut development. Maximal expression of the genes encoding the digestive enzymes lactase-phlorizin hydrolase and sucrase-isomaltase is spatially restricted to distinct segments along the anterior-posterior axis of the small intestine and is temporally regulated during postnatal maturation. Transcription factors capable of interacting with the intestinal lactase and sucrase gene promoters are candidate regulators of spatio-temporal patterning during gut development and maturation. We aimed to quantitatively examine and compare the relative expression levels of a set of intestine-specific transcription factors along the anterior-posterior gut axis during postnatal maturation. Our analysis was focused on the transcription factors capable of regulating the intestinal lactase and sucrase-isomaltase genes. A real-time PCR protocol was used to quantitatively examine and compare spatially and temporally the relative transcript abundance levels for intestine-specific factors during postnatal intestinal maturation. Distinct spatial expressions patterns were detected along the length of the small intestine for PDX-1, Cdx-2, GATA-4, GATA-5, GATA-6, HNF-1alpha, HNF-1beta and CDP transcription factor genes. There is a general decline in transcript abundance for the factor genes during postnatal maturation. Defining the spatio-temporal expression patterns for intestine-specific transcription factor genes contributes to investigation of the roles that factor gradients play in mediating gut development and differentiation.

    View details for DOI 10.1016/j.modgep.2005.09.003

    View details for Web of Science ID 000236434500012

    View details for PubMedID 16377257

  • Lactase gene promoter fragments mediate differential spatial and temporal expression patterns in transgenic mice DNA AND CELL BIOLOGY Wang, Z., Maravelias, C., Sibley, E. 2006; 25 (4): 215-222

    Abstract

    Lactase gene expression is spatiotemporally regulated during mammalian gut development. We hypothesize that distinct DNA control regions specify appropriate spatial and temporal patterning of lactase gene expression. In order to define regions of the lactase promoter involved in mediating intestine-specific and spatiotemporal restricted expression, transgenic mice harboring 100 bp, 1.3- and 2.0- kb fragments of the 5' flanking region of the rat lactase gene cloned upstream of a luciferase reporter were characterized. The 100-bp lactase promoter-reporter transgenic mouse line expressed maximal luciferase activity in the intestine with a posterior shift in spatial restriction and ectopic expression in the stomach and lung. The temporal pattern of expression mediated by the 1.3-kb promoter?reporter transgene increases with postnatal maturation in contrast with the postnatal decline mediated by the 2.0-kb promoter-reporter transgene and the endogenous lactase gene. The differential transgene expression patterns mediated by the lactase promoter fragments suggests that intestine-specific spatial and temporal control elements reside in distinct regions of the DNA sequences upstream of the lactase gene transcription start-site.

    View details for Web of Science ID 000237131800003

    View details for PubMedID 16629594

  • Proteasome modulating agents induce rAAV2-mediated transgene expression in human intestinal epithelial cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Tang, S. C., Sambanis, A., Sibley, E. 2005; 331 (4): 1392-1400

    Abstract

    Intestinal gene transfer offers promise as a therapeutic option for treatment of both intestinal and non-intestinal diseases. Recombinant adeno-associated virus serotype 2, rAAV2, based vectors have been utilized to transduce lung epithelial cells in culture and in human subjects. rAAV2 transduction of intestinal epithelial cells, however, is limited both in culture and in vivo. Proteasome-inhibiting agents have recently been shown to enhance rAAV2-mediated transgene expression in airway epithelial cells. We hypothesized that similar inhibition of proteasome-related cellular processes can function to induce rAAV2 transduction of intestinal epithelial cells. Our results demonstrate that combined treatment with proteasome-modulating agents MG101 (N-acetyl-L-leucyl-L-leucyl-L-norleucine) and Doxorubicin synergistically induces rAAV2-mediated luciferase transgene expression by >400-fold in undifferentiated Caco-2 cells. In differentiated Caco-2 monolayers, treatment with MG101 and Doxorubicin induces transduction preferentially from the basolateral cell surface. In addition to Caco-2 cells, treatment with MG101 and Doxorubicin also results in enhanced rAAV2 transduction of HT-29, T84, and HCT-116 human intestinal epithelial cell lines. We conclude that MG101 and Doxorubicin mediate generic effects on intestinal epithelial cells that result in enhanced rAAV2 transduction. Use of proteasome-modulating agents to enhance viral transduction may facilitate the development of more efficient intestinal gene transfer protocols.

