An autoregulatory loop controls peroxisome proliferator-activated receptor gamma coactivator 1alpha expression in muscle

Abstract

Skeletal muscle adapts to chronic physical activity by inducing mitochondrial biogenesis and switching proportions of muscle fibers from type II to type I. Several major factors involved in this process have been identified, such as the calcium/calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A (CnA), and the transcriptional component peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Transgenic expression of PGC-1alpha recently has been shown to dramatically increase the content of type I muscle fibers in skeletal muscle, but the relationship between PGC-1alpha expression and the key components in calcium signaling is not clear. In this report, we show that the PGC-1alpha promoter is regulated by both CaMKIV and CnA activity. CaMKIV activates PGC-1alpha largely through the binding of cAMP response element-binding protein to the PGC-1alpha promoter. Moreover, we show that a positive feedback loop exists between PGC-1alpha and members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. MEF2s bind to the PGC-1alpha promoter and activate it, predominantly when coactivated by PGC-1alpha. MEF2 activity is stimulated further by CnA signaling. These findings imply a unified pathway, integrating key regulators of calcium signaling with the transcriptional switch PGC-1alpha. Furthermore, these data suggest an autofeedback loop whereby the calcium-signaling pathway may result in a stable induction of PGC-1alpha, contributing to the relatively stable nature of muscle fiber-type determination.

Fig. 1.

Induction of PGC-1α by CaMKIV via CREB. (A) CaMKIV activation of PGC-1α is abolished by the dominant-negative ACREB. C2C12 cells were cotransfected with expression plasmids for CnA, CaMKIV, and ACREB together with reporter gene plasmids containing different fragments of the mouse PGC-1α promoter. After 48 h, cells were harvested and luciferase activity was determined. (B) Sequence of the CRE (depicted in bold and underlined) in the mouse PGC-1α promoter. Y = C or T.(C) Site-directed mutagenesis of the CRE inhibits CaMKIV-mediated activation of PGC-1α. C2C12 cells were cotransfected with expression plasmids for CnA, CaMKIV, and ACREB together with reporter gene plasmids containing 2 kb of wild-type PGC-1α promoter or a promoter with a mutation in the CRE site (ΔCRE), respectively. Cells subsequently were treated with either vehicle (0.1% DMSO) or 100 μM forskolin for 10 h and harvested 48 h after transfection before luciferase activity was determined. The values represent the average of three independent experiments, and bars represent SD. *, P < 0.5 between different treatments compared with the untreated control.

Fig. 2.

PGC-1α coactivates MEF2s on the PGC-1α promoter. (A) MEF2C and MEF2D activate the mouse PGC-1α promoter. C2C12 cells were cotransfected with expression plasmids for MEF2C, MEF2D, NFAT, and PGC-1α together with reporter gene plasmids containing 2 kb of the mouse PGC-1α promoter. After 48 h, cells were harvested and luciferase activity was determined. (B) MEF2 activity is increased by CnA. C2C12 cells were cotransfected with expression plasmids for MEF2C, MEF2D, NFAT, CnA, and PGC-1α together with reporter gene plasmids containing 6 kb of the mouse PGC-1α promoter. After 48 h, cells were harvested and luciferase activity was determined. The values represent the average of three independent experiments, and bars represent SD. *, P < 0.5 between different treatments compared with the untreated control.

Fig. 3.

MEF2C and MEF2D activate the PGC-1α promoter via a conserved binding site. (A) Identification of putative MEF2-binding site (depicted in bold and underlined) in the human and mouse PGC-1α promoter. N = A, T, C, or G; K = G or T; Y = C or T. (B) MEF2C and MEF2D activate an MEF-binding site in the PGC-1α promoter. C2C12 cells were cotransfected with expression plasmids for MEF2C, MEF2D, CnA, and PGC-1α together with reporter gene plasmids containing 2 kb of wild-type mouse PGC-1α promoter or 2 kb of the promoter with a mutation in the MEF2-binding site (ΔMEF2). After 48 h, cells were harvested and luciferase activity was determined. (C) MEF2C binds to the MEF-binding site in the PGC-1α promoter. Electrophoretic mobility-shift assays were performed by using in vitro transcribed/translated MEF2C and radiolabeled probe encoding the MEF-binding site from the PGC-1α promoter or the mutated binding site, ΔMEF. Antibodies against HNF3β and MEF2 were used to test for the specificity of the protein–DNA interactions. (D and E) PGC-1α interacts with MEF2C on the MEF-binding site. Increasing amounts of PGC-1α protein including amino acids 31–797 (0.8, 2, 4, and 6 μg, respectively) were added to the reactions, resulting in a larger protein–DNA complex and thus a supershift (D). As a control, 6 μg of PGC-1α protein including amino acids 1–180 that lacks the MEF2–interaction domain was not able to interact with MEF2C (E). The protein–DNA complex of PGC-1α, MEF2C, and the MEF-binding site could be supershifted further when adding anti-MEF2 antibody. The values represent the average of three independent experiments, and bars represent SD. *, P < 0.5 between different treatments compared with the untreated control.

Fig. 4.

A model for the autoregulatory loop regulating the PGC-1α promoter in muscle fiber-type determination. (A) Exercise and subsequently elevated intracellular calcium levels result in an activation of both CaMKIV and CnA in skeletal muscle. Activated CaMKIV can phosphorylate CREB, which then increases transcription of PGC-1α via a conserved CREB-binding site in the proximal promoter. Moreover, CaMKIV and CnA activate the transcriptional activity of MEF2s in part by promoting the dissociation of inhibitory HDACs and Cabin1. MEF2s, potentially in combination with NFAT, bind to at least one MEF2-binding site in the PGC-1α flanking region and increase transcriptional activity. Newly synthesized PGC-1α protein can coactivate MEF2s and thus positively regulate its own transcription. PGC-1α also might compete with the inhibitory HDACs and Cabin 1 for binding to MEF2s. Together, this positive feedback loop may ensure a stable transcription of PGC-1α, leading to muscle fiber-type I determination. (B) Endogenous PGC-1α expression is increased in transgenic mice expressing ectopic PGC-1α in skeletal muscle. Total RNA from wild-type and transgenic skeletal muscle was analyzed for the expression of transgenic and endogenous PGC-1α, GAPDH, myoglobin, and cytochrome c by using real-time PCR. Relative mRNA expression levels were normalized to 18S rRNA levels. nd, not detectable. The values represent the average of three independent experiments, and bars represent SD. *, P < 0.5 between different treatments compared with the untreated control.

Fig. 5.

PGC-1α autoregulation can be inhibited by dnMEF2C. (A) dnMEF2C completely blocks the activation of the PGC-1α promoter by PGC-1α. C2C12 cells were cotransfected with expression plasmids for CaMKIV, CnA, ACREB, dnMEF2C, and PGC-1α together with a reporter gene plasmid containing 2 kb of the mouse PGC-1α promoter. After 48 h, cells were harvested and luciferase activity was determined. (B–E) dnMEF2C adenovirus blocks induction of PGC-1α mRNA. C2C12 myoblasts were infected with adenovirus containing GFP, PGC-1α, MEF2C, dnMEF2C, or combinations thereof. After 5 days of differentiation into myotubes, cells were harvested and mRNA levels of endogenous PGC-1α (B), myoglobin (C), troponin I slow (TnI slow, D), and the glucose transporter GLUT4 (D) were determined. Relative expression levels were normalized to 18S rRNA levels. nd, not detectable. The values represent the average of at least three independent experiments, and bars represent SD. *, P < 0.5 between different treatments compared with the untreated control unless otherwise indicated.