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N.R. Perdue, E.H. Sage, Q. Yan; Absence of SPARC in Lens Epithelium Leads to Increased Deposition of Laminin–1 in Murine Lens Basement Membrane: . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1887.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To investigate the role of SPARC in the regulation of expression of the extracellular matrix protein laminin–1 in the lens capsule and lens epithelial cells. Methods:Wild–type (wt) and SPARC–null C57Bl6/129SVJ mice E14 to 3 months of age were studied. Transcript levels of laminin–1 were analyzed by RT–PCR from lens epithelial cells. Distribution of laminin–1 protein in the lens capsule and epithelium was analyzed by immunohistochemistry. For quantification of protein levels in the lens capsule, proteins extracted from the capsules with or without attached epithelial cells were analyzed by Western blot. Lens explant cultures were performed in 35–mm culture dishes coated with fibronectin. Cultured cells and secreted proteins were analyzed for laminin–1 expression under serum–free conditions. Results:Expression of laminin–1 in wt and SPARC–null lenses was revealed by immunostaining with anti–laminin–1 IgG. Laminin–1 was expressed at apparently high levels in the lens capsules of both wt and SPARC–null mice at ages E11 to E18, whereas the anterior and equatorial epithelial cells showed much less labeling of laminin–1 relative to the lens capsules. By immunohistochemistry there was no detectable difference for laminin–1 expression between wt and SPARC–null lenses up to E 18. From 5 days to 3 months of age, SPARC–null lens capsules exhibited higher levels of laminin–1 relative to their wt counterparts, as revealed by immunohistochemistry and immunoblotting. In addition, defects revealed by immunostaining with laminin antibody were apparent in the anterior region of SPARC–null lenses. Furthermore, lens capsules from 1–2 month–old SPARC–null mice attached to culture dishes much earlier than those from wt, and outgrowth of lens epithelial cells was more apparent in SPARC–null versus wt lens capsules. Under serum–free conditions, SPARC–null lens epithelial cells secreted more laminin–1 into their media in comparison to that secreted by wt counterparts. Transcripts of three chains of laminin–1 (α1, ß1 & γ1) showed similar levels between wt and SPARC–null lens epithelial cells. Conclusions:SPARC has the potential to regulate the secretion and deposition of laminin–1 protein in lens epithelial cells. SPARC is important in the regulation of the organization of lens basement membrane matrix. Increased deposition of laminin–1 in the lens basement membrane may influence lens epithelial cell adhesion, growth, and differentiation.
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