    View details for DOI 10.1016/j.bbrc.2005.03.245

    View details for Web of Science ID 000229234500069

    View details for PubMedID 15883029

  • Transcriptional regulation of the lactase-phlorizin hydrolase promoter by PDX-1 AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Wang, Z., Fang, R. X., Olds, L. C., Sibley, E. 2004; 287 (3): G555-G561

    Abstract

    Lactase-phlorizin hydrolase gene expression is spatially restricted along the anterior-posterior gut axis. Lactase gene transcription is maximal in the distal duodenum and jejunum in adult mammals and is barely detectable in the proximal duodenum. By contrast, pancreatic duodenal homeobox-1 (PDX-1) protein is expressed maximally in the proximal duodenum. This study aimed to determine the role of PDX-1 in regulating lactase gene promoter activity in intestinal epithelial cells. Caco-2 cells were cotransfected with lactase promoter-reporter constructs in the presence of a PDX-1 expression vector and assayed for luciferase activity. PDX-1 cotransfection results in repression of lactase promoter activity. Sequence analysis of the lactase promoter revealed a putative PDX-1 DNA binding site in the proximal 100-bp lactase gene promoter. EMSAs demonstrated that PDX-1 can interact with the lactase promoter binding site but not with a site in which the core PDX-1 binding sequence TAAT is mutated. Site-directed mutagenesis of the PDX-1 core binding site in the lactase promoter-reporter construct suggests that PDX-1 can function independently of DNA binding to its consensus binding site. Stable overexpression of PDX-1 results in repression of the endogenous human lactase gene in differentiated Caco-2 cells. Given the contrasting spatial expression pattern, PDX-1 may function to specify the anterior boundary of lactase expression in the small intestine and is thus a candidate regulator of anterior spatial restriction in the gut.

    View details for DOI 10.1152/ajpgi.00011.2004

    View details for Web of Science ID 000223446400009

    View details for PubMedID 15107297

  • PCR-RFLP genotyping assay for a lactase persistence polymorphism upstream of the lactase-phlorizin hydrolase gene GENETIC TESTING Chao, C. K., Sibley, E. 2004; 8 (2): 190-193

    Abstract

    The majority of the world's human population experiences a decline of lactase gene expression during maturation, so-called lactase nonpersistence. Thus, adults with lactase nonpersistence are susceptible to developing symptoms of lactose intolerance. By contrast, lactase persistence is an autosomal dominant heritable condition that results in a high level of lactase gene expression throughout adulthood and sustained lactose tolerance. Lactase persistence has recently been correlated with a single nucleotide genetic variant (a C --> T mutation) located 13,910 bases upstream from the lactase structural gene. We aimed to develop a restriction fragment length polymorphism (RFLP) method of detecting the C/T variants as a means of identifying individuals genetically inclined toward lactase persistence or nonpersistence. Genomic DNA in a 210-bp region surrounding the -13,910-bp variant site was PCR amplified with unique primers designed to avoid or mutate adjacent restriction sites. The amplified DNA was digested with a restriction enzyme, CviJI, that recognizes the base pair sequence generated by the lactase nonpersistence variant. Restriction digest gel analysis yielded DNA fragments of the expected diagnostic molecular weight sizes for individuals that were homozygote or heterozygote for the lactase persistence and nonpersistence variants. The genotypes predicted by the RFLP-based method were confirmed by DNA sequence analysis. The RFLP-based method provides a quick and noninvasive means of molecular detection of the presence or absence of the lactase persistence variant.

