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.
View: Text | PDF
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

×

Figure 1

Options: View larger image (or click on image) Download as PowerPoint
Effect of inhibition of PKC-δ by rottlerin on type I collagen production...
Effect of inhibition of PKC-δ by rottlerin on type I collagen production and biosynthesis. (a) Confluent cultures of normal (open squares) and SSc (filled squares) dermal fibroblasts were incubated for 24 hours with the indicated concentrations of rottlerin, as described in Methods. The culture media were analyzed for type collagen I using specific ELISA. Values represent the average of duplicate cultures determined in duplicate. (b) Confluent cultures of normal (diamonds) and SSc (squares) dermal fibroblasts were incubated under control conditions (open symbols) or with 3 μM rottlerin (filled symbols) and labeled with [14C]proline, as described in Methods. Two plates from each set were harvested 8 hours and 16 hours after labeling, and the amount of radiolabeled collagen representing newly synthesized protein present in the media was determined by a collagenase digestion assay.