Role of protein kinase C-δ in the regulation of collagen gene expression in scleroderma fibroblasts
Sergio A. Jimenez, … , William R. Abrams, Joel Rosenbloom
Sergio A. Jimenez, … , William R. Abrams, Joel Rosenbloom
Published November 1, 2001
Citation Information: J Clin Invest. 2001;108(9):1395-1403. https://doi.org/10.1172/JCI12347.
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Article

Role of protein kinase C-δ in the regulation of collagen gene expression in scleroderma fibroblasts

Abstract

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-δ (PKC-δ) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-δ, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70–90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides –804 to –675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-δ expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-δ participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-δ inhibitors could suppress fibrosis in this disease.

Authors

Sergio A. Jimenez, Svetlana Gaidarova, Biagio Saitta, Nora Sandorfi, David J. Herrich, Joan C. Rosenbloom, Umberto Kucich, William R. Abrams, Joel Rosenbloom

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Figure 8

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Cotransfection of COL1A1 promoter constructs and PKC-δ expression constr...
Cotransfection of COL1A1 promoter constructs and PKC-δ expression constructs into normal and SSc fibroblasts. Normal and SSc fibroblasts were cultured to 70% confluence and then cotransfected with 0.4 μg of either bp –675 or bp –804 COL1A1 deletion constructs and 0.4 μg of either a wild-type or a dominant-negative PKC-δ expression construct, as described in Methods. Following transfections, cell extracts were assayed for CAT activity. The upper panels show the autoradiograms of the CAT assays. The lower panels show the quantitation of CAT activity following normalization for the activity of the cotransfected β-galactosidase gene. Note that expression of the dominant-negative PKC-δ construct decreases the transcriptional activity of the bp –804 COL1A1 construct, but has no effect on that driven by the bp –675 COL1A1 construct. WT, wild-type; DN, dominant negative.