    View details for Web of Science ID 000223513900020

    View details for PubMedID 15345119

  • Carbohydrate intolerance CURRENT OPINION IN GASTROENTEROLOGY Sibley, E. 2004; 20 (2): 162-167

    Abstract

    The small intestinal mucosa is highly specialized for terminal digestion of nutrient polysaccharides and disaccharides and absorption of monosaccharides. However, in the case of digestive or absorptive deficiency, symptoms of carbohydrate intolerance result. Significant progress has been made toward defining the molecular genetic mechanisms responsible for several carbohydrate intolerances.This review summarizes monosaccharide and disaccharide intolerance conditions and recent clinical and basic science reports related to carbohydrate digestion and membrane transport. Genetic polymorphisms closely associated with lactase persistence/nonpersistence have been identified. Lactose intolerance is capable of preventing the achievement of adequate peak bone mass in susceptible young adults and may predispose to osteoporosis. Recent studies support previous reports that fructose malabsorption is associated with unexplained gastrointestinal symptoms. GLUT2 may be recruited from the basolateral to the apical membrane of enterocytes to facilitate small intestinal fructose absorption.Knowledge regarding the clinical aspects of and the physiologic mechanisms responsible for specific carbohydrate intolerances has allowed for improved diagnostic and treatment options and has contributed to continuing investigation of intestinal gene expression.

    View details for Web of Science ID 000189319200018

    View details for PubMedID 15703639

  • Genetic variation and lactose intolerance: detection methods and clinical implications. American journal of pharmacogenomics Sibley, E. 2004; 4 (4): 239-245

    Abstract

    The maturational decline in lactase activity renders most of the world's adult human population intolerant of excessive consumption of milk and other dairy products. In conditions of primary or secondary lactase deficiency, the lactose sugars in milk pass through the gastrointestinal tract undigested or are partially digested by enzymes produced by intestinal bacterial flora to yield short chain fatty acids, hydrogen, carbon dioxide, and methane. The undigested lactose molecules and products of bacterial digestion can result in symptoms of lactose intolerance, diarrhea, gas bloat, flatulence, and abdominal pain. Diagnosis of lactose intolerance is often made on clinical grounds and response to an empiric trail of dietary lactose avoidance. Biochemical methods for assessing lactose malabsorption in the form of the lactose breath hydrogen test and direct lactase enzyme activity performed on small intestinal tissue biopsy samples may also be utilized. In some adults, however, high levels of lactase activity persist into adulthood. This hereditary persistence of lactase is common primarily in people of northern European descent and is attributed to inheritance of an autosomal-dominant mutation that prevents the maturational decline in lactase expression. Recent reports have identified genetic polymorphisms that are closely associated with lactase persistence and nonpersistence phenotypes. The identification of genetic variants associated with lactase persistence or nonpersistence allows for molecular detection of the genetic predisposition towards adult-onset hypolactasia by DNA sequencing or restriction fragment length polymorphism analysis. The role for such genetic detection in clinical practice seems limited to ruling out adult-onset hypolactasia as a cause of intolerance symptoms but remains to be fully defined. Attention should be paid to appropriate interpretation of genetic detection in order to avoid potentially harmful reduction in dairy intake or misdiagnosis of secondary lactase deficiency.

    View details for PubMedID 15287817

  • Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element HUMAN MOLECULAR GENETICS Olds, L. C., Sibley, E. 2003; 12 (18): 2333-2340

    Abstract

    Lactase persistence is a heritable, autosomal dominant, condition that results in a sustained ability to digest the milk sugar lactose throughout adulthood. The majority of the world's human population experiences a decline in production of the digestive enzyme lactase-phlorizin hydrolase during maturation. However, individuals with lactase persistence continue to express high levels of the lactase gene into adulthood. Lactase persistence has been strongly correlated with single nucleotide genetic variants, C/T_(13910) and G/A_(22018), located 13.9 and 22 kb upstream from the lactase structural gene. We aimed to characterize a functional role for the polymorphisms in regulating lactase gene transcription. DNA in the region of the C/T_(13910) or G/A_(22018) human lactase variants was cloned upstream of the 3.0 kb rat lactase gene promoter in a luciferase reporter construct. Human intestinal Caco-2 cells were transfected with the lactase variant/promoter-reporter constructs and assayed for promoter activity. A 200 bp region surrounding the C_(13910) variant, associated with lactase non-persistence, results in a 2.2-fold increase in lactase promoter activity. The T_(13910) variant, associated with lactase persistence, results in an even greater 2.8-fold increase. The DNA sequence of the C/T_(13910) variants differentially interacts with intestinal cell nuclear proteins on EMSAs. AP2 co-transfection results in a similar repression of the C/T_(13910) variant/promoter-reporter constructs. The DNA region of the C/T_(13910) lactase persistence/non-persistence variant functions in vitro as a cis element capable of enhancing differential transcriptional activation of the lactase promoter. Such differential regulation by the C and T variants is consistent with a causative role in the mechanism specifying the lactase persistence/non-persistence phenotypes in humans.

