Cardiac fibrosis is marked by increased deposition of extracellular matrix components including fibrillar collagens, leading to impaired cardiac contractility and function. We recently demonstrated that the transcription factor scleraxis is expressed in collagen-producing cardiac fibroblasts and myofibroblasts, is up-regulated in the collagen-rich scar following myocardial infarction and is sufficient to transactivate the human collagen 1α2 (COL1A2) gene, suggesting a central role in fibrosis. Here we describe the mechanism of scleraxis-mediated regulation of the COL1A2 promoter. Using chromatin immunoprecipitation in primary human cardiac fibroblasts in combination with luciferase assays, we demonstrate that two E box sequences within the proximal COL1A2 promoter are required for scleraxis-mediated transactivation. Expression of scleraxis itself was induced by receptor Smad3, an effector of the pro-fibrotic growth factor TGF-β(1), and attenuated by inhibitory Smad7. TGF-β(1) augmented the effect of scleraxis on COL1A2 transactivation, an effect which was due to synergy between scleraxis and Smad3. Mutation of the COL1A2 Smad-binding element significantly attenuated the ability of scleraxis to transactivate the promoter, while mutation of the scleraxis-interacting E boxes attenuated the effect of Smad3, suggesting that these factors form a common signaling complex at the promoter. COL1A2 promoter transactivation and Col1α2 gene expression in cardiac fibroblasts were completely abrogated by a scleraxis basic domain deletion mutant in a dominant negative fashion, blocking the ability of TGF-β(1) to activate collagen synthesis and suggesting that scleraxis-DNA interaction is absolutely required for this process. Scleraxis thus appears to play a key role in the transcriptional regulation of type I collagen synthesis.

BackgroundResident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis.ResultsUsing explanted primary cardiac fibroblasts in gain- and loss-of-function studies, we found that scleraxis critically controls cardiac fibroblast/myofibroblast phenotype by direct transcriptional regulation of myriad genes that effectively define these cells, including extracellular matrix components and α-smooth muscle actin. Scleraxis furthermore potentiated the TGFβ/Smad3 signaling pathway, a key regulator of myofibroblast conversion, by facilitating transcription complex formation. While scleraxis promoted fibroblast to myofibroblast conversion, loss of scleraxis attenuated myofibroblast function and gene expression. These results were confirmed in scleraxis knockout mice, which were cardiac matrix-deficient and lost ~50 % of their complement of cardiac fibroblasts, with evidence of impaired epithelial-to-mesenchymal transition (EMT). Scleraxis directly transactivated several EMT marker genes, and was sufficient to induce mesenchymal/fibroblast phenotype conversion of A549 epithelial cells. Conversely, loss of scleraxis attenuated TGFβ-induced EMT marker expression.ConclusionsOur results demonstrate that scleraxis is a novel and potent regulator of cellular progression along the continuum culminating in the cardiac myofibroblast phenotype. Scleraxis was both sufficient to drive conversion, and required for full conversion to occur. Scleraxis fulfills this role by direct transcriptional regulation of key target genes, and by facilitating TGFβ/Smad signaling. Given the key role of fibroblast to myofibroblast conversion in fibrotic diseases in the heart and other tissue types, scleraxis may be an important target for therapeutic development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0243-8) contains supplementary material, which is available to authorized users.

Rationale: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown. Objective: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. Methods and Results: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-β (transforming growth factor-β) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin ( Postn )-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-β-mediated cardiac fibroblast activation. Conclusions: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4 . The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.