Joachim D. Meissner

Activation of the β 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) β promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCβ 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 MyHCβ promoter in calcium‐ionophore‐treated myotubes in a calcineurin‐dependent manner. To elucidate the mechanism involved in regulating MyHCβ gene expression, we analyzed the −2.4‐kb MyHCβ 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 MyHCβ promoter were shown to be calcineurin‐dependent. Together, our findings demonstrate calcium‐ionophore‐induced activation of the β MyHC promoter by NFATc1, MyoD, MEF‐2D, and p300 in a calcineurin‐dependent manner. J. Cell. Physiol. 211: 138–148, 2007.

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/β gene expression via acetylation of nuclear factor of activated T cells c1

The nuclear factor of activated T-cells (NFAT) c1 has been shown to be essential for Ca2+-dependent upregulation of myosin heavy chain (MyHC) I/β expression during skeletal muscle fiber type transformation. Here, we report activation of extracellular signal-regulated kinase (ERK) 1/2 in Ca2+-ionophore-treated C2C12 myotubes and electrostimulated soleus muscle. Activated ERK1/2 enhanced NFATc1-dependent upregulation of a −2.4 kb MyHCI/β promoter construct without affecting subcellular localization of endogenous NFATc1. Instead, ERK1/2-augmented phosphorylation of transcriptional coactivator p300, promoted its recruitment to NFATc1 and increased NFATc1–DNA binding to a NFAT site of the MyHCI/β promoter. In line, inhibition of ERK1/2 signaling abolished the effects of p300. Comparison between wild-type p300 and an acetyltransferase-deficient mutant (p300DY) indicated increased NFATc1–DNA binding as a consequence of p300-mediated acetylation of NFATc1. Activation of the MyHCI/β promoter by p300 depends on two conserved acetylation sites in NFATc1, which affect DNA binding and transcriptional stimulation. NFATc1 acetylation occurred in Ca2+-ionophore treated C2C12 myotubes or electrostimulated soleus. Finally, endogenous MyHCI/β gene expression in C2C12 myotubes was strongly inhibited by p300DY and a mutant deficient in ERK phosphorylation sites. In conclusion, ERK1/2-mediated phosphorylation of p300 is crucial for enhancing NFATc1 transactivation function by acetylation, which is essential for Ca2+-induced MyHCI/β expression.

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid

The activity of membrane‐bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL‐60 myeloblastic leukemia cells was studied at physiological pH using p‐nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4–8.5. The value of apparent Km for pNPP in P19 and HL‐60 cells was 120 μM. Hydrolytic activity of the ecto‐enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (Ki P19 = 57 μM; Ki HL‐60 = 50 μM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL‐60 cells, induced levamisole‐sensitive ecto‐phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto‐kinase activity, a 98‐kDa membrane protein in P19 cells was found to be sensitive to ecto‐ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole‐sensitive dephosphorylation of the membrane phosphoproteins, while (R)‐(−)‐epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole‐sensitive phosphohydrolase activity on the cell surface is consistent with ecto‐ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto‐ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL‐60 myeloblastic leukemia cells. J. Cell. Biochem. 76:420–436, 2000.

Mitogen-Activated Protein Kinase-Activated Protein Kinases 2 and 3 Regulate SERCA2a Expression and Fiber Type Composition To Modulate Skeletal Muscle and Cardiomyocyte Function

ABSTRACT The mitogen-activated protein kinase (MAPK)-activated protein kinases 2 and 3 (MK2/3) represent protein kinases downstream of the p38 MAPK. Using MK2/3 double-knockout (MK2/3−/−) mice, we analyzed the role of MK2/3 in cross-striated muscle by transcriptome and proteome analyses and by histology. We demonstrated enhanced expression of the slow oxidative skeletal muscle myofiber gene program, including the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α). Using reporter gene and electrophoretic gel mobility shift assays, we demonstrated that MK2 catalytic activity directly regulated the promoters of the fast fiber-specific myosin heavy-chain IId/x and the slow fiber-specific sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) gene. Elevated SERCA2a gene expression caused by a decreased ratio of transcription factor Egr-1 to Sp1 was associated with accelerated relaxation and enhanced contractility in MK2/3−/− cardiomyocytes, concomitant with improved force parameters in MK2/3−/− soleus muscle. These results link MK2/3 to the regulation of calcium dynamics and identify enzymatic activity of MK2/3 as a critical factor for modulating cross-striated muscle function by generating a unique muscle phenotype exhibiting both reduced fatigability and enhanced force in MK2/3−/− mice. Hence, the p38-MK2/3 axis may represent a novel target for the design of therapeutic strategies for diseases related to fiber type changes or impaired SERCA2 function.

