Dietary cholecalciferol regulates the recruitment and growth of skeletal muscle fibers and the expressions of myogenic regulatory factors and the myosin heavy chain in European sea bass larvae.

The aim of this study was to determine whether dietary cholecalciferol affects the recruitment and growth of axial skeletal muscle fibers in first-feeding European sea bass. Larvae were fed diets containing 0.28 (VD-L, low dose), 0.69 (VD-C, control dose), or 3.00 (VD-H, high dose) mg cholecalciferol/kg from 9 to 44 d posthatching (dph). Larvae were sampled at 44 dph for quantification of somatic growth, muscle growth, and muscle growth dynamics and at 22 and 44 dph for the relative quantification of transcripts encoded by genes involved in myogenesis, cell proliferation, and muscle structure. The weight increase of the VD-L-fed larvae was less than that of the VD-H-fed group, whereas that of VD-C-fed larvae was intermediate. The level of expression of genes involved in cell proliferation (PCNA) and early myogenesis (Myf5) decreased between 22 and 44 dph, whereas that of the myogenic determination factor MyoD1 and that of genes involved in muscle structure and function (myosin heavy chain, myosin light chains 2 and 3) increased. Dietary cholecalciferol regulated Myf5, MyoD1, myogenin, and myosin heavy chain gene expression, with a gene-specific shape of response. The maximum hypertrophy of white muscle fibers was higher in larvae fed the VD-C and VD-H diets than in larvae fed the VD-L diet. White muscle hyperplasia was highly stimulated in VD-H-fed larvae compared to VD-L- and VD-C-fed ones. These findings demonstrate a dietary cholecalciferol effect on skeletal muscle growth mechanisms of a Teleost species.

Sodium arsenite delays the differentiation of C2C12 mouse myoblast cells and alters methylation patterns on the transcription factor myogenin.

Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (-473 to +90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at -236 and -126 bp, whereas hypomethylated CpGs were found at -207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.

Expression profile and differential regulation of the Human I-mfa domain-Containing protein (HIC) gene in immune cells.

The Human I-mfa domain-Containing protein, HIC, is a 246 amino acid protein that functions as a transcriptional regulator. Although the precise function of HIC remains to be clarified, the association of the HIC gene locus with myeloid neoplasms, its interactions with lymphotropic viruses such as EBV, HIV-1 and HTLV-1 and its expression in immune tissues suggest that HIC might have a modulatory role in immune cells. To further characterise the HIC functional relationship with the immune system, we sought to analyse the HIC gene expression profile in immune cells and to determine if immunomodulatory cytokines, such as interleukin (IL)-2, could regulate the expression of HIC mRNA. Relative quantitative real-time RT-PCR revealed that HIC mRNA is highly expressed in PBMCs and in various hematopoietic cell lines. The immunomodulatory cytokine IL-2 up-regulated HIC gene expression in PBMCs, CEM, MT-2 and U937 but markedly reduced HIC gene expression in Raji. Addition of cycloheximide indicated that the IL-2 effects were independent of de novo protein synthesis and that the HIC gene is a direct target of IL-2. Two cell lines (Jurkat and BJAB) displayed a distinct loss in HIC gene expression. However, when these cell lines were subjected to a combination of DNA methyltransferase and histone-deacetylase inhibitors, (5-aza-2-deoxycytidine and trichostatin A, respectively), HIC expression was de-repressed, indicating possible epigenetic control of HIC expression. Overall, our study describes that the immune expression of HIC is cell-specific, dynamic, and identifies the HIC gene as an IL-2 responsive gene. Furthermore, our de-repression studies support the hypothesis that HIC might represent a candidate tumor suppressor gene. Overall, this report provides new insights for a putative role of HIC in the modulation of immune and inflammatory responses and/or hematological malignancies.

Metoprolol represses PGC1alpha-mediated carnitine palmitoyltransferase-1B expression in the diabetic heart.

We have previously shown that metoprolol decreases carnitine palmitoyltransferase-1 (CPT-1) activity, a mechanism which may partly explain its beneficial effects in heart failure. It is possible that this effect occurs as a result of repression of cardiac CPT-1B expression. CPT-1B is induced by the transcription factors peroxisome proliferator activated receptor-alpha (PPAR-alpha) and PPAR-gamma-coactivator 1alpha (PGC1alpha) and repressed by upstream stimulatory factor-2 (USF-2). We therefore hypothesized that metoprolol represses CPT-1B by increasing USF-2-mediated repression of PGC1alpha. Male Wistar Rats were divided into 4 groups: control, control treated with metoprolol for 5 weeks, diabetic and diabetic treated with metoprolol for 5 weeks. After termination, the expression of CPT-1 isoforms, PPAR-alpha, PGC1alpha USF-1 and USF-2, as well as downstream targets were measured. Binding of PPAR-alpha, PGC1alpha and USF-2 to PGC1alpha was measured using coimmunoprecipitation. The occupation of PPAR-alpha and MEF-2A consensus sites in the CPT-1B promoter was measured using chromatin immunoprecipitation assays. Chronic metoprolol treatment decreased the expression of CPT-1B in diabetic hearts. The expression of USF-2 was increased by metoprolol in both control and diabetic hearts, but the association of USF-2 with PGC1alpha was increased by metoprolol only in diabetic hearts. Metoprolol prevented the increase in PGC1alpha occupation of the CPT-1B promoter region observed in the diabetic heart without affecting PPAR-alpha occupation. Metoprolol decreases CPT-1B expression by decreasing PGC1alpha-mediated coactivation of PPAR-alpha and MEF-2A. This is associated with increased PGC1alpha/ USF-2 binding, suggesting that USF-2 mediates the metoprolol-induced repression of PGC1alpha.

