Regulation of scleraxis transcriptional activity by serine phosphorylation

Cardiac fibroblasts are the major extracellular matrix producing cells in the heart. Our laboratory was the first to demonstrate that the transcription factor scleraxis induces collagen 1 alpha 2 expression in both cardiac fibroblasts and myofibroblasts. Here we identify a novel post-translational mechanism by which scleraxis activity is regulated and determine its effect on transcription of genes targeted by scleraxis. Putative serine phosphorylation sites on scleraxis were revealed by in silico analysis using motif prediction software. Mutation of key serine residues to alanine, which cannot be phosphorylated, significantly attenuated the expression of fibrillar type I collagen and myofibroblast marker genes that are normally induced by scleraxis. Down-regulation of collagen 1 alpha 2 expression was due to reduced binding of the non-phosphorylated scleraxis mutant to specific E-box DNA-binding sites within the promoter as determined by chromatin immunoprecipitation in human cardiac myofibroblast cells and by electrophoretic mobility shift assay. This is the first evidence suggesting that scleraxis is phosphorylated under basal conditions. The phosphorylation sequence matched that targeted by Casein Kinase 2, and inhibition of this kinase activity disrupted the ability of scleraxis to modulate the expression of its target genes while also attenuating TGF beta-induced expression of type I collagen and myofibroblast phenotype conversion marker genes. These results demonstrate a novel mechanism for regulation of scleraxis activity, which may prove to be tractable for pharmacologic manipulation. (C) 2016 Elsevier Ltd. All rights reserved.