Calcineurin differentially regulates fast myosin heavy chain genes in oxidative muscle fibre type conversion

In skeletal muscle, calcineurin is crucial for myocyte differentiation and in the determination of the slow oxidative fibre phenotype, both processes being important determinants of muscle performance, metabolic health and meat-animal production. Fibre type is defined by the isoform identity of the skeletal myosin heavy chain (MyHC). We have examined the responses of the major MyHC genes to calcineurin signalling during fibre formation of muscle C2C12 cells. We have found that calcineurin acts as a signal to up-regulate the fast-oxidative MyHC2a gene and to down-regulate the faster MyHC2x and MyHC2b genes in a manner that appears to be NFAT-independent. Contrary to expectation, the up-regulation of MyHCslow by calcineurin seems to be time-dependent and is only detectable once the initial differential expression of the post-natal fast MyHC genes has been established. The simultaneous elevated expression of MyHC2a and the repression of MyHC2x and MyHC2b expression indicate that both processes (elevation and repression) are actively coordinated during oxidative fibre conversion. We have further determined that muscle LIM protein (MLP), a calcineurin-binding Z-line co-factor, is induced by calcineurin and that its co-expression with calcineurin has an additive effect on MyHCslow expression. Hence, post-natal fast MyHCs are important early effector targets of calcineurin, whereas MyHCslow up-regulation is mediated in part by calcineurin-induced MLP.

Chapter 2. Calcineurin signaling and the slow oxidative skeletal muscle fiber type.

Calcineurin, also known as protein phosphatase 2B (PP2B), is a calcium-calmodulin-dependent phosphatase. It couples intracellular calcium to dephosphorylate selected substrates resulting in diverse biological consequences depending on cell type. In mammals, calcineurins functions include neuronal growth, development of cardiac valves and hypertrophy, activation of lymphocytes, and the regulation of ion channels and enzymes. This chapter focuses on the key roles of calcineurin in skeletal muscle differentiation, regeneration, and fiber type conversion to an oxidative state, all of which are crucial to muscle development, metabolism, and functional adaptations. It seeks to integrate the current knowledge of calcineurin signaling in skeletal muscle and its interactions with other prominent regulatory pathways and their signaling intermediates to form a molecular overview that could provide directions for possible future exploitations in human metabolic health.

The p38α/β Mitogen-activated Protein Kinases Mediate Recruitment of CREB-binding Protein to Preserve Fast Myosin Heavy Chain IId/x Gene Activity in Myotubes

In skeletal muscle, the transformation of fast into slow fiber type is accompanied by shifts in fiber type-specific gene expression that includes down-regulation of the adult fast fiber myosin heavy chain IId/x (MyHCIId/x) gene. Here, we report that the mitogen-activated protein kinases (MAPKs) p38α/β regulate MyHCIId/x gene expression. Electrical stimulation of rabbit skeletal muscle cells with a slow fiber type activity pattern and treatment of C2C12 myotubes with Ca2+-ionophore inhibited p38α/β MAPKs and reduced fast fiber type MyHC protein expression and promoter activity. Pharmacological inhibition of p38α/β also down-regulated MyHCII gene expression. In controls, binding of the myocyte enhancer factor-2 (MEF-2) isoforms C and D as a heterodimer to a proximal consensus site within the MyHCIId/x promoter and recruitment of a transcriptional coactivator, the CREB-binding protein CBP, were observed. Overexpression of wild type MEF-2C but not of a MEF-2C mutant that cannot be phosphorylated by p38 induced promoter activity. Mutation of the MEF-2-binding site decreased the inducing effect of overexpressed CBP. Inhibition of p38α/β MAPKs abolished CBP binding, whereas enforced induction of p38 by activated MAPK kinase 6 (MKK6EE) enhanced binding of CBP and increased promoter activity. Furthermore, knockdown of endogenous CBP by RNA interference eliminated promoter activation by MEF-2C or MKK6EE. In electrical stimulated and Ca2+-ionophore-treated myotubes, CBP was absent in complex formation at that site. Taken together, the data indicate that p38α/β MAPKs-mediated coactivator recruitment at a proximal MEF-2 site is important for MyHCIId/x gene regulation in skeletal muscle.

