ABSTRACT
BACKGROUND: Caveolar raft domains, also termed caveolae, are flask shaped invaginations that require the expression of the structural protein caveolin-1 (cav-1). Matrix metalloproteinase 1 (MMP-1) is a collagenase capable of degrading insoluble triple helical collagens. Deregulation of MMP-1 contributes to various pathological processes, including tissue fibrosis and impaired wound healing. OBJECTIVE: In this study we investigated the role of cav-1 in MMP-1 gene regulation in human dermal fibroblasts. METHODS: Fibroblasts were isolated from healthy subjects. Western blot was used to analyze protein levels and quantitative real time RT-PCR was used to measure mRNA expression. Cells were transiently transfected with siRNA oligos against acid sphingomyelinase (ASMase) and cav-1, or transduced with adenoviruses overexpressing ASMase and cav-1. The specific pharmacological inhibitors UO126 and SP600125 were used to block Erk1/2 and JNK activity. RESULTS: This study shows that siRNA-mediated depletion of ASMase or cav-1, results in upregulation of MMP-1 gene expression. Similarly, MMP-1 expression was decreased after overexpresssion of cav-1 via an adenoviral vector. Depletion of cav-1 had no effect on JNK phosphorylation, while it resulted in an increase in Erk1/2 and Ets1 phosphorylation levels. Furthermore, in cav-1 depleted cells treated with the Erk inhibitor UO126, there was no increase in the levels of phospho-Erk1/2, phospho-Ets1, and MMP-1, suggesting that cav-1 mediated effects on MMP-1 and phospho-Ets1 are Erk1/2 dependent. CONCLUSIONS: In conclusion, this study has revealed an important role for cav-1 as a negative regulator of MMP-1 gene expression via inhibition of Erk1/2/Ets1 signaling. Cav-1 could potentially be a therapeutic target in diseases with deregulated extracellular matrix (ECM) turnover.
Subject(s)
Caveolin 1/metabolism , Dermis/enzymology , Fibroblasts/enzymology , Matrix Metalloproteinase 1/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Signal Transduction , Blotting, Western , Caveolin 1/genetics , Cells, Cultured , Dermis/cytology , Dermis/drug effects , Fibroblasts/drug effects , Gene Expression Regulation, Enzymologic , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Matrix Metalloproteinase 1/genetics , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Phosphorylation , Protein Kinase Inhibitors/pharmacology , RNA Interference , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/metabolism , TransfectionABSTRACT
BACKGROUND: During scleroderma (SSc) pathogenesis, fibroblasts acquire an activated phenotype characterized by enhanced production of extracellular matrix (ECM) and constitutive activation of several major signaling pathways including extracellular signal-related kinase (ERK1/2). Several studies have addressed the role of ERK1/2 in SSc fibrosis however the mechanism of its prolonged activation in SSc fibroblasts is still unknown. Protein phosphatase 2A (PP2A) is a key serine threonine phosphatase responsible for dephosphorylation of a wide array of signaling molecules. Recently published microarray data from cultured SSc fibroblasts suggests that the catalytic subunit (C-subunit) of PP2A is downregulated in SSc. In this study we examined the role and regulation of PP2A in SSc fibroblasts in the context of ERK1/2 phosphorylation and matrix production. RESULTS: We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression. Furthermore, transforming growth factor ß (TGFß), a major profibrotic cytokine implicated in SSc fibrosis, downregulates PP2A expression in healthy fibroblasts. PP2A-specific small interfering RNA (siRNA) was utilized to confirm the role of PP2A in ERK1/2 dephosphorylation in dermal fibroblasts. Accordingly, blockade of autocrine TGFß signaling in SSc fibroblasts using soluble recombinant TGFß receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression. In addition, we observed that inhibition of ERK1/2 in SSc fibroblasts increased PP2A expression suggesting that ERK1/2 phosphorylation also contributes to maintaining low levels of PP2A, leading to an even further amplification of ERK1/2 phosphorylation. CONCLUSIONS: Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFß signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.
