Qi Yan1, Nikole Perdue, David Blake, E Helene Sage. 1. Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101-2795, USA. qyan@benaroyaresearch.org
Abstract
PURPOSE: To investigate the role of SPARC in the regulation of the expression and deposition of extracellular matrix (ECM) proteins in the lens capsule. METHODS: Wild-type (SP+/+) and SPARC-null (SP-/-) mice of embryonic day (E)14 to 3 months of age were examined. Transcript levels of lens basement membrane (BM) components were analyzed by semiquantitative RT-PCR with mRNA from lens epithelia. Expression of ECM proteins in lens capsule and lens epithelium was analyzed by immunohistochemistry and Western blot analysis. Cell attachment was assessed by lens epithelial explant culture. Coimmunoprecipitation was performed to identify intracellular protein interactions. RESULTS: From postnatal day 5 to 3 months of age, SPARC-null lens capsules exhibited higher levels of laminin-1 deposition relative to their wild-type counterparts, as revealed by immunohistochemistry and immunoblot analysis. An uneven and aggregated distribution of laminin-1 protein was apparent in the anterior region of SPARC-null lens capsules. SPARC and laminin-1 were expressed abundantly in the endoplasmic reticulum (ER) of lens epithelial cells. Coimmunoprecipitation identified that SPARC associates with laminin-1 before laminin secretion. Furthermore, increased laminin-1 in lens capsule promoted the attachment of lens epithelial explants in culture. CONCLUSIONS: SPARC affects the secretion and deposition of laminin-1 protein in lens epithelial cells. Because abnormal deposition of laminin-1 in the lens BM could influence lens epithelial cell adhesion and fiber cell differentiation, the authors propose that SPARC is important to lens homeostasis through its regulation of lens BM matrix organization.
PURPOSE: To investigate the role of SPARC in the regulation of the expression and deposition of extracellular matrix (ECM) proteins in the lens capsule. METHODS: Wild-type (SP+/+) and SPARC-null (SP-/-) mice of embryonic day (E)14 to 3 months of age were examined. Transcript levels of lens basement membrane (BM) components were analyzed by semiquantitative RT-PCR with mRNA from lens epithelia. Expression of ECM proteins in lens capsule and lens epithelium was analyzed by immunohistochemistry and Western blot analysis. Cell attachment was assessed by lens epithelial explant culture. Coimmunoprecipitation was performed to identify intracellular protein interactions. RESULTS: From postnatal day 5 to 3 months of age, SPARC-null lens capsules exhibited higher levels of laminin-1 deposition relative to their wild-type counterparts, as revealed by immunohistochemistry and immunoblot analysis. An uneven and aggregated distribution of laminin-1 protein was apparent in the anterior region of SPARC-null lens capsules. SPARC and laminin-1 were expressed abundantly in the endoplasmic reticulum (ER) of lens epithelial cells. Coimmunoprecipitation identified that SPARC associates with laminin-1 before laminin secretion. Furthermore, increased laminin-1 in lens capsule promoted the attachment of lens epithelial explants in culture. CONCLUSIONS:SPARC affects the secretion and deposition of laminin-1 protein in lens epithelial cells. Because abnormal deposition of laminin-1 in the lens BM could influence lens epithelial cell adhesion and fiber cell differentiation, the authors propose that SPARC is important to lens homeostasis through its regulation of lens BM matrix organization.
Authors: Matt Weaver; Gail Workman; Chad R Schultz; Nancy Lemke; Sandra A Rempel; E Helene Sage Journal: J Cell Biochem Date: 2011-11 Impact factor: 4.429