CREB-1α Is Recruited to and Mediates Upregulation of the Cytochrome c Promoter during Enhanced Mitochondrial Biogenesis Accompanying Skeletal Muscle Differentiation

Abstract

To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energydemanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1⌬ in myoblasts to phosphorylated CREB-1␣ protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1␣ in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis.In mammals, mitochondria are composed of at least 1,000 proteins, including components of the inner membrane electron transport and oxidative phosphorylation system (OXPHOS), metabolite carriers, matrix enzymes, subunits of the protein import machineries, factors necessary for replication and expression of the small mitochondrial DNA (mtDNA) genome, and components of the mitochondrial protein biosynthesis machinery (5). To synthesize these proteins in a reasonably economical way, it is essential to orchestrate the expression of their genes, which are predominantly located on nuclear chromosomes, and coordinate it with the expression of mtDNA. As both ATP demand and mitochondrial content are very different in the various cell types of the body and can change even in terminally differentiated cells, these regulatory mechanisms must operate during developmental programs as well as in adaptation processes in the adult. Indeed, cells are able to adjust energy metabolism by altering the architecture and dynamics of the mitochondrial reticulum (10), by modifying its enzyme equipment and/or the level of proton leak, or by adjusting total mitochondrial respiratory capacity when changes in energy demand persist for long periods (23). Among the factors known to strongly stimulate mitochondrial biogenesis in vivo, the most prominent examples are high levels of thyroid (67) and glucocorticoid (55, 66) hormones and also conditions like endurance exercise of muscle (1) and cold adaptation in brown fat tissue (31).Wh...

Mitochondrial H2O2 generated from electron transport chain complex I stimulates muscle differentiation

“…TFAM is encoded by nuclear DNA and regulates mitochondrial biogenesis and mtDNA transcription [1,28]. In this study, mitochondrial alterations in response to induced TFAM, such as mtDNA activation and mitochondrial biogenesis, were found to be critical for muscle differentiation ( Figure 5).…”

“…Mitochondrial transcription factor A (TFAM) is encoded by nuclear DNA and regulates mitochondrial biogenesis and mitochondrial DNA (mtDNA) transcription [1,28]. The mitochondrial genome encodes some mETC complex subunits, such as ND1-6 and ND4L of complex I, Cyt b of complex III, COX I-III of complex IV, and ATPase 6 and 8 of complex V, along with mitochondrial rRNA and tRNA [29].…”

An active mitochondrial biogenesis occurs during dendritic cell differentiation

“…1A), proteins derived from an equal number of nuclei were loaded. This WB analysis method, currently used by others (Franko et al, 2008), can indeed take into account the protein synthesis occurring during DC differentiation and demonstrate the actual increase in mitochondrial proteins per nuclear gene copy. From this analysis, a statistically significant increase in the protein level of NDFUS3 of Complex I (1.79 fold), Core I of Complex III (2.28 fold), and Cox IV of Complex IV (2.07 fold) (Fig.…”

Regulation of SIK1 abundance and stability is critical for myogenesis

“…Although cAMP signaling and CREB-dependent transcription are dynamically regulated during myogenesis in vivo and in vitro (2,13,(35)(36)(37)(38), relatively little is known about molecular mechanisms by which cAMP-dependent transcription contributes to normal responses in myoblasts and adult skeletal muscle (5). We previously identified one cAMP effector pathway in myocytes mediated by the CREB target gene SIK1, which we showed inhibits class II HDACs and allows activation of MEF2-dependent transcription in skeletal muscle cells (14).…”