Doctor of Philosophy, Universita Degli Studi Di Roma (2011)
Nidhi Bhutani, Postdoctoral Faculty Sponsor
To investigate the role of the newly discovered epigenetic mark 5-hydroxymethylcytosine (5hmC) and its regulators in altered gene expression in osteoarthritis (OA).Cartilage was obtained from OA patients undergoing total knee arthroplasty and from control patients undergoing anterior cruciate ligament reconstruction. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene expression changes were monitored by quantitative polymerase chain reaction (PCR) analysis. Levels of locus-specific 5hmC and 5mC at CpG sites in the matrix metalloproteinase 1 (MMP-1), MMP-3, ADAMTS-5, and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT-1) promoters were quantified using a glucosylation and enzyme digestion-based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were monitored in normal chondrocytes stimulated with inflammatory cytokines to identify the effect of joint inflammation.A global 5-6-fold increase in 5hmC concomitant with a loss of TET1 was observed in human OA chondrocytes compared to normal chondrocytes. Enrichment of 5hmC was observed in promoters of enzymes critical to OA pathology, MMP-1 and MMP-3. Short-term treatment of normal chondrocytes with inflammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrichment.This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accumulation. Our experiments identify 5hmC and its regulators as potential diagnostic and therapeutic targets in OA.
View details for DOI 10.1002/art.38200
View details for PubMedID 24449578
A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization.
View details for DOI 10.1073/pnas.1103873108
View details for Web of Science ID 000292635200039
View details for PubMedID 21709251
Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and ?1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.
View details for DOI 10.1091/mbc.E10-10-0821
View details for Web of Science ID 000289558600025
View details for PubMedID 21346196