Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

Abstract

IntroductionExtracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.MethodsThe steady-state mRNA levels of COL1A1, COL1A2, TIMP-1, MMP-1, and MMP-2 were assessed by quantitative PCR in human dermal fibroblasts cultured in the presence of TGF-β, bortezomib, or both. Transient fibroblast transfection was performed with wild-type and mutated COL1A1 and MMP-1 promoters. Chromatin immunoprecipitation, electrophoretic mobility shift assay (EMSA), and DNA pull-down assays were used to assess the binding of c-Jun, SP1, AP2, and Smad2 transcription factors. Immunoblotting and immunofluorescent microscopy were performed for identifying phosphorylated transcription factors and their cellular localization.ResultsBortezomib decreased the steady-state mRNA levels of COL1A1 and COL1A2, and abrogated SP1 binding to the promoter of COL1A2 in both untreated and TGF-β-activated fibroblasts. Reduced COL1A2 expression was not due to altered TGF-β-induced Smad2 phosphorylation, nuclear translocation, or binding to the COL1A2 promoter. In contrast to collagen, bortezomib specifically increased the steady-state mRNA levels of MMP-1 and enhanced the binding of c-Jun to the promoter of MMP-1. Furthermore, disruption of the proximal AP-1-binding site in the promoter of MMP-1 severely impaired MMP-1 transcription in response to bortezomib.ConclusionsBy altering the binding of at least two transcription factors, c-Jun and SP1, proteasome inhibition results in increased production of MMP-1 and decreased synthesis of type I collagen in human dermal fibroblasts. Thus, the antifibrotic phenotype observed in fibroblasts submitted to proteasome inhibition results from profound modifications in the binding of key transcription factors. This provides a novel rationale for assessing the potential of drugs targeting the proteasome for their anti-fibrotic properties.

Proteasome Inhibition Reduces Proliferation, Collagen Expression, and Inflammatory Cytokine Production in Nasal Mucosa and Polyp Fibroblasts

“…We now report that moderately low concentrations of MG262 (10 -50 nM) significantly reduce the mRNA expression of the three collagen types in both unstimulated and TGF-␤-stimulated nasal mucosa and polyp fibroblasts. Likewise, a decrease in collagen mRNA expression has been reported after proteasome inhibition of human dermal fibroblasts (Fineschi et al, 2006;Goffin et al, 2010) and murine lung fibroblasts (Fineschi et al, 2008). Proteasome inhibition in rat cardiac fibroblasts also decreased collagen mRNA expression and expression of matrix metalloproteinases 2 and 9 (Meiners et al, 2004).…”

“…Mutlu et al (2012) similarly found that bortezomib did not inhibit TGF-␤-induced Smad3 phosphorylation or nuclear translocation, although it inhibited the transcription of a Smad-responsive luciferase reporter construct. It is worth noting that bortezomib abrogated the binding of SP1 to the collagen 1␣2 promoter in both untreated and TGF-␤-stimulated fibroblasts (Goffin et al, 2010). Analysis of the possible role of SP1 will be the subject of future experiments but are beyond the scope of the current article.…”

“…We found that MG262 increased c-Jun phosphorylation and p53 levels in both unstimulated and TGF-␤-stimulated nasal fibroblasts, but none of these effects could explain the marked suppressive effect of MG262 on collagen expression. With regard to other transcription factors involved in TGF-␤-induced collagen expression, Goffin et al (2010) reported that bortezomib did not affect TGF-␤-induced Smad2 phosphorylation, nuclear translocation, or binding to the collagen 1␣2 promoter. In fact, Fineschi et al (2008) reported an increase in TGF-␤-induced Smad2 phosphorylation by proteasome inhibitors.…”

Targeting the Tumor Proteasome as a Mechanism to Control the Synthesis and Bioactivity of Matrix Macromolecules

“…Proteasome blockade by PIs has been shown to result in a marked modification of gene expression in dermal fibroblasts by altering the binding of at least two transcription factors (c-Jun and SP1) on various matrix effectors promoter regions leading to the reduction of ECM synthesis and increase of ECM degradation. This was due to the down-regulation of Col-I and tissue inhibitor of metalloproteinase-1 (TIMP-1) and the up-regulation of MMP-1 protein production and collagenolytic activity [140,141]. Specifically, the effect of PIs on Col-I synthesis was mediated, at least in part, by their capacity to reduce the binding of SP1, known to be a crucial cis-acting element for basal COL1A2 transcription, to the promoter of COL1A2.…”

Inhibition of Matrix Metalloproteinase-1 Induced by Oxidative Stress in Human Keratinocytes by Mangiferin Isolated fromAnemarrhena asphodeloides

“…PCR products were separated on 2% agarose gels, and DNA bands were visualized using the Image program (NIH, Bethesda, MD). 18) Cell viability. The cells were treated with mangiferin.…”