ATF-1 is a hypoxia-responsive transcriptional activator of skeletal muscle mitochondrial-uncoupling protein 3

Abstract

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.

FIGURE 1.

Effects of hypoxia on UCP3 expression and on reactive oxygen species. A, mitochondria-related genes mRNA expression level in C2C12 myotubes exposed to 5% O₂ for 24 h. Results are relative to normoxia content and represent the mean ± S.D. of three experiments. B, representative Western blot of UCP3 steady-state protein in C2C12 myotubes exposed to 5% O₂ for 48 h. C, representative Western blot of UCP3 protein levels in gastrocnemius tissues from C57bl/6 mice subjected to 10% O₂ versus normoxia for 12 and 24 h respectively. D, skeletal muscle primary cells from UCP3 wild-type and knock-out mice were incubated in condition of normoxia or hypoxia (5% O₂) for 48 h. H₂O₂ level was detected by H₂DCFDA. E, the rate of H₂O₂ production following 24 h of hypoxia was determined by measuring amplex red oxidation rates in wild-type and UCP3 knock-out primary myocytes compared with normoxic controls. F, aconitase activity, as an indirect measure of oxidative damage induced inhibition of enzyme activity was quantified under the same experimental conditions described in E. ^*, within normoxia or hypoxia group, p < 0.05. #, between groups, p < 0.05. PGC-1α, peroxisome proliferator-activated receptor γ co-activator 1α; NRF-1, nuclear respiratory factor-1; ND1-NADPH dehydrogenase subunit 1; Cytb, cytochrome b; UCP2, uncoupling protein 2; and UCP3, uncoupling protein 3.

FIGURE 2.

Effects of myoD and ATF-1 on UCP3 murine promoter activity. A, luciferase reporter constructs of mouse UCP3 5′-end promoter region. Numerical annotation is relative to the transcriptional start site designation as +1. B, reporter constructs transfected into C2C12 myoblasts with MyoD or pcDNA empty control vector. Luciferase activities were normalized to the activity of 2k-luc co-transfected with the empty vector (value = 1) and represent mean ± S.D. of three independent experiments in this and all subsequent cotransfection experiments. C, similar study to B, with the exception that the luciferase activity in response to ATF-1 is assessed instead of that of myoD. D, cotransfection experiments to evaluate the combined ex vivo activation capacity of myoD and ATF-1 using the full-length and 2-luc UCP3 promoter-luciferase reporter constructs.

FIGURE 3.

Identification of the ATF-1-responsive region within UCP3 promoter. A, reporter constructs of deletion and point mutation of UCP3 promoter employed to identify region required for ATF-1 transactivation. The three mutation constructs of 2-luc are shown immediately below the index construct. Mutated nucleotides are underlined and deleted nucleotides shown as dashes. B, luciferase activity of deletion/mutation reporter constructs in response to the cotransfection of ATF-1 compared with the empty vector control in C2C12 cells. Results are normalized to 2k-luc activity co-transfected with empty vector (value = 1) and represent mean ± S.D. of three independent experiments. C, representative ChIP analysis of ATF1 binding to UCP3 promoter region corresponding to the region -1556 to -1342.

FIGURE 4.

Effects of hypoxia on ATF-1 phosphorylation and of ATF-1 knockdown on hypoxia-mediated UCP3 up-regulation. A, representative Western blot of temporal levels of ATF-1 and phosphorylated ATF-1 levels in nuclear protein from C2C12 myotubes exposed to 5% O₂ from 3 to 24 h compared with levels in normoxic control cells. B, representative Western blot showing knockdown of ATF-1 in C2C12 myotubes, 5 days after insertion into myoblasts and growth in differentiation media. Nuclear protein loading is normalized to TATA-binding protein (Tbp). Histogram shows mean and S.D. nuclear protein levels of ATF-1 comparing cells electroporated with the control vector construct versus the shATF-1 construct. C, representative Western blot showing expression of UCP3 protein levels in C2C12 myotubes transfected with pLKO-shATF-1 or control vector in response to 48 h of normoxia or hypoxia (5% O₂), respectively. pLKO, control vector; pLKO-shATF-1, vector harboring the ATF-1 shRNA.

FIGURE 5.

Signal transduction pathways of ATF-1-mediated UCP3 up-regulation. A, a histogram showing the transcript levels of UCP3 under a normoxic and hypoxic environment for 48 h under control conditions compared with myotubes treated with vehicle or the PKA inhibitors KT5720 (5uM) or H89 (5uM). B, representative Western blot comparing UCP3 levels under a normoxic and hypoxic environment for 48 h under control conditions compared with myotubes treated with vehicle or the PKA inhibitors KT5720 (5 μm) or H89 (5 μm). C, representative Western blot comparing phospho-ATF-1 and UCP3 levels under a normoxic and hypoxic environment for 48 h comparing myotubes treated with vehicle or the p38 MAP kinase inhibitor SB203580 (30 μm). Quantification of the protein levels of phospho-ATF-1 (D) and UCP3 (E) protein levels under the same conditions described in C. DMSO, dimethyl sulfoxide. F, representative Western blot of HIF-1α and UCP3 levels in response to cobalt chloride administration (0.5 mm) or vehicle control for 48 h. Equal protein loading is shown by immunoblot of actin levels. G, histogram showing UCP3 transcript levels in control and cobalt chloride-treated C2C12 myotubes under the same conditions described in F.^*, p < 0.05.