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N.R. Perdue, L. Cheng, Q. Yan; Expression of Caveolin–1 in Murine Ocular Lens and Primary Lens Epithelial Cells: Role in SPARC Endocytosis? . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4101.
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© ARVO (1962-2015); The Authors (2016-present)
SPARC, an important matricellular protein in mammalian lens, was internalized by lens epithelium and cultured lens epithelial cells (LEC) in vitro. Caveolae–mediated endocytosis is an important endocytic pathway for the entry of macromolecules into cells. Caveolae have recently been implicated in organizing the cell’s signal transduction pathways and can play an important role in cell function. Caveolin–1 is the major coat protein of caveolae. Our goal is to characterize the expression of caveolin–1 in epithelial and fiber cells of the murine ocular lens and the cultured murine LEC, and to determine whether caveolae are involved in SPARC endocytosis.
Eyes from C57BL6/129SVJ wt and SPARC–null mice (1–4 month–old) were prepared for paraffin sections and frozen sections. LEC were cultured from wt and SPARC–null 1–2 month–old mice. The distribution of caveolin–1 in murine lens and cultured LEC was analyzed by immunohistochemistry and immunoblotting with antibodies against caveolin–1. Transcript level of caveolin–1 was determined by RT–PCR. SPARC endocytosis was examined in the presence and absence of the caveolae–inhibiting drug filipin.
Of all 1–4 month–old wt and SPARC–null lenses examined, caveolin–1 expression was undetectable in lens epithelium and lens fibers by immunohistochemistry. Western blotting with antibody against caveolin–1 confirmed this observation. Furthermore, RT–PCR analysis showed that lens epithelium or lens fiber cells expressed very low levels of caveolin–1 mRNA. In contrast, primary LEC and LEC at later passages expressed abundant caveolin–1 at both the mRNA and protein levels. Exogenous SPARC protein was efficiently internalized into wt or null LEC after 15 min incubation at 37C. Pretreatment of LEC with cholesterol depletion agent filipin did not affect uptake of SPARC by LEC.
Caveolin–1 was not detected in murine lens epithelium and lens fiber cells. Caveolin–1 expression was abundant in cultured LEC. SPARC endocytosis by LEC appears to be mediated via a caveolae–independent endocytic pathway.
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