    View details for DOI 10.1093/hmg/ddg244

    View details for Web of Science ID 000185389200009

    View details for PubMedID 12915462

  • Regulation of intestine-specific spatiotemporal expression by the rat lactase promoter JOURNAL OF BIOLOGICAL CHEMISTRY Lee, S. Y., Wang, Z., Lin, C. K., Contag, C. H., Olds, L. C., Cooper, A. D., Sibley, E. 2002; 277 (15): 13099-13105

    Abstract

    Lactase gene transcription is spatially restricted to the proximal and middle small intestine of the developing mouse. To identify regions of the lactase gene involved in mediating the spatiotemporal expression pattern, transgenic mice harboring 0.8-, 1.3-, and 2.0-kb fragments of the 5'-flanking region cloned upstream of a firefly-luciferase reporter were generated. Transgene expression was assessed noninvasively in living mice using a sensitive low light imaging system. Two independent, 1.3- and 2.0-kb, lactase promoter-reporter transgenic lines expressed appropriate high levels of luciferase activity in the small intestine (300-3,000 relative light units/microg) with maximal expression in the middle segments. Post-weaned 30-day transgenic offspring also demonstrated an appropriate 4-fold maturational decline in luciferase expression in the small intestine. The pattern of the 2.0-kb promoter transgene mRNA abundance most closely mimicked that of the endogenous lactase gene with respect to spatiotemporal restriction. In contrast, a 0.8-kb promoter-reporter construct expressed low level luciferase activity (<25 relative light units/microg) in multiple organs and throughout the gastrointestinal tract in transgenic mice. Thus, a distinct 5'-region of the lactase promoter directs intestine-specific expression in the small intestine of transgenic mice, and regulatory sequences have been localized to a 1.2-kb region upstream of the lactase transcription start site. In addition, we have demonstrated that in vivo bioluminescence imaging can be utilized for assessment of intestinal expression patterns of a luciferase reporter gene driven by lactase promoter regions in transgenic mice.

    View details for DOI 10.1074/jbc.M112152200

    View details for Web of Science ID 000175036300078

    View details for PubMedID 11812796

  • Subarachnoid hemorrhage and isolated atresia of the aortic arch JOURNAL OF EMERGENCY MEDICINE Sibley, E., Doroshow, R. W., Milliken, J. C., Anand, S. K. 2002; 22 (2): 179-183

    Abstract

    The case of a 14 year old boy with subarachnoid hemorrhage and atresia of the aorta without patent ductus arteriosus or intracardiac shunt is described. This case calls attention to the possibility of aortic obstruction in adolescents or young adults with hypertensive stroke. The clinical symptoms, radiographic findings, and surgical repair of isolated aortic interruption, including atresia, are discussed.

    View details for Web of Science ID 000174061800011

    View details for PubMedID 11858924

  • Lactase gene transcription is activated in response to hypoxia in intestinal epithelial cells MOLECULAR GENETICS AND METABOLISM Lee, S. Y., Madan, A., Furuta, G. T., Colgan, S. P., Sibley, E. 2002; 75 (1): 65-69

