ATF-1 is a hypoxia-responsive transcriptional activator of skeletal muscle mitochondrial-uncoupling protein 3.
Hypoxia induces oxidative damage in skeletal muscle. Uncoupling protein 3 (UCP3) is the skeletal muscle enriched uncoupling protein and has previously been shown to confer resistance against oxidative stress. We show that hypoxia robustly up-regulates skeletal muscle UCP3 and that the absence of UCP3 in primary skeletal myocytes exacerbates hypoxia-induced reactive oxygen species generation. In this context, we reasoned that the investigation of the regulation of UCP3 may identify novel hypoxia-responsive regulatory pathways that modulate intrinsic anti-oxidant defenses. By screening a transcription factor array of 704 full-length cDNAs in murine C2C12 myoblasts following cotransfection of a murine UCP3 promoter-luciferase construct and myoD we identified numerous candidate regulatory factors that up-regulate UCP3. Active transcription factor-1 (ATF-1) was identified, and as this transcription factor is a known component of a multiprotein hypoxia-induced regulatory complex, we explored its role in hypoxia-mediated UCP3 up-regulation. Site-directed mutagenesis and chromatin immunoprecipitation assays identify a 10-bp region required for ATF-1 induction of UCP3 promoter activity. Hypoxia promotes the phosphorylation of ATF-1, and the knockdown of ATF-1 by shRNA prevents hypoxia-mediated up-regulation of UCP3. Pharmacologic inhibition of p38 MAP kinase prevents both hypoxia-mediated ATF-1 phosphorylation and UCP3 up-regulation. PKA signaling does not modulate hypoxia-induced UCP3 up-regulation and neither does HIF-1alpha activation by cobalt chloride. In conclusion, ATF-1, via p38 MAP kinase activation, functions as a novel regulatory pathway driving UCP3 expression. These data reinforce the role of ATF-1 as a hypoxia-responsive trans-activator and identifies a novel regulatory program that may modulate cellular responses to oxygen-deficit.
Lu
Zhongping
Z
Translational Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1454, USA.
Sack
Michael N
MN
D051697:Activating Transcription Factor 1 / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D000860:Hypoxia; D007473:Ion Channels / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D000071247:Uncoupling Protein 3; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D051697:Activating Transcription Factor 1; D000975:Antioxidants; D007473:Ion Channels; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; C000604393:Ucp3 protein, mouse; D000071247:Uncoupling Protein 3; D003035:Cobalt; D048051:p38 Mitogen-Activated Protein Kinases; C018021:cobaltous chloride