Bhlhe40 Represses PGC-1α Activity on Metabolic Gene Promoters in Myogenic Cells

Abstract

c PGC-1␣ is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1␣ might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1␣ directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1␣-targeted gene promoters/enhancers, which in turn repressed PGC-1␣ transactivational activity. Bhlhe40 repressed PGC-1␣ activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1␣ intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1␣ target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1␣-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1␣-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1␣ activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM. Skeletal muscle (SKM) is one of the major metabolic organs, and it has the ability to adapt to various physiological conditions, such as cold, exercise, and sedentary life, by adjusting the balance between glycolytic and oxidative metabolism to meet the energy requirement under these conditions (1). Recent studies suggest that this adaptation relies much on the expression and activity of PGC-1␣, a transcriptional coactivator highly expressed in tissues with high energy metabolism (1, 2). PGC-1␣ enhances the activity of many nuclear receptors (NR), including peroxisome proliferator-activated receptor ␥ (PPAR␥), estrogen receptor (ER), and glucocorticoid receptor (GR), and non-NR transcription factors, such as MEF2, Sox9, FoxO1, and SREBP1 (reviewed in reference 3 and references therein), to promote oxidative metabolism, metabolic thermogenesis adaptation, biogenesis of mitochondria, gluconeogenesis, and fatty acid oxidation in various tissues (4-6). Overexpression of PGC-1␣ can promote metabolic switch from anaerobic glycolysis to oxidative metabolism (7,8). Current studies also show that PGC-1␣ plays critical roles in orchestrating the cellular response to hypoxia (9).In SKM, PGC-1␣ is preferentially expressed in oxidative metabolism-dependent slow-twitch fibers (10), and its overexpression can convert putative fast-twitch fibers into slow-twitch fibers (10). The activation of oxidative and slow-twitch muscle-specific genes by PGC-1␣ is mediated through its coactivation of Mef2 and PPAR binding to the upstream regulatory sites of these target genes (11). PGC-1␣ null mice sho...

Bhlhe40 differentially regulates the function and number of peroxisomes and mitochondria in myogenic cells

“…The plasmids pEGFP-N1-roGFP-PTS1 and –KillerRed-PTS1 were generous gifts from Dr. Marc Fransen (Katholieke Universiteit Leuven, Belgium) and their construction can be found in Dr. Fransen's publication [20] . The expression vectors of PGC-1α and Bhlhe40 and their target promoters driven reporters have been described in our previous studies [19] , [21] . A TET-off expression vector, pCEGFP-TRE, was created by inserting the Sal I/ Mlu I fragment of pBI-EGFP vector into the Bgl II/ Hind III sites of pCDNA3 vector.…”

“…Relief of this repression by knockdown of Bhlhe40 expression increased levels of ROS, fatty acid oxidation, mitochondria DNA, and the expression of PGC-1α target genes. These observations suggest that Bhlhe40 should be an important regulator of mitochondrial and peroxisome biogenesis and functions [19] . Therefore, more endeavor should be devoted to decipher the regulation of these two organelles by Bhlhe40.…”

“…It has been shown that PGC-1α play critical roles in the regulation of the biogenesis and function of mitochondria and peroxisomes through coactivating the activity of various DNA-binding transcription factors, such as PPARγ, ERRα, and NRF1 [1] , [18] . Recently we found that PGC-1α directly interacted with Bhlhe40 and they co-occupied PGC-1α targeted gene promoters/enhancers, which in turn repressed PGC-1α transactivational activity [19] . Relief of this repression by knockdown of Bhlhe40 expression increased levels of ROS, fatty acid oxidation, mitochondria DNA, and the expression of PGC-1α target genes.…”

MEHP interferes with mitochondrial functions and homeostasis in skeletal muscle cells

“…The detailed protocol of qRT-PCR has been described in our previous works [24,25]. Briefly, myotubes incubated in differentiation for 3 days (DM3) were treated with DMSO or 100 μM MEHP for 2 days.…”

“…The detailed protocols for these assays have been described in our previous study [24]. Briefly, cells were incubated in KRPH buffer containing 30 μM H2-DCFDA at 37 • C for 30 min.…”