Serum response factor neutralizes Pur alpha- and Pur beta-mediated repression of the fetal vascular smooth muscle alpha-actin gene in stressed adult cardiomyocytes.

Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle alpha-actin (SM alpha A) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SM alpha A gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur alpha, an mRNA-binding protein and known negative modulator of SRF-activated SM alpha A gene transcription. Activation of fetal SM alpha A gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur alpha to a purine-rich DNA element in the SM alpha A promoter called SPUR, previously shown to be required for induction of SM alpha A gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SM alpha A-positive cardiomyocytes in parallel with enhanced Pur alpha:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SM alpha A gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SM alpha A promoter as well as the appearance of novel Pur alpha protein complexes in both the nuclear and cytosolic compartments of these cells.

YB-1 coordinates vascular smooth muscle alpha-actin gene activation by transforming growth factor beta1 and thrombin during differentiation of human pulmonary myofibroblasts.

Profibrotic regulatory mechanisms for tissue repair after traumatic injury have developed under strong evolutionary pressure to rapidly stanch blood loss and close open wounds. We have examined the roles played by two profibrotic mediators, transforming growth factor beta1 (TGFbeta1) and thrombin, in directing expression of the vascular smooth muscle alpha-actin (SMalphaA) gene, an important determinant of myofibroblast differentiation and early protein marker for stromal cell response to tissue injury. TGFbeta1 is a well known transcriptional activator of the SMalphaA gene in myofibroblasts. In contrast, thrombin independently elevates SMalphaA expression in human pulmonary myofibroblasts at the posttranscriptional level. A common feature of SMalphaA up-regulation mediated by thrombin and TGFbeta1 is the involvement of the cold shock domain protein YB-1, a potent repressor of SMalphaA gene transcription in human fibroblasts that also binds mRNA and regulates translational efficiency. YB-1 dissociates from SMalphaA enhancer DNA in the presence of TGFbeta1 or its Smad 2, 3, and 4 coregulatory mediators. Thrombin does not effect SMalphaA gene transcription but rather displaces YB-1 from SMalphaA exon 3 coding sequences previously shown to be required for mRNA translational silencing. The release of YB-1 from promoter DNA coupled with its ability to bind RNA and shuttle between the nucleus and cytoplasm is suggestive of a regulatory loop for coordinating SMalphaA gene output in human pulmonary myofibroblasts at both the transcriptional and translational levels. This loop may help restrict organ-destructive remodeling due to excessive myofibroblast differentiation.