Lens Epithelial Cells from SPARC-null Mice Exhibit Enhanced Invasion of the Lens Basement Membrane

Abstract

Abstract: : Purpose: We previously demonstrated that targeted deletion of SPARC (secreted protein, acidic and rich in cysteine) in mice resulted in a compromised lens basement membrane, and was associated with cataract formation. The present study was undertaken to investigate whether SPARC affects lens epithelial cell behavior in vitro and might thereby contribute to the structural integrity of the lens capsule. Methods: SPARC and basal lamina components were analyzed by RT-PCR, immunohistochemistry, and immunoblotting to study their distribution and expression in lenses of wt and SPARC-null mice. Lens epithelial cells were cultured from wt and SPARC-null lenses. The response of lens epithelial cells to growth factors (FGF-2, EGF, and IGF-1) and SPARC was measured by 3H-thymidine incorporation. Cell invasion was assessed through an 8-um pore polycarbonate membrane, precoated with a reconstituted basement membrane. Invading cells were counted on the lower surface of the membrane. Results: SPARC was not found to be a structural component of the lens capsule, but was abundant in lens epithelial cells and newly-differentiated fiber cells. Levels of laminin 1 and collagen IV mRNA were similar between wt and SPARC-null lenses. Genetic ablation of SPARC was associated with cellular protrusions into the basement membrane of lens cells situated beneath the capsule. In vitro, there was a 4-fold difference in the number of SPARC-null lens epithelial cells that invaded through the reconstituted basement membrane, in comparison to wt lens epithelial cells, over a period of 20 hr. Although SPARC is anti-proliferative on many types of cells, recombinant human SPARC did not inhibit growth factor-stimulated DNA synthesis in lens epithelial cells cultured from wt and SPARC-null mice to a significant degree. Conclusions: SPARC does not appear to be a stable component of the lens basement membrane, nor does it seem to regulate the levels of laminin 1 and collagen IV mRNA in the lens. SPARC appears to mediate lens cell invasion but not proliferation.

Keywords: extracellular matrix • cell adhesions/cell junctions • proliferation