Alterations in the expression level of genes may contribute to the development and pathophysiology of obesity. To find genes differentially expressed in adipose tissue during obesity, we performed suppression subtractive hybridization on epididymal fat mRNA from goldthioglucose (GTG) obese mice and from their lean littermates. We identified the secreted protein acidic and rich in cysteine (SPARC), a protein that mediates cell-matrix interactions and plays a role in modulation of cell adhesion, differentiation, and angiogenesis. SPARC mRNA expression in adipose tissue was markedly increased (between 3-and 6-fold) in three different models of obesity, i.e. GTG mice, ob/ob mice, and AKR mice, after 6 weeks of a high fat diet. Immunoblotting of adipocyte extracts revealed a similar increase in protein level. Using a SPARC-specific ELISA, we demonstrated that SPARC is secreted by isolated adipocytes. We found that insulin administration to mice increased SPARC mRNA in the adipose tissue. Food deprivation had no effect on SPARC expression, but after high fat refeeding SPARC mRNA levels were significantly increased. Our results reveal both hormonal and nutritional regulation of SPARC expression in the adipocyte, and importantly, its alteration in obesity. Finally, we show that purified SPARC increased mRNA levels of plasminogen activator inhibitor 1 (PAI-1) in cultured rat adipose tissue suggesting that elevated adipocyte expression of SPARC might contribute to the abnormal expression of PAI-1 observed in obesity. We propose that SPARC is a newly identified autocrine/paracrine factor that could affect key functions in adipose tissue physiology and pathology.
Several lines of evidence suggest a role for laminin-5 in skin wound healing. We report here that transforming growth factor- (TGF-), which elicits various responses during cutaneous healing, stimulates transcription of the mouse laminin ␣3A (lama3A) gene. To identify the TGF--responsive elements (TGF-REs) on the lama3A promoter, we have generated a series of 5-deletions of the promoter upstream of the -galactosidase reporter gene. Transient cell transfection assays using mouse PAM212 keratinocytes revealed that TGF-REs lie between nucleotides ؊297 and ؊54 relative to the transcription start site. Insertion of the TGF-RE in front of the unresponsive minimal SV40 promoter conferred TGF- inducibility. Computer analysis of the promoter sequence identified three canonical activator protein-1 (AP-1) sites located at nucleotides ؊277 (AP-1A), ؊125 (AP-1B), and ؊69 (AP-1C). Site-directed mutagenesis of either the AP-1A or AP-1C site did not drastically alter the basal activity of the lama3A promoter, but reduced TGF- responsiveness by 50%. Simultaneous mutation of these two AP-1 sites resulted in a 65% decline in the response to TGF-, suggesting a cooperative contribution of each site to the overall promoter activity. In contrast, mutation of the AP-1B site markedly reduced the basal activity of the lama3A promoter, indicating that this AP-1 site is essential for gene expression. Mobility shift assays demonstrated specific binding of Fra-2 and JunD to the AP-1 sites, suggesting for the first time a possible regulatory function for the Fra-2⅐JunD AP-1 complex in a basal keratinocyte-specific gene.Laminin-5 is the major adhesion ligand present in the basement membranes of stratified squamous epithelia (1, 2). In the skin, this adhesive protein is secreted by the basal keratinocytes and colocalize with the anchoring filaments of the lamina lucida of the dermal epidermal junction (3-5). Laminin-5 binds to integrin ␣ 3  1 in focal adhesions and interacts with hemidesmosomes via ␣ 6  4 to form a stable anchorage complex (6, 7). Laminin-5 is a heterotrimeric glycoprotein composed of the ␣3A, 3, and ␥2 polypeptide chains that are products of different genes. Mutations in the genes encoding laminin ␣3 (LA-MA3), 3 (LAMB3), and ␥2 (LAMC2) have been shown to underlie the Herlitz or non-Herlitz forms of junctional epidermolysis bullosa, characterized by blister formation and erosions of the skin and mucosas that frequently lead to neonatal death (8 -13).Several lines of evidence suggest a role for laminin-5 in the re-epithelialization of wound skin repair. Laminin-5 is found at the epidermal-dermal junction at sites and times that coincide with actively migrating or rapidly proliferating basal keratinocytes (14, 15). Moreover, enhancement of laminin-5 transcription is observed at low cell densities in vitro in migrating and proliferating keratinocytes, similar to what happens at the wound edge (15). Although the function of laminin-5 in wound healing remains to be clarified, its major dual contribution would be to all...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.