    Abstract

    Lactase-phlorizin hydrolase, a brush-border membrane disaccharidase, is a marker of intestinal epithelial cell differentiation and digestive function. The intestine is susceptible to conditions of hypoxia resulting from vascular perfusion deficits. We hypothesized that lactase gene induction may provide a mechanism to efficiently increase nutrient energy substrates during gut hypoxia. These studies sought to characterize expression of the lactase gene in response to hypoxia and to characterize a role for hypoxia-inducible factor (HIF-1) in mediating the hypoxic response. Microarray analysis and confirmatory RT-PCR identified a 4-fold induction of lactase mRNA abundance in intestinal epithelial Caco-2 cells exposed to hypoxia. Lactase promoter activity was similarly induced by hypoxia in cells stably transfected with a 2.0-kb 5' flanking region of the rat lactase gene linked to a reporter gene. Transient cotransfection with HIF-1alpha and beta stimulated lactase promoter activity 2.4- and 3.5-fold under conditions of normoxia and hypoxia, respectively. We conclude that HIF-1 can activate the lactase promoter in intestinal epithelial cells exposed to hypoxia. Induction of lactase transcription may represent an adaptive response to gut hypoxia.

    View details for DOI 10.1006/mgme.2001.3263

    View details for Web of Science ID 000173752000007

    View details for PubMedID 11825065

  • GATA family transcription factors activate lactase gene promoter in intestinal Caco-2 cells AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Fang, R. X., Olds, L. C., Santiago, N. A., Sibley, E. 2001; 280 (1): G58-G67

    Abstract

    The GATA family of transcription factors regulate tissue-specific patterns of gene expression during development. We have characterized the interaction between GATA proteins and the lactase gene promoter. Nuclear protein bound to the lactase gene GATA region cis element (-97 to -73) was analyzed by electrophoretic mobility shift assays (EMSA) and supershift assays with GATA antibodies. Lactase promoter activities were assayed in Caco-2 cells transfected with wild-type and mutated luciferase promoter-reporter constructs and GATA-4/5/6 expression constructs. EMSA with the GATA region probe yields a specific DNA-protein complex that requires the GATA factor binding site WGATAR. The complex is recognized by GATA-4- and GATA-6-specific antibodies. GATA-4/5/6 expression constructs are able to activate transcription driven by the wild-type promoter, but not by a promoter in which the GATA binding site is mutated, in Caco-2 and nonintestinal QT6 cells. GATA factor binding to the lactase cis element correlates with functional promoter activation. We conclude that each of the GATA family zinc finger proteins expressed in the intestine, GATA-4, -5, and -6, can interact with the lactase promoter GATA element and can function to activate the promoter in Caco-2 cells.

    View details for Web of Science ID 000165948200008

    View details for PubMedID 11123198

  • The homeodomain protein Cdx2 regulates lactase gene promoter activity during enterocyte differentiation GASTROENTEROLOGY Fang, R. X., Santiago, N. A., Olds, L. C., Sibley, E. 2000; 118 (1): 115-127

    Abstract

    Lactase is the intestinal disaccharidase responsible for digestion of lactose, the predominant carbohydrate in milk. Transcription of the lactase gene is activated during enterocyte differentiation. We have characterized the interaction between the lactase promoter and Cdx2, a homeodomain protein involved in regulating intestinal development and differentiation.Nuclear protein bound to the lactase gene cis element, CE-LPH1, was analyzed by electrophoretic mobility shift assays and supershifts with Cdx2 antibody. Lactase promoter activities were assayed in cells transfected with luciferase reporter constructs and a Cdx2 expression construct.Electrophoretic mobility shift assay with CE-LPH1 yields a specific DNA/protein complex that requires the caudal-related protein binding site, TTTAC. The complex is recognized by Cdx2 antibody and is more abundant in differentiated enterocytes. A Cdx2 expression construct is able to activate transcription driven by the wild-type, but not a mutated, promoter and results in increased endogenous lactase messenger RNA.The homeodomain protein Cdx2 interacts with the lactase promoter and is capable of activating transcription of the endogenous gene. In contrast to a previous report, Cdx2 interaction with the lactase promoter correlates with enterocyte differentiation. These conclusions are consistent with the role of Cdx2 in regulating intestinal cell differentiation.

