Regulation of Egr-1, SRF, and Sp1 mRNA expression in contracting skeletal muscle cells

The early cellular signals associated with contractile activity initiate the activation and induction of transcription factors that regulate changes in skeletal muscle phenotype. The transcription factors Egr-1, Sp1, and serum response factor (SRF) are potentially important mediators of mitochondrial biogenesis based on the prevalence of binding sites for them in the promoter regions of genes encoding mitochondrial proteins, including PGC-1α, the important regulator of mitochondrial biogenesis. Thus, to further define a role for transcription factors at the onset of contractile activity, we examined the time-dependent alterations in Egr-1, Sp1, and SRF mRNA and the levels in electrically stimulated mouse C2C12 skeletal muscle cells. Early transient increases in Egr-1 mRNA levels within 30 min ( P < 0.05) of contractile activity led to threefold increases ( P < 0.05) in Egr-1 protein by 60 min. The increase in Egr-1 mRNA was not because of increased stability, as Egr-1 mRNA half-life after 30 min of stimulation showed only a 58% decline. Stimulation of muscle cells had no effect on Sp1 mRNA but led to progressive increases ( P < 0.05) in SRF mRNA by 30 and 60 min. This was not matched by increases in SRF protein but occurred coincident with increases ( P < 0.05) in SRF-serum response element DNA binding at 30 and 60 min as a result of SRF phosphorylation on serine-103. To assess the importance of the recovery period, 12 h of continuous contractile activity was compared with four successive 3-h bouts, with an intervening 21-h recovery period after each bout. Continuous contractile activity led to a twofold increase ( P < 0.05) in Egr-1 mRNA, no change in SRF mRNA, and a 43% decrease in Sp1 mRNA expression. The recovery period prevented the decline in Sp1 mRNA, produced a decrease in Egr-1 mRNA, and had no effect on SRF mRNA. Thus continuous and intermittent contractile activity evoked different specific transcription factor expression patterns, which may ultimately contribute to divergent qualitative, or temporal patterns of, phenotypic adaptation in muscle.

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Invited Review: Contractile activity-induced mitochondrial biogenesis in skeletal muscle

Journal of Applied Physiology ◽

10.1152/jappl.2001.90.3.1137 ◽

2001 ◽

Vol 90 (3) ◽

pp. 1137-1157 ◽

Cited By ~ 464

Author(s):

David A. Hood

Keyword(s):

Transcription Factors ◽

Respiratory Chain ◽

Mitochondrial Biogenesis ◽

Adenine Nucleotide ◽

Contractile Activity ◽

Recovery Period ◽

Atp Synthesis ◽

Nucleotide Metabolism ◽

Significant Shift ◽

Mrna Levels

Chronic contractile activity produces mitochondrial biogenesis in muscle. This adaptation results in a significant shift in adenine nucleotide metabolism, with attendant improvements in fatigue resistance. The vast majority of mitochondrial proteins are derived from the nuclear genome, necessitating the transcription of genes, the translation of mRNA into protein, the targeting of the protein to a mitochondrial compartment via the import machinery, and the assembly of multisubunit enzyme complexes in the respiratory chain or matrix. Putative signals involved in initiating this pathway of gene expression in response to contractile activity likely arise from combinations of accelerations in ATP turnover or imbalances between mitochondrial ATP synthesis and cellular ATP demand, and Ca2+ fluxes. These rapid events are followed by the activation of exercise-responsive kinases, which phosphorylate proteins such as transcription factors, which subsequently bind to upstream regulatory regions in DNA, to alter transcription rates. Contractile activity increases the mRNA levels of nuclear-encoded proteins such as cytochrome c and mitochondrial transcription factor A (Tfam) and mRNA levels of upstream transcription factors like c- junand nuclear respiratory factor-1 (NRF-1). mRNA level changes are often most evident during the postexercise recovery period, and they can occur as a result of contractile activity-induced increases in transcription or mRNA stability. Tfam is imported into mitochondria and controls the expression of mitochondrial DNA (mtDNA). mtDNA contributes only 13 protein products to the respiratory chain, but they are vital for electron transport and ATP synthesis. Contractile activity increases Tfam expression and accelerates its import into mitochondria, resulting in increased mtDNA transcription and replication. The result of this coordinated expression of the nuclear and the mitochondrial genomes, along with poorly understood changes in phospholipid synthesis, is an expansion of the muscle mitochondrial reticulum. Further understanding of 1) regulation of mtDNA expression, 2) upstream activators of NRF-1 and other transcription factors, 3) the identity of mRNA stabilizing proteins, and 4) potential of contractile activity-induced changes in apoptotic signals are warranted.

