Glucocorticoids Alter CRTC-CREB Signaling in Muscle Cells: Impact on PGC-1α Expression and Atrophy Markers

July Glucocorticoids Alter CRTC-CREB Signaling in Muscle Cells: Impact on PGC-1α Expression and Atrophy Markers Jill A. Rahnert 1 2 3 Bin Zheng 0 1 2 3 Matthew B. Hudson 0 1 2 3 Myra E. Woodworth-Hobbs 1 2 3 S. Russ Price 0 1 2 3 0 Atlanta Veterans Affairs Medical Center , Decatur, Georgia , United States of America 1 Institutes of Health grants to SRP (RO1DK95610) and to JAR and MBH (T32DK007656), and a Merit Review Award from the US Department of Veterans Affairs Biomedical Development Laboratory Research and Development Service to SRP , I01BX001456 2 Renal Division, Department of Medicine, Emory University , Atlanta, Georgia , United States of America 3 Editor: Ashok Kumar, University of Louisville School of Medicine , UNITED STATES Muscle wasting associated with chronic diseases has been linked to decreased expression of PGC-1α and overexpression of PGC-1α counters muscle loss. CREB, in conjunction with the CREB-regulated transcription coactivator (CRTC2), is a positive modulator of PGC-1α transcription. We previously reported that PGC-1α expression is decreased in skeletal muscle of diabetic rats despite a high level of CREB phosphorylation (i.e., activation), suggesting that CRTC2-CREB signaling may be dysregulated. In this study, the relationship between CREB/CRTC signaling and PGC-1α expression was examined in L6 myotubes treated with dexamethasone (Dex, 48h) to induce atrophy. Dex decreased PGC-1α mRNA and protein as well as the levels of CRTC1 and CRTC2 in the nucleus. Dex also altered the nuclear levels of two known regulators of CRTC2 localization; the amount of calcinuerin catalytic A subunit (CnA) was decreased whereas SIK was increased. To assess PGC-1α transcription, muscle cells were transfected with a PGC-1α luciferase reporter plasmid (PGC1α-Luc). Dex suppressed PGC-1α luciferase activity while both isobutylmethylxanthine (IBMX) and over-expression of CRTC1 or CRTC2 increased PGC-1α-Luc activity. Mutation of the CRE binding site from PGC-1α-Luc reporter attenuated the responses to both IBMX and the CRTC proteins. Consistent with the reporter gene results, overexpression of CRTC2 produced an increase in CRTC2 in the nucleus and in PGC-1α mRNA and PGC-1α protein. Overexpression of CRTC2 was not sufficient to prevent the decrease in PGC-1α mRNA or protein by Dex. In summary, these data suggest that attenuated CREB/CRTC signaling contributes to the decrease in PGC-1α expression during atrophy. Data Availability Statement; All relevant data are within the paper - Competing Interests: The authors have declared that no competing interests exist. The contents do not Introduction Accelerated protein degradation contributes to the loss of skeletal muscle mass in a variety of catabolic conditions including sepsis, cancer cachexia, kidney disease and diabetes. Chronic systemic diseases (e.g., chronic kidney disease and diabetes) are often associated with represent the views of the U.S. Department of Veterans Affairs or the United States Government. prolonged increases in circulating glucocorticoids that contribute to protein degradation as adrenalectomy or treatment with glucocorticoid receptor antagonist attenuates muscle loss in these conditions [ 1–4 ]. These reports underscore the permissive role that glucocorticoids play in the activation of various proteolytic systems (e.g., ubiquitin-proteasome, autophagy) [ 5–9 ]. Muscle atrophy during chronic diseases has been linked to a decrease in the level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) [ 10–14 ]. In skeletal muscle, PGC-1α is a transcriptional coactivator that regulates energy and other aspects of metabolism, in part, by facilitating a genetic program that drives the oxidative fiber phenotype, mitochondrial biogenesis, and fatty acid oxidation. In recent studies, overexpression of PGC1α was reported to maintain muscle mass in several models of atrophy by a proposed mechanism that involves inhibition of the FoxO transcription factors. FoxO1 and FoxO3 are key regulators of a number of atrophy-related genes (i.e., atrogenes) [ 10,14–17 ]. Sandri et al [ 14 ] reported that overexpression of PGC-1α attenuated FoxO3a activity, thereby providing a protective effect against atrophy. In other studies, overexpression of PGC-1α prevented the induction of FoxO-mediated atrogenes, MuRF-1 and Atrogin-1/MAFbx, and the reduction in fiber size during atrophy [ 10,15 ]. Thus, maintenance of the level of PGC-1α in muscle appears to be important for sustaining muscle health and function. The level of PGC-1α protein in cells is regulated by both transcriptional and post-transcriptional mechanisms. Consistent with its role as a “master” regulator, the PGC-1α promoter region has binding sites for a variety of transcription factors, thus providing inputs for various signaling pathways. Many of the contraction-induced metabolic adaptations are a result of increased PGC-1α expression that is mediated by MEF2 (...truncated)