Hypoxia acts through multiple signaling pathways to induce metallothionein transactivation by the metal-responsive...

Metal-responsive transcription factor-1 (MTF-1) is essential for the induction of genes encoding metallothionein by metals and hypoxia. Here, we studied the mechanism controlling the activation of MTF-1 by hypoxia. Hypoxia activation of Mt gene transcription is dependent on the presence of metal regulatory elements (MREs) in the promoter of Mt genes. We showed that MREa and MREd are the main elements controlling mouse Mt-1 gene induction by hypoxia. Transfection experiments in Mtf-1-null cells showed that MTF-1 is essential for induction by hypoxia. Chromatin immunoprecipitation analysis showed that MTF-1 DNA-binding activity was strongly enhanced in the presence of zinc but not by hypoxia. Notably, hypoxia inducible factor- (HIF) 1α was recruited to the Mt-1 promoter in response to hypoxia but not to zinc. MTF-1 activation was inhibited by PKC, JNK, and PI3K inhibitors and by the electron transport chain inhibitors rotenone and myxothiazol, but not by the antioxidant N-acetylcysteine. We showed that prolyl-hydroxylase inhibitors can activate MTF-1, but this activation requires the presence of HIF-1α. Finally, HIF-dependent transcription is enhanced in the presence of MTF-1 and induction of an MRE promoter is stimulated by HIF-1α, thus indicating cooperation between these 2 factors. However, coimmunoprecipitation experiments did not suggest direct interaction between MTF-1 and HIF-1α.