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All rights reserved. http://resource.belframework.org/belframework/1.0/knowledge/large_corpus.bel http://purl.org/dc/elements/1.1/title BEL Framework Large Corpus Document http://resource.belframework.org/belframework/1.0/knowledge/large_corpus.bel http://purl.org/pav/authoredBy http://www.tkuhn.ch/bel2nanopub/RAe4v6ara99B6xzds1yjprTYfLDTxGOiOnLtDS14FyEzI#_8 http://resource.belframework.org/belframework/1.0/knowledge/large_corpus.bel http://purl.org/pav/version 1.4 http://www.tkuhn.ch/bel2nanopub/RAe4v6ara99B6xzds1yjprTYfLDTxGOiOnLtDS14FyEzI#_7 http://www.w3.org/ns/prov#value This, in turn, would lead to activation of the CREB and MEF2 proteins, which then would bind to and potentially activate the PGC-1-alpha promoter. Of course, MEF2 bound to the PGC-1-alpha promoter is also a target for PGC-1-alpha coactivation, resulting in a stable, feed-forward regulatory loop. A key feature of this model is that PGC-1-alpha should be a regulator of its own expression in skeletal muscle. To critically test this hypothesis, we examined whether ectopic PGC-1-alpha expressed in the muscle of transgenic mice activates expression of the endogenous PGC-1-alpha gene. Real-time PCR primers were designed for the PGC-1-alpha 3 untranslated region that allows for determination of the levels of ectopically expressed and endogenous PGC-1-alpha . As shown in Fig. 4B and as expected, ectopic PGC-1-alpha is detected only in the muscle of transgenic mice. Strikingly, a 7-fold elevation of endogenous PGC-1-alpha mRNA was seen in the transgenic animals as compared with wild-type mice (Fig. 4B). A robust increase in the transcript levels of cytochrome c and myoglobin also was observed in the RNA isolated from skeletal muscle of the transgenic mice, whereas GAPDH transcript levels remained unchanged (Fig. 4B). These results indicate a regulation of PGC-1-alpha transcription by PGC-1-alpha protein in an apparent autoregulatory loop. As a second critical test of our proposed model, we examined whether inhibiting MEF2 andor CREB activity in transfections can decrease the transcription of the PGC-1-alpha promoter stimulated by CaMKIV, CnA, and PGC-1-alpha . To do this, we used a previously characterized dominant-negative allele of MEF2C in addition to ACREB (26). As shown in Fig. 5A, dnMEF2C reduces the effects of the combination of CnA and CaMKIV, and completely eliminates the effect of PGC-1-alpha on the PGC-1-alpha Fig. 2. PGC-1-alpha coactivates MEF2s on the PGC-1-alpha promoter. (A) MEF2C and MEF2D activate the mouse PGC-1-alpha promoter. C2C12 cells were cotransfected with expression plasmids for MEF2C, MEF2D, NFAT, and PGC-1-alpha together with reporter gene plasmids containing 2 kb of the mouse PGC-1-alpha 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-1-alpha together with reporter gene plasmids containing 6 kb of the mouse PGC-1-alpha 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. promoter. In contrast, the dominant-negative allele of CREB (ACREB) reduces the activation by CnA and CaMKIV but does not affect the relative fold activation by PGC-1-alpha . The effects of MEF2C, PGC-1-alpha , and dnMEF2C on the expression of endogenous PGC-1-alpha mRNA and on other fiber type-I specific genes also were examined. C2C12 cells were infected with adenoviruses encoding GFP, PGC-1-alpha , MEF2C, and dnMEF2C in various combinations. Subsequently, gene expression levels were determined by using real-time PCR. Levels of endogenous PGC-1-alpha mRNA were elevated only slightly in cells infected with PGC-1-alpha alone, and no induction was observed when MEF2C alone was expressed (Fig. 5B). In contrast, a large induction of endogenous PGC-1-alpha mRNA (4-fold) was seen in cells that received a combination of PGC-1-alpha and MEF2C. The induction of endogenous PGC-1-alpha mRNA could be blocked completely by coexpression of dnMEF2C. In all cases, similar amounts of the virally expressed PGC-1-alpha and MEF2C mRNAs were observed (data not shown). Expression of myoglobin in C2C12 myotubes also required infection of both PGC-1-alpha and MEF2C (Fig. 5C). 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