rottlerin and Scleroderma--Systemic

Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases.

Topics: Acetophenones; Benzopyrans; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Humans; Protein Kinase C-delta; Scleroderma, Systemic; Skin

To examine the mechanism of collagen induction by connective tissue growth factor (CTGF), a profibrotic cytokine overexpressed in the skin of patients with systemic sclerosis (SSc).. Dermal fibroblasts from 7 SSc patients and 7 matched healthy adult donors were stimulated with CTGF in the presence or absence of the culture-medium supplement, insulin-transferrin-selenium (ITS). Expression of collagen protein was analyzed by a (3)H-proline incorporation assay. To identify the signaling pathways mediating CTGF induction of collagen, pharmacologic inhibitors were used, including rottlerin, a protein kinase C delta (PKC delta) inhibitor.. Collagen levels in both SSc and normal fibroblasts were increased after treatment with transforming growth factor beta in serum-free medium, whereas no stimulation was observed following addition of CTGF. In the presence of ITS, CTGF (2.5 ng/ml) potently stimulated collagenous protein levels in SSc cell lines (n = 5); however, CTGF was not stimulatory in the majority of normal fibroblasts (n = 6). ITS alone induced collagen levels in normal fibroblasts to the levels observed in SSc skin fibroblasts, thereby diminishing the hallmark difference in basal collagen levels in these cell types. Insulin was the ITS component responsible for promoting the basal and CTGF stimulation of collagenous proteins. Rottlerin, the PKC delta inhibitor, down-regulated collagen synthesis in normal and SSc fibroblasts cultured in ITS, and inhibited the stimulatory effects of CTGF in cooperation with insulin or of insulin (500 ng/ml) alone.. Increased responsiveness of SSc fibroblasts to CTGF-mediated collagen synthesis requires the costimulatory activation of insulin signaling pathways to induce matrix production. Blockade of this effect via rottlerin may suggest that PKC delta is a downstream signaling molecule necessary for CTGF stimulation of collagen synthesis.

Topics: Acetophenones; Benzopyrans; Cells, Cultured; Collagen; Connective Tissue Growth Factor; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Fibroblasts; Humans; Immediate-Early Proteins; Infant, Newborn; Insulin; Intercellular Signaling Peptides and Proteins; Proline; Protein Kinase C; Protein Kinase C-delta; Scleroderma, Systemic; Selenium; Signal Transduction; Skin; Transferrin

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, 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-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease.

Topics: Acetophenones; Benzopyrans; Blotting, Western; Cell Nucleus; Cells, Cultured; Cloning, Molecular; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type III; Dose-Response Relationship, Drug; Fibroblasts; Gene Expression Regulation; Genes, Dominant; Humans; Isoenzymes; Microscopy, Fluorescence; Plasmids; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C-delta; RNA, Messenger; Scleroderma, Systemic; Time Factors; Transcription, Genetic; Transfection