Abstract
Abstract: :
Introduction:Maspin, a tumor–suppressor protein that regulates cell migration, invasion, and adhesion, is expressed in human corneal epithelial, endothelial and stromal keratocytes. When keratocytes are converted to the fibroblast phenotype, maspin synthesis is downregulated. Incubation of these cells with maspin increases their ability to adhere to type I and type IV collagens, fibronectin and laminin. This study was initiated to identify extracellular matrix and adhesion genes differentially expressed by corneal stromal fibroblast cells cultured in the presence of exogenous maspin. Methods: Human corneal stromal fibroblast cells were serum starved for 18 hr and then incubated in the presence or absence of exogenous maspin for 1, 4, 8 or 24 hrs. RNA was extracted, reverse transcribed into cDNA, biotin labeled, and differential gene expression determined using Superarray’s Extracellular Matrix arrays. The data was analyzed using Scanalyze and GEAnalyzer. A change in gene expression of 2–fold or greater in the triplicate samples was considered significant. Proteins in cell–conditioned mediums were separated by SDS–PAGE and SPARC and PAI–1 levels determined using Western Blots. Results:Maspin treatment of the corneal fibroblasts did not change the phenotype of the cells. mRNA levels of CD44 antigen (CD44), cathepsin L (CTSL), integrin ß1 (ITGB1), laminin ß1 (LAMB1), tissue plasminogen activator (tPA), plasminogen activator inhibitor, type I (PAI–1), and secreted protein, acidic, cysteine–rich (SPARC) were elevated greater than 2 fold at 1hr while at 4hr, mRNA levels of carcinoembryonic antigen–related cell adhesion molecule 5 (CEA), collagen, type I, α–1 (COL1A1), extracellular matrix protein 1 (ECM1), LAMB1, tPA, matrix metalloproteinase 2 (MMP–2), and SPARC were decreased greater than two fold. Western blots revealed that protein levels of SPARC secreted into the media by all cells increased over time and the addition of maspin elevated SPARC levels by 5 fold at 8 hrs and 3 fold at 24 hrs relative to controls. PAI–1 levels also increased over time and the addition of maspin elevated PAI–1 levels by 3 fold at 8 hrs and 5 fold at 24 hrs.Conclusions:These results suggest that at the mRNA level, genes that induce wound healing are initially upregulated by maspin while at later times maspin decreases expression of these genes. Maspin may stimulate the synthesis of SPARC and PAI–1, anti–adhesive proteins, as a control mechanism to balance the increase in adhesion of corneal stromal cells to extracellular matrix molecules induced by maspin. PAI–1 may be directly regulated or indirectly through SPARC.
Keywords: cornea: stroma and keratocytes • cornea: basic science • extracellular matrix