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NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00195.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. C715-C725 ◽

Cited By ~ 26

Author(s):

Juan Antonio Valdés ◽

Eduardo Gaggero ◽

Jorge Hidalgo ◽

Nancy Leal ◽

Enrique Jaimovich ◽

...

Keyword(s):

Skeletal Muscle ◽

Transcription Factors ◽

Electrical Stimulation ◽

Stimulus Frequency ◽

Muscle Cells ◽

C2c12 Cells ◽

Skeletal Muscle Cells ◽

Mrna Levels ◽

Activated T Cells ◽

Nfat Activation

Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c- fos, c- jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-κB activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-κB activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-κB and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels.

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Selected Contribution: Effects of contractile activity on mitochondrial transcription factor A expression in skeletal muscle

Journal of Applied Physiology ◽

10.1152/jappl.2001.90.1.389 ◽

2001 ◽

Vol 90 (1) ◽

pp. 389-396 ◽

Cited By ~ 102

Author(s):

Joe W. Gordon ◽

Arne A. Rungi ◽

Hidetoshi Inagaki ◽

David A. Hood

Keyword(s):

Skeletal Muscle ◽

Transcription Factor ◽

Mitochondrial Biogenesis ◽

Contractile Activity ◽

Regulatory Protein ◽

Mrna Levels ◽

Mitochondrial Transcription ◽

Mitochondrial Transcription Factor ◽

Mitochondrial Transcription Factor A ◽

Mitochondrial Transcript

Mitochondrial transcription factor A (Tfam) is a nuclear-encoded gene product that is imported into mitochondria and is required for the transcription of mitochondrial DNA (mtDNA). We hypothesized that conditions known to produce mitochondrial biogenesis in skeletal muscle would be preceded by an increase in Tfam expression. Therefore, rat muscle was stimulated (10 Hz, 3 h/day). Tfam mRNA levels were significantly elevated (by 55%) at 4 days and returned to control levels at 14 days. Tfam import into intermyofibrillar (IMF) mitochondria was increased by 52 and 61% ( P < 0.05) at 5 and 7 days, respectively. This corresponded to an increase in the level of import machinery components. Immunoblotting data indicated that IMF Tfam protein content was increased by 63% ( P < 0.05) at 7 days of stimulation. This was associated with a 49% ( P < 0.05) increase in complex formation at the mtDNA promoter and a 65% ( P< 0.05) increase in the levels of a mitochondrial transcript, cytochrome- c oxidase (COX) subunit III. Similarly, COX enzyme activity was elevated by 71% ( P < 0.05) after 7 days of contractile activity. These results indicate that early events in mitochondrial biogenesis include increases in Tfam mRNA, followed by accelerations in mitochondrial import and increased Tfam content, which correspond with increased binding to the mtDNA promoter region. This was accompanied by increased mitochondrial transcript levels and elevated COX activity. These data support the role of Tfam as a regulatory protein involved in contractile activity-induced mitochondrial biogenesis.

