Abstract
Abstract: :
Purpose: Absence of SPARC in lens epithelium resulted in damaged lens capsules in SPARC–null mice at 1–2 months of age. The thickness of the damaged anterior lens capsule increased significantly by 4–5 months of age. This study was to investigate the mechanism(s) underlying this phenotype. Methods: Wild–type (wt) and SPARC–null C57Bl6/129SVJ mice 1 to 13 months of age were studied. Transcript levels of extracellular matrix (ECM) molecules and TGF–beta 1 and 2, and TGF–beta RI and II were analyzed by semi–quantitative RT–PCR using mRNA from lens epithelia and lens fibers. Expression of ECM proteins and EMT markers in the lens epithelium was analyzed by immunohistochemistry and immunoblotting. A spontaneous immortalized lens epithelial cell line (ML.SP–10/02) was characterized with overexpression of SPARC protein. Expression of EMT markers was analyzed in this cell line. Results: Lens epithelium of 5–month–old SPARC–null mice presented a multilayered, irregular morphology that was not observed in younger mice. RT–PCR data showed that the mRNAs of alpha–smooth muscle actin, collagen type I, connective tissue growth factor, and fibronectin (EMT markers) were expressed in SPARC–null lens epithelial cells as early as 2–3 months of age in SPARC–null mice. Levels of alpha–smooth muscle actin and fibronectin proteins increased with age, as identified by immunoblotting. Wild–type counterparts did not show any EMT markers up to 7 months of age in SPARC–null mice. To evaluate whether enhanced TGF–beta response was involved in lens EMT in SPARC–null mice, we examined expression of TGF–beta and its cognate receptors. Expression of mRNA for TGF–beta 1 and 2, and TGF–beta type I and II receptors, was slightly greater in SPARC–null lens epithelium. Levels of activated TGF–beta protein in wt and SAPRC–null lenses are under investigation. Furthermore, cells from the ML.SP–10/02 cell line overexpress SPARC protein. These cells produced a significantly diminished level of alpha–smooth muscle actin, suggesting that SPARC might inhibit lens EMT. Conclusions: Lens epithelium lacking SPARC transdifferentiated to mesenchyme (EMT) in 3 month–old (or older) SPARC–null mice. SPARC–null lens epithelium produced ECM proteins not normally expressed by lens cells, might lead to a thicker anterior lens capsule. SPARC appears to be a key protein for the maintenance of lens epithelial characteristics and normal expression/organization of ECM proteins in the lens basement membrane. Supported by EY14150.
Keywords: extracellular matrix • growth factors/growth factor receptors • cytoskeleton