Effects of retinoic acid on N-glycosylation and mRNA stability of the liver/bone/kidney alkaline phosphatase in neuronal cells†

Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We investigated the effects of RA on N‐linked glycosylation of the tissue nonspecific liver/bone/kidney‐ type of ALP (L/B/K ALP), on ALP transcripts, and on total protein glycosylation in two neuronal cell lines, P19 and NG108CC15, and in primary cultures of neonatal rat brain. ALP activity was determined in cell extracts and found to be induced by RA. Tunicamycin was used at various concentrations to inhibit protein N‐glycosylation. After treatment of cells with low concentrations (0.1 and 0.125 μg/ml) of tunicamycin for 48 h, uninduced and RA‐induced ALP activity declined while incubation with a protease inhibitor restored activity, indicating that the L/B/K ALP bear N‐linked oligosaccharide chains important for maintaining enzymatic activity. Interestingly, ALP activity in RA‐treated cultures was less inhibited by tunicamycin compared to untreated controls suggesting that RA may have an impact on ALP N‐glycosylation. To investigate effects of RA on ALP glycosylation further, incorporation of [14C]mannose and [35S]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mannosylation and biosynthesis demonstrate that incubation of cells with RA increased [14C]mannose incorporation into ALP molecules. Also, the release of free [14C]mannose from ALP molecules relative to the amount of protein by N‐Glycanase was increased in RA‐treated cultures. In addition, mannosylation of total protein was found to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosylation of the polypeptides. Furthermore, tunicamycin decreased the stability of ALP mRNA, an effect that was reduced by cotreatment with RA. Thus, the degree of N‐glycosylation of the L/B/K ALP as well as mRNA and protein levels of this enzyme are affected by RA. The P19 cell line provides a useful model system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further. J. Cell. Physiol. 182:50–61, 2000.

Gene regulation mediating fiber-type transformation in skeletal muscle cells is partly glucose- and ChREBP-dependent.

Adaptations in the oxidative capacity of skeletal muscle cells can occur under several physiological or pathological conditions. We investigated the effect of increasing extracellular glucose concentration on the expression of markers of energy metabolism in primary skeletal muscle cells and the C2C12 muscle cell line. Growth of myotubes in 25mM glucose (high glucose, HG) compared with 5.55mM led to increases in the expression and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a marker of glycolytic energy metabolism, while oxidative markers peroxisome proliferator-activated receptor γ coactivator 1α and citrate synthase decreased. HG induced metabolic adaptations as are seen during a slow-to-fast fiber transformation. Furthermore, HG increased fast myosin heavy chain (MHC) IId/x but did not change slow MHCI/β expression. Protein phosphatase 2A (PP2A) was shown to mediate the effects of HG on GAPDH and MHCIId/x. Carbohydrate response element-binding protein (ChREBP), a glucose-dependent transcription factor downstream of PP2A, partially mediated the effects of glucose on metabolic markers. The glucose-induced increase in PP2A activity was associated with an increase in p38 mitogen-activated protein kinase activity, which presumably mediates the increase in MHCIId/x promoter activity. Liver X receptor, another possible mediator of glucose effects, induced only an incomplete metabolic shift, mainly increasing the expression of the glycolytic marker. Taken together, HG induces a partial slow-to-fast transformation comprising metabolic enzymes together with an increased expression of MHCIId/x. This work demonstrates a functional role for ChREBP in determining the metabolic type of muscle fibers and highlights the importance of glucose as a signaling molecule in muscle.

Passive mechanical forces upregulate the fast myosin heavy chain IId/x via integrin and p38 MAP-Kinase activation in a primary muscle cell culture.

We have studied the mechanism by which a previously described primary muscle culture growing on microcarriers predominantly expresses fast myosin heavy chain (MHC) IId/x. We have measured MHC IId/x mRNA and protein levels, mRNA of MHC I and markers of muscle metabolism, insulin-like growth factor (IGF)-1 and mechano-growth factor (MGF) transcripts, indicators of the activation of the Akt-mammalian target of rapamycin (mTOR) axis, the p38-, ERK1/2-, and JNK-mitogen-activated protein kinase (MAP) kinase pathways, and of protein phosphatase PP2A, and we have assessed the involvement of integrin. By placing the culture flasks on a rotary shaker, we induce a continuous motion of the culture medium in which the carrier-myotube aggregates are suspended. This motion exerts passive forces on the myotubes that are decisive for the predominance of MHC II expression. These forces act via integrin, which transduces the mechanical signal into activation of PP2A and of p38 MAP-Kinase. The latter presumably is directly responsible for a drastic upregulation of MHC IId/x, whereas MHC I and metabolic markers remain unaffected. At the same time, despite an elevated level of IGF-1 transcription under passive forces, the IGF-1 receptor-Akt-mTOR axis is switched off as evident from the lack of an effect of inhibition of the IGF-1 receptor and from the PP2A-mediated low degree of phosphorylation of Akt and 4E-BP1. Similarly, the ERK1/2- and JNK-MAP kinase pathways are repressed. We conclude that passive stretch exerted on the myotubes by the rotary fluid motion induces a rather selective upregulation of fast MHC II, which goes along with a mild muscle hypertrophy as judged from the amount of protein per cell and is caused by p38 MAP kinase activity elevated via integrin sensing. The direct link between passive stretch and MHC II expression constitutes a novel mechanism, which is expected to become effective physiologically under passive stretch and eccentric contractions of skeletal muscles.