Myogenic Basic Helix-Loop-Helix Proteins Regulate the Expression of Peroxisomal Proliferator Activated Receptor-γ Coactivator-1α

Endocrinology, Jun 2006

Chang, Ju Hui, Lin, Kwang Huei, Shih, Chung Hsuan, Chang, Yu Jung, Chi, Hsiang Chung, Chen, Shen Liang

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Myogenic Basic Helix-Loop-Helix Proteins Regulate the Expression of Peroxisomal Proliferator Activated Receptor-γ Coactivator-1α

Endocrinology Myogenic Basic Helix-Loop-Helix Proteins Regulate the Expression of Peroxisomal Proliferator Activated Receptor- Coactivator-1 Ju Hui Chang 0 Kwang Huei Lin 0 Chung Hsuan Shih 0 Yu Jung Chang 0 Hsiang Chung Chi 0 Shen Liang Chen 0 0 Department of Life Sciences, National Central University (J.H.C. , C.H.S., Y.J.C., H.C.C., S.L.C.), Jhongli 32054 , Taiwan; and Department of Biochemistry, Chang Gung University (K.H.L.) , Kweisan 333 , Taiwan Peroxisomal proliferator-activated receptor- coactivator-1 (PGC-1 ), a transcriptional coactivator, is selectively expressed in slow-twitch fibers in skeletal muscle. Ectopic expression of the PGC-1 gene in either a cell or an animal has been shown to promote fast to slow fiber-type switch. The expression of PGC-1 in muscle is regulated by myocyte enhancer factor 2 and Forkhead in rhabdomyosarcoma, two transcription factors implicated in terminal muscle differentiation. In this study we found that PGC-1 expression was activated during terminal muscle differentiation in both C2C12 and Sol8 myoblasts. Using retrovirus-mediated MyoD overexpression in C3H10T1/2 cells, we also demonstrated that MyoD, the master regulator of terminal differentiation, could activate PGC-1 expression in vivo. Our transient transfection results also show that myogenic basic helix-loop-helix - P EROXISOMAL PROLIFERATOR-activated receptor(PPAR ) coactivator-1 (PGC1 ) is a transcriptional coactivator identified in the screening for proteins associating with PPAR . It is highly expressed in tissues requiring high-energy metabolism, such as heart, skeletal muscle, and brown fat ( 1, 2 ). Its expression in brown fat is highly induced when animals are exposed to a cold environment, implying its role in metabolic thermogenesis adaptation. Later studies have proven its function in the biogenesis of mitochondria, gluconeogenesis, and fatty acid oxidation ( 3, 4 ). Since its identification, PGC-1 has been found to coactivate the function of many nuclear hormone receptors, including thyroid hormone, estrogen, and glucocorticoid receptors, and many more ( 1, 5– 8 ). The wide variety of its partners suggests that PGC-1 is not only implicated in the energy metabolism, but is also involved in many other physiological processes. First Published Online March 9, 2006 Abbreviations: bHLH, Basic helix-loop-helix; CMB, confluent; Ct, maximum cycle threshold; DTT, dithiothreitol; FKHR, Forkhead in rhabdomyosarcoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GFP, green fluorescence protein; GST, glutathione-S-transferase; h, human; MEF2, myocyte enhancer factor 2; MRF, myogenic regulatory factor; MT, myotube; Mt, mutant; Pfu, DNA polymerase-derived from Pyrococcus furiosus; PGC-1, peroxisome proliferation activation receptor- coactivator 1; PGC1 , peroxisomal proliferator activated receptor- coactivator 1 ; PMB, proliferating; PNK, polynucleotide kinase; SDS, sodium dodecyl sulfate. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. (bHLH) proteins, especially MyoD, can activate PGC-1 expression by targeting its promoter. Myogenic bHLH protein target sites on PGC-1 promoter were localized to a short region ( 49 to 2) adjacent to the transcription start site, which contains two putative E boxes. Mutation of either site significantly reduced MyoD-mediated transactivation in the cells, suggesting that both sites are required for myogenic bHLH protein-mediated activation. However, only one site, the E2 box, was directly bound by glutathione-S-transferaseMyoD protein in EMSAs. Our results indicate that myogenic bHLH proteins not only are involved in lineage determination and terminal differentiation, but also are directly implicated in activation of the key fiber-type and metabolic switch gene, PGC-1 . (Endocrinology 147: 3093–3106, 2006) In muscle, PGC-1 is preferentially expressed in slowtwitch fibers, which are much higher in mitochondria content and are more dependent on oxidative metabolism than fasttwitch fibers ( 9 ). In transgenic mice, as the expression of PGC-1 is driven by muscle creatine kinase promoter, the putative fast-twitch fibers are converted into slow-twitch fibers, which are characterized by the expression of troponin I slow, myoglobin, and genes of mitochondria oxidative metabolism ( 9 ). The activation of slow-twitch muscle-specific genes by PGC-1 is mediated through MEF2 transcription factors binding to the upstream enhancer sites. MEF2 proteins also activate the transcription of PGC-1 gene and thus establish a positive feedback loop ( 10 ). Hormones both activating, such as thyroid hormone, and inhibiting, such as insulin, oxidation metabolism can also regulate the expression of PGC-1 either positively or negatively ( 11–13 ). The signals of insulin in the nucleus are mediated by Forkhead in rhabdomyosarcoma (FKHR), a forkhead transcription factor that b (...truncated)


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Chang, Ju Hui, Lin, Kwang Huei, Shih, Chung Hsuan, Chang, Yu Jung, Chi, Hsiang Chung, Chen, Shen Liang. Myogenic Basic Helix-Loop-Helix Proteins Regulate the Expression of Peroxisomal Proliferator Activated Receptor-γ Coactivator-1α, Endocrinology, 2006, pp. 3093-3106, Volume 147, Issue 6, DOI: 10.1210/en.2005-1317