Identification and characterization of an alternative promoter of the human PGC-1a gene Toyo Yoshioka a,2 , Kenjiro Inagaki a,2 , Tetsuya Noguchi a, * , Mashito Sakai a , Wataru Ogawa a , Tetsuya Hosooka a,1 , Haruhisa Iguchi b , Eijiro Watanabe b , Yasushi Matsuki b , Ryuji Hiramatsu b , Masato Kasuga a,c a Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Genomic Science Laboratories, DainipponSumitomo Pharma Co. Ltd., 4-2-1 Takatsukasa, Takarazuka 665-8555, Japan c Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan article info Article history: Received 13 February 2009 Available online 20 February 2009 Keywords: PGC-1a gene Alternative promoter Skeletal muscle Exercise cAMP response element E-box Muscle-specific transcription factor CREB abstract The transcriptional regulator peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a) con- trols mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1a expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1a transcript (designated PGC-1a-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1a-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this pro- moter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca 2+ - and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated pro- tein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1a-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1a expression in contracting muscle. Ó 2009 Elsevier Inc. All rights reserved. Introduction Peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a) is a master regulator that directs mitochondrial biogene- sis and the formation of insulin-sensitive type I (slow-twitch) myofibers in skeletal muscle [1]. Endurance exercise, which im- proves insulin sensitivity, has been shown to induce PGC-1a expression in muscle and to enhance oxidative metabolism in rodents and humans [2–4], implying that feasible measures to up-regulate PGC-1a expression or activity selectively in skeletal muscle may prove effective for the prevention or treatment of insulin resistance. Several molecular pathways have been postulated to mediate the stimulatory effect of exercise on PGC-1a expression in skeletal muscle. Activation of Ca 2+ - and calmodulin-dependent protein ki- nase IV (CaMKIV) and the protein phosphatase calcineurin A (CnA) in response to an increase in the cytosolic Ca 2+ concentration may thus result in transactivation of the PGC-1a gene through cAMP response element (CRE)-binding protein (CREB) and the myocyte enhancer factor 2 family of transcription factors, respec- tively [5]. The p38 mitogen-activated protein kinase (p38 MAPK) and b 2 -adrenergic receptor, both of which are activated by physical exercise, have also been implicated in the induction of muscle PGC- 1a expression [6,7]. We have identified an alternative first exon (designated exon 1b) of the mouse PGC-1a gene that is spliced to the canonical exon 2 and contributes both the 5 0 untranslated region and the first 12 codons to a novel isoform of PGC-1a transcript (PGC-1a-b mRNA) (H. Iguchi, E. Watanabe, and Y. Matsuki, unpublished observation). The same splicing variant was independently described by another laboratory [8]. The PGC-1a-b mRNA was found to produce func- tional protein with transcriptional activity; it was up-regulated in skeletal muscle in response to exercise to a much greater extent than was the previously described PGC-1a-a transcript [8]. These findings suggested that the molecular mechanism by which exer- cise induces PGC-1a expression in skeletal muscle may be more complex than that proposed on the basis of previous studies with 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.077 * Corresponding author. Fax: +81 78 382 2080. E-mail address: noguchi@med.kobe-u.ac.jp (T. Noguchi). 1 Present address: Division of Endocrinology, Diabetes, and Metabolism, Depart- ment of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA. 2 These authors contributed equally to this work. Biochemical and Biophysical Research Communications 381 (2009) 537–543 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc