Inhibitors of oxygen sensing prolyl hydroxylases regulate nuclear localization of the transcription factors Smad2 and YAP/TAZ involved in CTGF synthesis.

Pharmacological inhibition of oxygen sensing prolyl hydroxylase domain enzymes (PHDs) has been shown to preserve renal structure and function in various models of kidney disease. Since transforming growth factor β-1 (TGFβ-1) is one of the major mediators of kidney injury, we investigated if inhibition of PHDs with subsequent stabilization of hypoxia inducible transcription factors (HIF) might interfere with TGFβ-1 signaling with special emphasis on its target gene connective tissue growth factor (CTGF). Overnight incubation of human renal tubular cells, primary cells and cell lines, with the PDH inhibitor DMOG increased Smad3 expression, but barely affected Smad2. Both Smads were translocated into the nucleus upon activation of the cells with TGFβ-1. Interestingly, Smad3 nuclear localization was enhanced upon pretreatment of the cells with DMOG for several hours, whereas nuclear Smad2 was reduced. This differential localization was independent of Smad2/3 phosphorylation. Reduced nuclear Smad2 correlated with impaired CTGF secretion in DMOG-treated cells and transient downregulation of Smad2 interfered with TGFβ-1-induced CTGF synthesis. Furthermore, YAP was confirmed as indispensable transcription factor involved in CTGF synthesis. Nuclear localization of YAP and TAZ was reduced in DMOG-treated cells. Our data thus provide evidence for DMOG-mediated reduction of CTGF expression by regulating the nuclear localization of the transcription factors Smad2, YAP and TAZ. Prolonged inhibition of PHDs was necessary to achieve alterations in cellular localization suggesting an indirect HIF-mediated effect. This mechanism might be extended to other transcription factors and target genes, and may thus represent a novel mechanism of negative regulation of gene expression by PHD inhibition.