scholarly journals Actions of 1,25(OH)2-vitamin D3 on the cellular cycle depend on VDR and p38 MAPK in skeletal muscle cells

2014 ◽  
Vol 53 (3) ◽  
pp. 331-343 ◽  
Author(s):  
Ana P Irazoqui ◽  
Ricardo L Boland ◽  
Claudia G Buitrago
Keyword(s):  
Skeletal Muscle ◽  
Vitamin D ◽  
P38 Mapk ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Cyclin D3 ◽  
C2c12 Myoblast ◽  
G1 Arrest ◽  
Dependent Manner

Previously, we have reported that 1,25(OH)2-vitamin D3(1,25D) activates p38 MAPK (p38) in a vitamin D receptor (VDR)-dependent manner in proliferative C2C12 myoblast cells. It was also demonstrated that 1,25D promotes muscle cell proliferation and differentiation. However, we did not study these hormone actions in depth. In this study we have investigated whether the VDR and p38 participate in the signaling mechanism triggered by 1,25D. In C2C12 cells, the VDR was knocked down by a shRNA, and p38 was specifically inhibited using SB-203580. Results from cell cycle studies indicated that hormone stimulation prompts a peak of S-phase followed by an arrest in the G0/G1-phase, events which were dependent on VDR and p38. Moreover, 1,25D increases the expression of cyclin D3 and the cyclin-dependent kinase inhibitors, p21Waf1/Cip1and p27Kip1, while cyclin D1 protein levels did not change during G0/G1 arrest. In all these events, p38 and VDR were required. At the same time, a 1,25D-dependent acute increase in myogenin expression was observed, indicating that the G0/G1 arrest of cells is a pro-differentiative event. Immunocytochemical assays revealed co-localization of VDR and cyclin D3, promoted by 1,25D in a p38-dependent manner. When cyclin D3 expression was silenced, VDR and myogenin levels were downregulated, indicating that cyclin D3 was required for 1,25D-induced VDR expression and the concomitant entrance into the differentiation process. In conclusion, the VDR and p38 are involved in control of the cellular cycle by 1,25D in skeletal muscle cells, providing key information on the mechanisms underlying hormone regulation of myogenesis.

scholarly journals Extracellular-regulated kinase and p38 mitogen-activated protein kinases are involved in the antiapoptotic action of 17β-estradiol in skeletal muscle cells

2010 ◽  
Vol 206 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Ana Carolina Ronda ◽  
Andrea Vasconsuelo ◽  
Ricardo Boland
Keyword(s):  
Skeletal Muscle ◽  
P38 Mapk ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Mitogen Activated Protein Kinases ◽  
Mitogen Activated Protein ◽  
17Β Estradiol ◽  
Mapk Inhibitors ◽  
Extracellular Regulated Kinase

17β-Estradiol (E2) stimulates the mitogen-activated protein kinases (MAPKs) in various cellular types. We have shown that the hormone activates extracellular-regulated kinase (ERK) and p38 MAPK in skeletal muscle cells. However, the functions of MAPK modulation by the estrogen in muscle cells have not been studied yet. We have recently reported antiapoptotic actions of E2 in C2C12 cells. Here, the role of MAPKs mediating the hormone effect in muscle cells was investigated. The results showed that cells exposed to 0.5 mM hydrogen peroxide (H2O2) presented cytoskeleton disorganization, mitochondrial redistribution, and picnotic/fragmented nuclei. Pretreatment with 10−8 M E2 prevented these morphological apoptotic characteristics, except in the presence of ERK or p38 MAPK inhibitors, U0126 and SB203580 respectively. Mitochondrial membrane integrity was also studied. Preincubation of cultures with 10−8 M E2 abrogated H2O2 effects such as Janus Green oxidation, presence of cytochrome c oxidase activity in the cytoplasm, and SMAC/DIABLO release from mitochondria. When MAPKs were inhibited, the hormone could not prevent mitochondrial membrane damage exerted by oxidative stress. Blocking experiments with small interfering RNAs confirmed that both ERK and p38 MAPKs mediate the antiapoptotic effects of the hormone at the mitochondrial level. Further, some of the molecular mechanisms involved were also investigated. Thus, E2 was able to induce AKT (Ser473) and BAD (Ser112) phosphorylation in C2C12 cells in the absence or in the presence of H2O2 but not when the cultures were incubated with H2O2 and MAPK inhibitors. Altogether, these results show that E2 exerts a survival action in skeletal muscle cells involving ERK and p38 MAPK activation.

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

2013 ◽  
Vol 304 (2) ◽  
pp. C128-C136 ◽  
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.

scholarly journals 17β-Estradiol abrogates apoptosis in murine skeletal muscle cells through estrogen receptors: role of the phosphatidylinositol 3-kinase/Akt pathway

2007 ◽  
Vol 196 (2) ◽  
pp. 385-397 ◽  
Author(s):  
Andrea Vasconsuelo ◽  
Lorena Milanesi ◽  
Ricardo Boland
Keyword(s):  
Skeletal Muscle ◽  
Protective Action ◽  
Muscle Cells ◽  
Cellular Systems ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Akt Pathway ◽  
Akt Activation ◽  
17Β Estradiol ◽  
Murine Skeletal Muscle

Estrogens can regulate apoptosis in various cellular systems. The present study shows that 17β-estradiol (E2), at physiological concentrations, abrogates DNA damage, poly (ADP-ribose) polymerase cleavage, and mitochondrial cytochrome c release induced by H2O2 or etoposide in mouse skeletal muscle C2C12 cells. This protective action, which involved PI3K/Akt activation and Bcl-2 associated death agonist (BAD) phosphorylation, was inhibited by antibodies against the estrogen receptor (ER) α or β isoforms, or transfecting siRNA specific for each isoform. The inhibition of the antiapoptotic action of E2 at the mitochondrial level was more pronounced when ER-β was immunoneutralized or suppressed by mRNA silencing, whereas transfection of C2C12 cells with either ER-α siRNA or ER-β siRNA blocked the activation of Akt by E2, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway evaluated. These results indicate that E2 exerts antiapoptotic effects in skeletal muscle cells which are mediated by ER-β and ER-α and involve the PI3K/Akt pathway.

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

2008 ◽  
Vol 294 (3) ◽  
pp. C715-C725 ◽  
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.

scholarly journals Alignment of Skeletal Muscle Cells Cultured in Collagen Gel by Mechanical and Electrical Stimulation

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Takara Tanaka ◽  
Noriko Hattori-Aramaki ◽  
Ayano Sunohara ◽  
Keisuke Okabe ◽  
Yoshiaki Sakamoto ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tissue Engineering ◽  
Electrical Stimulation ◽  
Collagen Gel ◽  
Muscle Cells ◽  
Mechanical Force ◽  
C2c12 Cells ◽  
Skeletal Muscle Cells ◽  
Collagen Gels ◽  
Cells Cultured

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method.

scholarly journals Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

2021 ◽  
Vol 22 (16) ◽  
pp. 8610
Author(s):  
Nives Škorja Milić ◽  
Klemen Dolinar ◽  
Katarina Miš ◽  
Urška Matkovič ◽  
Maruša Bizjak ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cancer Cells ◽  
Pyruvate Dehydrogenase ◽  
Hypoxia Inducible Factor ◽  
Muscle Cells ◽  
Pyruvate Dehydrogenase Kinase ◽  
Skeletal Muscle Cells ◽  
Dependent Manner ◽  
Protein Levels ◽  
L6 Myotubes

Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.

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