Fra-2 mediates oxygen-sensitive induction of transforming growth factor β in cardiac fibroblasts

In the ischaemia-reperfused heart, transforming growth factor beta (TGF beta) proteins trigger the differentiation of cardiac fibroblasts (CFs) contributing to fibrosis. Reoxygenation of the heart, in addition to being a trigger for reperfusion injury, induces tissue remodelling by hyperoxia-sensitive signalling processes involving TGF beta. Here, we sought to characterize the molecular mechanisms responsible for the O-2-sensitive transcriptional induction of TGF beta in murine CF and to test the significance of such findings in the infarcted myocardium in vivo using laser capture microdissection. All three isoforms of TGF beta were induced in the CF-rich peri-infarct tissue as well as in CF exposed to hyperoxic challenge. Reporter studies demonstrated that TGF beta transcription is hyperoxia inducible. Deletion of any one or both of the activating protein-1 (AP-1) binding sites in the TGF beta reporter construct resulted in loss of O-2 sensitivity, demonstrating that AP-1 confers O-2 sensitivity to TGF beta transcription. Fos-related AP-1 transcription factor (Fra-2) and Ask-1 (apoptosis signal-regulating kinase-1) were identified as key mediators of AP-1-dependent O-2-sensitive TGF beta transcription. Knockdown of Fra-2 significantly blunted O-2-induced expression of TGF beta 1 as well as TGF beta 3 in CF. Knockdown of Ask-1 blunted hyperoxia-induced Fra-2 gene expression and nuclear localization in CF. Collectively, these observations point towards a central role of Ask-1 and Fra-2 in O-2-inducible AP-1 activation and induction of TGF beta. Taken together with the observation that Fra-2-regulated genes are implicated in fibrosis, identification of Fra-2 as an O-2-sensitive transcriptional regulator of inducible TGF beta expression positions Fra-2 as an important player in reoxygenation-induced fibrosis.