Role of protein kinase C-δ in the regulation of collagen gene expression in scleroderma fibroblasts
Sergio A. Jimenez, Svetlana Gaidarova, Biagio Saitta, Nora Sandorfi, David J. Herrich, Joan C. Rosenbloom, Umberto Kucich, William R. Abrams, Joel Rosenbloom
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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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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In vitro nuclear transcription of control or rottlerin-treated SSc cells...
In vitro nuclear transcription of control or rottlerin-treated SSc cells. Confluent cultures of SSc fibroblasts (S1, S2) were incubated in T-175 flasks in MEM supplemented with 10% (vol/vol) FBS and ascorbic acid (50 μg/ml) with or without rottlerin (3 μM) for 24 hours. Nuclei were prepared and in vitro transcription assays were performed as described in Methods. Aliquots of 32P-labeled RNA containing equal cpm were hybridized to duplicate dot-blotted cDNAs for α1(I) and α1(III) procollagens, GAPDH, or the plasmid pUC19. After hybridization these were analyzed using storage phosphor technology. The values obtained were corrected for the transcription of GAPDH after subtraction of the background represented by pUC19. The values shown represent the corrected pixel densities from the average of two separate experiments.