Department of Medicine, Division of Rheumatology and Immunology and the Hollings Cancer Center, Laboratory of Cancer Genomics, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Extracellular matrix (ECM) production and turnover are tightly controlled under normal physiological conditions. Ets factors regulate matrix turnover by activating transcription of several metalloproteinases (MMPs) and are frequently overexpressed in aggressive tumors and arthritis. Because of the prominent role of transforming growth factor beta (TGF-beta) in ECM synthesis, this study was undertaken to determine the possible interactions between Ets1 and the TGF-beta pathway. Experiments using adenoviral delivery of Ets1 in human fibroblasts have established that Ets1 strongly suppresses TGF-beta induction of collagen type I and other matrix-related genes and reverses TGF-beta-dependent inhibition of MMP-1. Subsequent experiments utilizing COL1A2 promoter demonstrated that Ets1 in the presence of TGF-beta signaling interferes with the stimulatory role of p300. To gain further insight into the mechanism of Ets1 inhibition of the TGF-beta signaling, the protein levels and post-translational modifications of Ets1 after TGF-beta treatment were analyzed. The level of total Ets1 protein was not affected after 24 h of TGF-beta stimulation. Moreover, TGF-beta did not affect either serine or threonine phosphorylation levels of Ets1. However, TGF-beta induced rapid and prolonged lysine acetylation of Ets1. In addition, analyses of endogenous p300.Ets1 complexes revealed that acetylated Ets1 is preferentially associated with the p300/CBP complexes. TGF-beta treatment leads to dissociation of Ets1 from the CBP/p300 complexes. Together, these findings suggest that elevated expression of Ets1 in fibroblasts fundamentally alters their responses to TGF-beta in favor of matrix degradation and away from matrix deposition as exemplified by arthritis and cancer.
"There are several key positive and negative regulator proteins of collagen transcription in the setting of fibrotic disease that interact with these sites. The Sp1 family of proteins activates collagen transcription through G/C rich sites   , while the Ets domain family of proteins both activate and repress collagen gene expression in fibroblasts   . Fig. 2 Model of collagen repression by CIITA during IFN-γ. "
[Show abstract][Hide abstract] ABSTRACT: Extracellular matrix (ECM) within the vascular network provides both a structural and regulatory role. The ECM is a dynamic composite of multiple proteins that form structures connecting cells within the network. Blood vessels are distended by blood pressure and, therefore, require ECM components with elasticity yet with enough tensile strength to resist rupture. The ECM is involved in conducting mechanical signals to cells. Most importantly, ECM regulates cellular function through chemical signaling by controlling activation and bioavailability of the growth factors. Cells respond to ECM by remodeling their microenvironment which becomes dysregulated in vascular diseases such hypertension, restenosis and atherosclerosis. This review examines the cellular and ECM components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.