    View details for Web of Science ID 000084405100019

    View details for PubMedID 10611160

  • EVOLVING ASYMMETRIC HYPERTROPHIC PYLORIC-STENOSIS ASSOCIATED WITH HISTOLOGIC EVIDENCE OF EOSINOPHILIC GASTROENTERITIS PEDIATRIC RADIOLOGY Blankenberg, F. G., Parker, B. R., Sibley, E., Kerner, J. A. 1995; 25 (4): 310-311

    Abstract

    The most frequently occurring and important cause of gastric outlet obstruction in the neonate and young infant is infantile hypertrophic pyloric stenosis (IHPS). A reported association of IHPS and eosinophilic gastroenteritis [1] raises interesting questions about the possible etiologic relationship between the two entities. It is plausible that the observed sonographic pyloric muscular wall thickness in IHPS may in part be dependent on the degree and duration of an allergic gastroenteropathy. A recent report suggests that endoscopy may be a more reliable diagnostic method than sonography in the patient with evolving IHPS [2]. Our recent experience with a patient with evolving IHPS supports the findings described in these prior reports.

    View details for Web of Science ID A1995RF84300025

    View details for PubMedID 7567248

  • SUBSTRATE PHOSPHORYLATION CATALYZED BY THE INSULIN-RECEPTOR TYROSINE KINASE - KINETIC CORRELATION TO AUTOPHOSPHORYLATION OF SPECIFIC SITES IN THE BETA-SUBUNIT JOURNAL OF BIOLOGICAL CHEMISTRY FLORESRIVEROS, J. R., Sibley, E., Kastelic, T., Lane, M. D. 1989; 264 (36): 21557-21572

    Abstract

    The kinetics of insulin-stimulated autophosphorylation of specific tyrosines in the beta subunit of the mouse insulin receptor and activation of receptor kinase-catalyzed phosphorylation of a model substrate were compared. The deduced amino acid sequence of the mouse proreceptor was determined to locate tyrosine-containing tryptic peptides. Receptor was first incubated with unlabeled ATP to occupy nonrelevant autophosphorylation sites, after which [32P]autophosphorylation at relevant sites and attendant activation of substrate phosphorylation were initiated with [gamma-32P]ATP and insulin. Activation of substrate phosphorylation underwent an initial lag of 10-20 s during which there was substantial 32P-autophosphorylation of tryptic phosphopeptides p2 and p3, but not p1. Following the lag, incorporation of 32P into p1 and the activation of substrate phosphorylation increased abruptly and exhibited identical kinetics. The addition of substrate to the receptor prior to ATP inhibits insulin-stimulated autophosphorylation, and consequently substrate phosphorylation. Insulin-stimulated autophosphorylation of the receptor in the presence of substrate inhibited primarily the incorporation of 32P into p1 and drastically inhibited substrate phosphorylation. From Edman radiosequencing of 32P-labeled p1, p2, and p3 and the amino acid sequence of the mouse receptor, the location of each phosphopeptide within the beta subunit was determined. Further characterization of these phosphopeptides revealed that p1 and p2 represent the triply and doubly phosphorylated forms, respectively, of the region within the tyrosine kinase domain containing tyrosines 1148, 1152, and 1153. The doubly phosphorylated forms contain phosphotyrosines either at positions 1148 and 1152/1153 or positions 1152 and 1153. These results indicate that insulin stimulates sequential autophosphorylation of tyrosines 1148, 1152 and 1153, and that the transition from the doubly to the triply phosphorylated forms is primarily responsible for the activation of substrate phosphorylation.

    View details for Web of Science ID A1989CE48800025

    View details for PubMedID 2557333

  • CHARACTERIZATION OF THE MOUSE INSULIN-RECEPTOR GENE PROMOTER PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sibley, E., Kastelic, T., Kelly, T. J., Lane, M. D. 1989; 86 (24): 9732-9736

    Abstract

    The 5' flanking region of the mouse insulin proreceptor gene was isolated, and the 5' boundary of the minimal promoter was mapped. Genomic clones encompassing greater than 30 kilobases of the gene contain the promoter and exons 1 and 2 interrupted by an approximately 20-kilobase intron at the codon for amino acid 7 of the alpha subunit. The nucleotide sequence of a 1.3-kilobase fragment containing 766 base pairs of the 5' flanking region and the entire first exon was determined. Two major transcription start sites were mapped by S1 nuclease analysis to sites located 469 and 424 nucleotides upstream from the initiation codon for translation. The 5' terminus of an insulin proreceptor cDNA, isolated from a mouse 3T3-L1 adipocyte cDNA library, corresponds to the 3'-most major start site of transcription. The 5' deletion mutants of the 5' flanking region of the proreceptor gene, linked upstream of the bacterial chloramphenicol acetyltransferase reporter gene, were transfected into 3T3-L1 preadipocytes and assayed for promoter activity. The 5' boundary of the minimal promoter, which directs unexpectedly high levels of reporter gene expression, maps to a region 22 base pairs upstream from the 3'-most major transcription start site.

