Abstract
Abstract: :
Purpose: Matricellular glycoprotein SPARC (secreted protein acidic and rich in cysteine) displays complex biological activities, and is an important protein in the regulation of lens epithelial cell function and cataractogenesis. To understand the mechanism(s) of SPARC–mediated activities on lens epithelial cells, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. Methods:Murine lens epithelial cells were cultured from C57Bl6/129SVJ wild–type and SPARC–null mice at 1–2 month of age. Primary cultures and spontaneously immortalized cultures of lens epithelial cells were established. Subcellular localization of SPARC was identified by immunofluorescent staining and by immunoblotting of cytoplasmic and nuclear subcellular fractions. Recombinant human (rh) SPARC was added to murine lens epithelial cells to track the distribution of rhSPARC in the murine cells. Results:Endogenous intracellular SPARC is located in the cytoplasm but not in the nucleus of primary cultured lens epithelial cells. An immortalized lens epithelial cell line was subcultured for 5 months, during which a rapid down–regulation of endogenous SPARC was observed. Over this period of time, cytoplasmic SPARC was translocated to the nuclei; eventually SPARC protein was not detectable either within nuclei or the cytoplasm by immunostaining and western blotting. Exogenous rhSPARC was internalized into the cytosol of the cultured murine lens epithelial cells. In addition, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. Conclusions:Cytoplasmic SPARC is translocated into the nuclei of cultured lens epithelial cells when cells no longer make endogenous SPARC or exposure to exogenous SPARC is terminated. Nuclear SPARC might act as a transcriptional regulator modulating the expression of other genes in response to the loss of SPARC. Alternatively, SPARC nuclear translocation might be used as a rescue strategy for the cells to preserve SPARC from proteolytic degradation in the cytosol.
Keywords: extracellular matrix • cell–cell communication • transcription factors