"This confirms previous in vitro studies that showed competition of Ets1 and Fli1 for the Ets binding site on the COL1A2 promoter . While Ets1 has been shown to be a positive mediator of fibrosis , , its direct role in collagen gene regulation has not been fully defined, and surprisingly overexpression of Ets1 in dermal fibroblasts leads to inhibition of the COL1A2 gene . Interestingly, recent ChIP-chip analysis of the Smad2/3 binding sites in HaCaT cells has revealed that the binding elements for Ets are significantly enriched in the Smad2/3 binding sites and knockdown of Ets1 results in overall alteration of TGF-β-induced transcription, suggesting that Ets contributes to the induction of the TGF-β-Smad pathways . "
[Show abstract][Hide abstract] ABSTRACT: Fli1, a member of the Ets transcription factor family, is a key repressor of the human α2(I) collagen (COL1A2) gene. Although our previous studies have delineated that TGF-β induces displacement of Fli1 from the COL1A2 promoter through sequential post-translational modifications, the detailed mechanism by which Fli1 functions as a potent transcriptional repressor of the COL1A2 gene has not been fully investigated. To address this issue, we carried out a series of experiments especially focusing on protein-protein interaction and epigenetic transcriptional regulation. The combination of tandem affinity purification and mass spectrometry identified HDAC1 as a Fli1 interacting protein. Under quiescent conditions, HDAC1 induced deacetylation of Fli1 resulting in an increase of Fli1 DNA binding ability and p300 enhanced this process by promoting the formation of a Fli1-HDAC1-p300 complex. TGF-β-induced phosphorylation of Fli1 at threonine 312 led to disassembly of this protein complex. In quiescent dermal fibroblasts Fli1, HDAC1, and p300 occupied the -404 to -237 region, including the Fli1 binding site, of the COL1A2 promoter. TGF-β induced Fli1 and HDAC1 dissociation from the COL1A2 promoter, while promoting Ets1 and p300 recruitment. Furthermore, acetylation levels of histone H3 around the Fli1 binding site in the COL1A2 promoter inversely correlated with the DNA occupancy of Fli1 and HDAC1, while positively correlating with that of Ets1 and p300. In the functional studies, HDAC1 overexpression magnified the inhibitory effect of Fli1 on the COL1A2 promoter. Moreover, pharmacological blockade of HDAC1 by entinostat enhanced collagen production in dermal fibroblasts. Collectively, these results indicate that under quiescent conditions Fli1 recruits HDAC1/p300 to the COL1A2 promoter and suppresses the expression of the COL1A2 gene by chromatin remodeling through histone deacetylation. TGF-β-dependent phosphorylation of Fli1 at threonine 312 is a critical step regulating the remodeling of the Fli1 transcription repressor complex, leading to transcriptional activation of the COL1A2 gene.
PLoS ONE 09/2013; 8(9):e74930. DOI:10.1371/journal.pone.0074930 · 3.23 Impact Factor
"Previous studies have identified an essential role for p300 acetyltransferase and its interaction with Smads in TGF-binduced profibrotic responses (Ghosh et al., 2000, 2001, 2004, 2009; Czuwara-Ladykowska et al., 2002). Despite its critical role in modulating profibrotic responses elicited by TGF-b and other mediators, the regulation of p300 expression and activity, as well as the mechanisms underlying their derangement in fibrotic diseases, have received scant attention to date. "
[Show abstract][Hide abstract] ABSTRACT: Fibrosis, the hallmark of systemic sclerosis (SSc), is characterized by persistent fibroblast activation triggered by transforming growth factor-β (TGF-β). As the acetyltransferase p300 has a key role in fibrosis and its availability governs the intensity of fibrotic responses, we investigated p300 expression in SSc and the molecular basis of its regulation. We found that expression of p300 was markedly elevated in SSc skin biopsies and was induced by TGF-β in explanted normal skin fibroblasts. Stimulation of p300 by TGF-β was independent of Smads and involved the early-immediate transcription factor Egr-1 (early growth response 1), a key regulator of profibrotic TGF-β signaling. Indeed, Egr-1 was both sufficient and necessary for p300 regulation in vitro and in vivo. Increased p300 accumulation in TGF-β-treated fibroblasts was associated with histone hyperacetylation, whereas p300 depletion, or selective pharmacological blockade of its acetyltransferase activity, attenuated TGF-β-induced responses. Moreover, TGF-β enhanced both p300 recruitment and in vivo histone H4 acetylation at the COL1A2 (collagen, type I, α2) locus. These findings implicate p300-mediated histone acetylation as a fundamental epigenetic mechanism in fibrogenesis and place Egr-1 upstream in TGF-β-driven stimulation of p300 gene expression. The results establish a firm link between fibrosis with aberrant p300 expression and epigenetic activity that, to our knowledge, is previously unreported. Targeted disruption of p300-mediated histone acetylation might therefore represent a viable antifibrotic strategy.Journal of Investigative Dermatology advance online publication, 10 January 2013; doi:10.1038/jid.2012.479.
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