    View details for Web of Science ID A1989CE97600020

    View details for PubMedID 2602374

  • DIFFERENTIATION-INDUCED GENE-EXPRESSION IN 3T3-L1 PREADIPOCYTES - CHARACTERIZATION OF A DIFFERENTIALLY EXPRESSED GENE ENCODING STEAROYL-COA DESATURASE JOURNAL OF BIOLOGICAL CHEMISTRY Ntambi, J. M., BUHROW, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., Lane, M. D. 1988; 263 (33): 17291-17300

    Abstract

    Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the activation of transcription of an unidentified gene which encodes a 4.9-kilobase (kb) mRNA. Several cDNAs that include the complete sequence of this mRNA were obtained and used to isolate and characterize the gene. Analysis of the nucleotide and amino acid sequences of both cDNA and genomic clones revealed that the gene encodes the mouse stearoyl-CoA desaturase (SCD), an enzyme known to be expressed upon differentiation of 3T3-L1 preadipocytes. The predicted amino acid sequence (355 residues) of the mouse 3T3-L1 adipocyte SCD exhibits 92% identity to that of the rat liver SCD. There is also a high degree of nucleotide sequence identity between the mouse and rat mRNAs in their unusually long approximately 3.5-kb 3'-untranslated regions. Mice fed a diet containing unsaturated triacylglycerides express SCD mRNA only in adipose tissue, whereas mice starved and refed a fat-free diet, express SCD mRNA in both liver and adipose tissue. The mouse gene for the desaturase spans approximately 15 kb and contains 6 exons and 5 introns with all intron-exon junctions conforming to the GT/AG splicing rule. As determined by S1 nuclease mapping and primer extension analysis, the transcriptional initiation site maps 152 nucleotides upstream from the initiation methionine codon. A canonical promoter "TATA" box is located 30 base pairs upstream of the Cap site. A typical "CCAAT" box sequence is not present in the adjacent 5'-flanking region; however, there is a GC-rich sequence (at nucleotide -215) similar to the binding site for the nuclear transcription factor Sp1. Upstream from the transcriptional initiation site are elements with homology (approximately 75%) to the putative fat-specific transcriptional element FSE2 and core consensus sequences for cAMP and glucocorticoid regulatory elements. A chimeric construct, containing 363 base pairs of 5'-flanking sequence and 30 nucleotides of 5'-untranslated sequence of the mouse SCD gene ligated to the bacterial chloramphenicol acetyltransferase gene, was transfected into 3T3-L1 cells. When cells were induced to differentiate into adipocytes, expression of the SCD chloramphenicol acetyltransferase gene increased approximately 63-fold, suggesting that the SCD promoter region contains elements that mediate the response to adipogenic agents which induce differentiation.

    View details for Web of Science ID A1988R004800020

    View details for PubMedID 2903162

  • EXPRESSION OF THE DIFFERENTIATION-INDUCED GENE FOR FATTY ACID-BINDING PROTEIN IS ACTIVATED BY GLUCOCORTICOID AND CAMP PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cook, J. S., Lucas, J. J., Sibley, E., BOLANOWSKI, M. A., Christy, R. J., Kelly, T. J., Lane, M. D. 1988; 85 (9): 2949-2953

