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Regulation of Fibronectin Gene Expression by Scleraxis

The glycoprotein fibronectin is a key component of the extracellular matrix (ECM), linking myriad ECM components such as collagen to one another as well as to cell surface integrins and contributing to basal tissue structure. Up‐regulation of fibronectin occurs in response to injury, and increased fibronectin expression is an important feature of pathological cardiac fibrosis, however the molecular regulators of this process are incompletely defined. We previously found that the transcription factor scleraxis regulates the expression of numerous ECM components, and that scleraxis is up‐regulated by pro‐fibrotic stimuli, thus our objective was to determine the effect of scleraxis on fibronectin expression. Transfection of NIH‐3T3 fibroblasts with scleraxis resulted in a three‐fold increase in fibronectin gene expression. In silico analysis of the human fibronectin gene promoter sequence revealed two putative E‐box sequences to which scleraxis may bind. Using chromatin immunoprecipitation assays, we confirmed the occupancy of these sites by scleraxis in human cardiac myofibroblasts, and demonstrated the ability of scleraxis to bind these E‐boxes using electrophoretic mobility shift assays. By luciferase gene reporter experiments, we found that scleraxis transactivates the fibronectin gene promoter. Site‐directed mutagenesis of single E‐boxes significantly reduced promoter transactivation, with complete abrogation of scleraxis‐mediated reporter activity when both E‐boxes were mutated. Our data demonstrates that scleraxis is a potent transcriptional regulator of fibronectin gene expression, reveals the mechanism by which this regulation is achieved, and suggests that scleraxis‐mediated fibronectin expression may contribute to fibrosis.