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Effect of protein kinase C delta (PKC-?) inhibition on the transcriptome of normal and systemic sclerosis human dermal fibroblasts in vitro.

ABSTRACT: Previous studies demonstrated that protein kinase C- ? (PKC-?) 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-? to regulate collagen production in fibroblasts, we examined the effects of PKC-? 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-? activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-? inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-? 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-? inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NF?B played an important role in the transcriptome changes induced by PKC-? 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-? inhibition. These results indicate that isoform-specific inhibition of PKC-? profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases.

SUBMITTER: Wermuth PJ

PROVIDER: S-EPMC3214051 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Effect of protein kinase C delta (PKC-δ) inhibition on the transcriptome of normal and systemic sclerosis human dermal fibroblasts in vitro.

Wermuth Peter J PJ Addya Sankar S Jimenez Sergio A SA

PloS one 20111111 11

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 produc ...[more]

PMID: 22096525

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Project description:Systemic sclerosis (SSc) is characterized by severe and often progressive fibrosis of the skin and multiple internal organs. The mechanisms responsible for these alterations remain obscure, although excessive reactive oxygen species (ROS)-mediated oxidative stress has been implicated. NOX-4 is 1 of 7 isoforms of NADPH oxidase responsible for the generation of ROS. The purpose of this study was to examine NOX-4 expression in skin and cultured dermal fibroblasts from SSc patients and to examine its regulation by transforming growth factor ?1 (TGF?1).NOX-4 was assessed in normal and SSc skin by immunohistologic analysis and in normal and SSc cultured dermal fibroblasts by quantitative polymerase chain reaction analysis, fluorescence microscopy, and Western blotting. ROS levels were assessed by fluorescence measurement of H2 O2 production. Specific kinase inhibitors were used to study the TGF?1 signaling involved in NOX-4 stimulation. NOX-4 inhibition/down-regulation was induced with a selective NOX-4 small-molecule inhibitor and NOX-4 small interfering RNA (siRNA).In contrast with normal skin fibroblasts, those from SSc skin showed intense NOX-4 staining. Cultured SSc fibroblasts displayed increased NOX-4 expression. TGF?1 caused potent NOX-4 protein and messenger RNA stimulation in normal and SSc fibroblasts, which was mediated by the protein kinase C? (PKC?) and Smad2/3 pathways. NOX-4 knockdown in SSc fibroblasts reduced the production of ROS and lowered the expression of type I collagen.NOX-4 expression and production were found to be constitutively elevated in SSc skin and cultured SSc dermal fibroblasts. TGF?1 stimulated NOX-4 expression in normal and SSc fibroblasts through PKC? and Smad2/3 signaling pathways. A small-molecule NOX-4 inhibitor decreased collagen and fibronectin production by normal and SSc fibroblasts, and NOX-4 siRNA knockdown reduced ROS and collagen production by SSc fibroblasts. These results demonstrate the involvement of NOX-4 in SSc-associated fibrosis and indicate NOX-4 inhibitors as novel therapeutic approaches for SSc.

Dataset Information

Effect of protein kinase C delta (PKC-?) inhibition on the transcriptome of normal and systemic sclerosis human dermal fibroblasts in vitro.

Publications

Effect of protein kinase C delta (PKC-δ) inhibition on the transcriptome of normal and systemic sclerosis human dermal fibroblasts in vitro.

Similar Datasets

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