    Abstract

    We have isolated and characterized a fragment of the gene encoding adipose fatty acid-binding protein (gene 422) from a 3T3-L1 adipocyte genomic library. The 5'-flanking sequence of the 422 gene contains potential regulatory regions for adipose-specific expression. At position -120 there is a fat-specific element that occurs in several genes expressed as preadipocytes differentiate, and at position -393 there is a glucocorticoid regulatory element core sequence. Chimeric constructs were prepared by ligating 858 base pairs or 248 base pairs of 5'-flanking sequence and 22 nucleotides of 5'-untranslated sequence of the 422 gene to the bacterial gene encoding chloramphenicol acetyltransferase (CAT); these constructs (delta 858.CAT and delta 248.CAT) were transfected into 3T3-L1 preadipocytes. When differentiation was initiated by the adipogenic agents methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin, expression of both constructs increased, reaching maximal levels within 24 hr. Both constructs were maximally induced 48 hr before appreciable accumulation of the endogenous 422 mRNA. Expression of delta 858.CAT, but not of delta 248.CAT, was induced by dexamethasone, which correlates with deletion of the potential glucocorticoid regulatory element. Expression of both constructs was induced by 8-bromoadenosine 3',5'-cyclic monophosphate, thus implicating the first 248 base pairs of 5'-flanking sequence of the 422 gene in the response to cAMP. Indirect effects by the adipogenic factors on CAT protein or mRNA synthesis and turnover were ruled out, since replacing the 5'-flanking region of the 422 gene constructs with viral promoters abolished the effects of dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate on CAT expression. We conclude that the first 858 base pairs of 5'-flanking sequence of the 422 gene contains elements that mediate activation by dexamethasone and cAMP.

    View details for Web of Science ID A1988N304400015

    View details for PubMedID 2452440

  • HIGH-LEVEL EXPRESSION OF A CLONED HLA HEAVY-CHAIN GENE INTRODUCED INTO MOUSE CELLS ON A BOVINE PAPILLOMAVIRUS VECTOR MOLECULAR AND CELLULAR BIOLOGY DiMaio, D., Corbin, V., Sibley, E., Maniatis, T. 1984; 4 (2): 340-350

    Abstract

    A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

    View details for Web of Science ID A1984SB97600018

    View details for PubMedID 6321959

Conference Proceedings


  • Thyroid hormone and the D-type cyclins interact in regulating enterocyte gene transcription Meng, S. F., Badrinarain, J., Sibley, E., Fang, R. X., Hodin, R. SPRINGER. 2001: 49-55

    Abstract

    Thyroid hormone (T3) is an important regulator of gut mucosal development and differentiation, inducing intestinal alkaline phosphatase (IAP) and repressing lactase gene transcription. In contrast, cyclin D1 (CD1) appears to be a growth promoter in the gut, functioning to maintain the undifferentiated state. The present studies were designed to examine the effects of CD1 on T3 action within intestinal epithelia. Caco-2 cells were maintained in hypothyroid medium and transiently transfected with either rat lactase (3.0 kb) or human IAP (2.4 kb) luciferase (Luc) reporter plasmids. Cotransfections were carried out using two T3 receptor (TR) isoforms, TR"-1 and TR$-1, as well as plasmids expressing CD1, CD3, CA, or CB1. Cells were then treated +/- 10 nmol/L T3 for 24 hours and luciferase activity was determined. With T3 treatment, IAP-Luc activity was induced (TR"-1 = eightfold, TR$-1 = ninefold), but these effects were dramatically inhibited (> 50%) by CD1 and CD3. In contrast, CA and CB1 did not alter T3-mediated IAP gene activation. The ability of CD1 and CD3 to inhibit T3 action was also tested in the context of the lactase gene, which is negatively regulated by T3. As expected, lactase reporter gene activity was repressed by T3 treatment in the case of both receptor isoforms, TR"-1 = 30% and TR$-1 = 40%. In contrast to its effects on the IAP gene, CD1 did not inhibit T3-mediated changes in lactase reporter gene activity. The D-type cyclins (CD1 and CD3), but not CA or CB1, specifically inhibit T3-mediated activation of the IAP gene. In contrast, the D-type cyclins do not inhibit T3-mediated repression of the lactase gene. These studies have identified a novel molecular interaction that exists between the pathways of growth and differentiation within intestinal epithelia.

    View details for Web of Science ID 000167919800014

    View details for PubMedID 11309648

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