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Roles of lactate in lactate oxidation complex, mitochondrial biogenesis and cell signaling in cultured L6 skeletal muscle cells

Japanese Journal of Physical Fitness and Sports Medicine ◽

10.7600/jspfsm.57.83 ◽

2008 ◽

Vol 57 (1) ◽

pp. 83-83 ◽

Cited By ~ 1

Author(s):

Takeshi Hashimoto ◽

Rajaa Hussien ◽

Saji Oommen ◽

Kishorchandra Gohil ◽

George A. Brooks

Keyword(s):

Skeletal Muscle ◽

Cell Signaling ◽

Mitochondrial Biogenesis ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Lactate Oxidation ◽

L6 Skeletal Muscle Cells

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Interleukin-6 promotes myogenic differentiation of mouse skeletal muscle cells: role of the STAT3 pathway

AJP Cell Physiology ◽

10.1152/ajpcell.00025.2012 ◽

2013 ◽

Vol 304 (2) ◽

pp. C128-C136 ◽

Cited By ~ 55

Author(s):

Miriam Hoene ◽

Heike Runge ◽

Hans Ulrich Häring ◽

Erwin D. Schleicher ◽

Cora Weigert

Keyword(s):

Skeletal Muscle ◽

Mrna Expression ◽

Myogenic Differentiation ◽

Muscle Cells ◽

C2c12 Cells ◽

Skeletal Muscle Cells ◽

Wild Type ◽

C2c12 Myoblasts ◽

Sequence Of Events ◽

Primary Mouse

Myogenic differentiation of skeletal muscle cells is characterized by a sequence of events that include activation of signal transducer and activator of transcription 3 (STAT3) and enhanced expression of its target gene Socs3. Autocrine effects of IL-6 may contribute to the activation of the STAT3-Socs3 cascade and thus to myogenic differentiation. The importance of IL-6 and STAT3 for the differentiation process was studied in C2C12 cells and in primary mouse wild-type and IL-6−/− skeletal muscle cells. In differentiating C2C12 myoblasts, the upregulation of IL-6 mRNA expression and protein secretion started after increased phosphorylation of STAT3 on tyrosine 705 and increased mRNA expression of Socs3 was observed. Knockdown of STAT3 and IL-6 mRNA in differentiating C2C12 myoblasts impaired the expression of the myogenic markers myogenin and MyHC IIb and subsequently myotube fusion. However, the knockdown of IL-6 did not prevent the induction of STAT3 tyrosine phosphorylation. The IL-6-independent activation of STAT3 was verified in differentiating primary IL-6−/− myoblasts. The phosphorylation of STAT3 and the expression levels of STAT3, Socs3, and myogenin during differentiation were comparable in the primary myoblasts independent of the genotype. However, IL-6−/− cells failed to induce MyHC IIb expression to the same level as in wild-type cells and showed reduced myotube formation. Supplementation of IL-6 could partially restore the fusion of IL-6−/− cells. These data demonstrate that IL-6 depletion during myogenic differentiation does not reduce the activation of the STAT3-Socs3 cascade, while IL-6 and STAT3 are both necessary to promote myotube fusion.

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Palmitate Induces Tumor Necrosis Factor-α Expression in C2C12 Skeletal Muscle Cells by a Mechanism Involving Protein Kinase C and Nuclear Factor-κB Activation

Endocrinology ◽

10.1210/en.2005-0440 ◽

2006 ◽

Vol 147 (1) ◽

pp. 552-561 ◽

Cited By ~ 110

Author(s):

Mireia Jové ◽

Anna Planavila ◽

Rosa M. Sánchez ◽

Manuel Merlos ◽

Juan Carlos Laguna ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Kinase C ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Mrna Levels ◽

Nuclear Factor ◽

Kinase C ◽

Tnf Α ◽

Fold Induction ◽

C2c12 Skeletal Muscle Cells

The mechanisms responsible for increased expression of TNF-α in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-α expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-α. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-α expression. Palmitate increased nuclear factor (NF)-κB activation and incubation of the cells with the NF-κB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-α expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-α expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCθ, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IκBα and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-α expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.

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