Abstract
The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis (Pichler, R. H., Bassuk, J. A., Hugo, C., Reed, M. J., Eng, E., Gordon, K. L., Pippin, J., Alpers, C. E., Couser, W. G., Sage, E. H., and Johnson, R. J. (1997) Am. J. Pathol. 148, 1153-1167). A potential mechanism by which SPARC controls both cell cycle and matrix production has been attributed to its regulation of a pleiotropic growth factor. In this study we used primary mesangial cell cultures from wild-type mice and from mice with a targeted disruption of the SPARC gene. SPARC-null cells displayed diminished expression of collagen type I mRNA and protein, relative to wild-type cells, by the criteria of immunocytochemistry, immunoblotting, and the reverse transcription-polymerase chain reaction. The SPARC-null cells also showed significantly decreased steady-state levels of transforming growth factor-beta1 (TGF-beta1) mRNA and secreted TGF-beta1 protein. Addition of recombinant SPARC to SPARC-null cells restored the expression of collagen type I mRNA to 70% and TGF-beta1 mRNA to 100% of wild-type levels. We conclude that SPARC regulates the expression of collagen type I and TGF-beta1 in kidney mesangial cells. Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus.
Highlights
The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis
Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus
Since SPARC [14], collagen type I [20], and transforming growth factor-1 (TGF-1) [22] were among the proteins shown to be augmented during mesangioproliferative glomerulonephritis, we examined the effect of SPARC on the expression of collagen type I and TGF-1 in mesangial cells cultured from wild-type and SPARC-null mice
Summary
Preparation and Characterization of Murine Glomerular Mesangial Cells—129/SvJ ϫ C57BL/6J wild-type and SPARC-null mice [31] were maintained in a specific pathogen-free facility. For the TGF-1 assays, wild-type and SPARC-null cells were grown to 80% confluence in growth medium, as described above. To measure the amounts of TGF-1 protein in the conditioned culture media of wild-type and SPARC-null cells, we used an enzymelinked immunosorbent assay (ELISA) kit (R & D Systems Inc., Minneapolis, MN) according to the manufacturer’s instructions. Preparation of RNA and Cellular Protein—For the mRNA preparation, wild-type and SPARC-null cells were grown to 80% confluence in growth medium. For preparation of cellular protein, wild-type and SPARC-null cells were grown to 80% confluence in growth medium. Western Blot Analysis and Metabolic Labeling—Primary mesangial cell cultures from wild-type and SPARC-null mice were grown to 80%. Immunocytochemistry—Primary mesangial cell cultures from wildtype and SPARC-null mice were plated on glass coverslips, incubated with sodium ascorbate (50 g/ml) for 24 h, and fixed in 2% paraformaldehyde for 30 min. Files were processed and analyzed by NIH Image softwareTM and are presented as composite figures
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