Activation of the beta myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway.
Calcium is a key element in intracellular signaling in skeletal muscle. Changes in intracellular calcium levels are thought to mediate the fast-to-slow transformation of muscle fiber type. One factor implicated in gene regulation in adult muscle is the nuclear factor of activated T-cells (NFAT) isoform c1, whose dephosphorylation by the calcium/calmodulin-dependent phosphatase calcineurin facilitates its nuclear translocation. Here, we report that differentiated C2C12 myotubes predominantly expressing fast-type MyHCII protein undergo fast-to-slow transformation following calcium-ionophore treatment, with several transcription factors and a transcriptional coactivator acting in concert to upregulate the slow myosin heavy chain (MyHC) beta promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCbeta promoter activation during transformation of C2C12 myotubes but is not sufficient for complete fast MyHCIId/x promoter inhibition. Along with NFATc1, myocyte enhancer factor-2D (MEF-2D) and the myogenic transcription factor MyoD transactivated the MyHCbeta promoter in calcium-ionophore-treated myotubes in a calcineurin-dependent manner. To elucidate the mechanism involved in regulating MyHCbeta gene expression, we analyzed the -2.4-kb MyHCbeta promoter construct for cis-regulatory elements. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation assays (ChIP), and nuclear complex coimmunoprecipitation (NCcoIP) assays, we demonstrated calcium-ionophore-induced binding of NFATc1 to a NFAT consensus site adjacent to a MyoD-binding E-box. At their respective binding sites, both NFATc1 and MyoD recruited the transcriptional coactivator p300, and in turn, MEF-2D bound to the MyoD complex. The calcium-ionophore-induced effects on the MyHCbeta promoter were shown to be calcineurin-dependent. Together, our findings demonstrate calcium-ionophore-induced activation of the beta MyHC promoter by NFATc1, MyoD, MEF-2D, and p300 in a calcineurin-dependent manner.
Meissner
Joachim D
JD
Department of Physiology, Hannover Medical School, Hannover, Germany. meissner.joachim@mh-hannover.de
Umeda
Patrick K
PK
Chang
Kin-Chow
KC
Gros
Gerolf
G
Scheibe
Renate J
RJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D016384:Consensus Sequence; D007476:Ionophores / Q000494:pharmacology; D064326:MEF2 Transcription Factors; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects; D017570:MyoD Protein / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects; D018006:Myogenic Regulatory Factors / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D050880:p300-CBP Transcription Factors / Q000378:metabolism*
D007476:Ionophores; D064326:MEF2 Transcription Factors; C578044:Mef2d protein, mouse; D017570:MyoD Protein; D018006:Myogenic Regulatory Factors; D050778:NFATC Transcription Factors; D012333:RNA, Messenger; D050880:p300-CBP Transcription Factors; D019703:Calcineurin; D018995:Myosin Heavy Chains; D002118:Calcium