Brain-derived neurotrophic factor stimulates the transcriptional and neuroprotective activity of myocyte-enhancer factor 2C through an ERK1/2-RSK2 signaling cascade.

Neurotrophin activation of myocyte-enhancer factor (MEF) 2C is one of the strongest pro-survival signaling pathways in developing neurons. To date, neurotrophin stimulation of MEF2C has been largely attributed to its direct phosphorylation by extracellular signal-regulated kinase (ERK) 5. Because MEF2C is not directly phosphorylated by ERK1/2 in vitro, it is generally assumed that the ERK1/2 signaling cascade does not regulate MEF2C. Surprisingly, we discovered that ERK1/2 are required for both the transcriptional and neuroprotective activity of MEF2C in cortical neurons stimulated by brain-derived neurotrophic factor. ERK1/2 stimulation of MEF2C is mediated by p90 ribosomal S6 kinase 2 (RSK2), a Ser/Thr protein kinase downstream of ERK1/2. RSK2 strongly phosphorylates purified recombinant MEF2C protein in vitro. Furthermore, RSK2 can directly phosphorylate MEF2C on S192, a consensus RSK2-phosphorylation site located in the transactivation domain of MEF2C. Substitution of S192 with a non-phosphorylatable alanine diminishes both the transcriptional and neuroprotective activity of MEF2C to an extent similar to mutation on S387, an established activating phosphorylation site. Together, our data identifies ERK1/2-RSK2 signaling as a novel mechanism by which neurotrophins activate MEF2C and promote neuronal survival.

A pharmacodynamic study of 5-azacytidine in the P39 cell line.

OBJECTIVE: 5-azacytidine (azacytidine), a DNA hypomethylating agent, was recently approved as the first therapeutic agent for the treatment of myelodysplastic syndromes. The present subcutaneous dosing schedule, 75 mg/m(2) for 7/28 days, is based on early clinical studies and may constitute a practical problem for patients. The present in vitro study aimed at evaluating the pharmacodynamics of azacytidine, thereby providing a rationale for clinical dose-finding studies. METHODS: P39 cells were incubated with 0.1, 0.5, and 1 microM azacytidine daily for 24, 48, and 72 hours, followed by 48 hours in drug-free medium. The effects of azacytidine on cell growth, proliferation, apoptosis, cell cycle status, and promoter methylation of E-cadherin, ER, and HIC genes were studied. RESULTS: Azacytidine decreased cell growth and proliferation, increased apoptosis, and affected cell cycle status in a dose-dependent manner. However, the exposure time, 24 to 72 hours, at doses between 0.5 and 1 microM, did not significantly affect any of these variables. Using first-order exponential pharmacokinetic model, we found that the effect of 1, 2, or 3 microM over 24 hours did not differ from that of 0.5 to 1 microM given over 48 to 72 hours. Induction of promoter hypomethylation was observed already after 24 hours of exposure with >or=0.5 microM azacytidine with no clear dose-effect relationship. CONCLUSION: Our results indicate that optimal cellular effects of azacytidine might be achieved by shorter exposure times. The model provides information about the relation between azacytidine dose intensity and exposure time on malignant myeloid cells, which could serve as a rationale for further clinical development of practical, safe, and cost-effective dosing schedules.

Phosphorylation of pRb is required for HGF-induced muscle cell proliferation and is p27kip1-dependent.

Hepatocyte growth factor (HGF) plays a crucial role in the differentiation of skeletal muscle cells, where a process in which the retinoblastoma protein (pRb) has been implicated. We addressed the role of pRb in HGF-mediated effects on the proliferation and differentiation of adult skeletal muscle myoblasts. HGF shifted pRb to its hyperphosphorylation forms and increased the transactivation of E2F1, a transcription factor required for S phase entry. A constitutively active pRb mutant blocked HGF-dependent pRb phosphorylation and transactivation of E2F1 and increased cell proliferation. Accordingly, this mutant reversed the inhibitory effects of HGF on the expression of the cyclin-dependent kinase (CDK) inhibitor p27 and myogenic differentiation markers. HGF-mediated pRb phosphorylation was reversed by ectopic expression of p27, but neither the myogenic regulatory factor, MEF2, nor the myogenic inhibitory protein Twist had that effect. These results suggest that in response to HGF signaling, there is a decrease in p27 expression that results in an accumulation of hyperphosphorylated Rb protein, and subsequent progression of myoblasts into the G1 phase of the cell cycle.

Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family.

The muscle regulatory factors (MRF) gene family regulate muscle fibre development. Several hormones and drugs also affect muscle development. Glucocorticoids are the only drugs reported to have a beneficial effect on muscle degenerative disorders. We investigated the glucocorticoid-related effects on C2C12 myoblast proliferation rate, morphological differentiation, and subsequent mRNA expression patterns of the MRF genes. C2C12 cells were incubated with the glucocorticoids dexamethasone or alpha-methyl-prednisolone. Both glucocorticoids showed comparable effects. Glucocorticoid treatment of C2C12 cells during the proliferative phase reduced the proliferation rate of the cells dose dependently, especially during the third and fourth day of culture, increased MyoD1, myf-5, and MRF4 mRNA levels, and reduced myogenin mRNA level, compared to untreated control cells. Thus, the mRNA level of proliferation-specific MyoD1 and myf-5 expression does not seem to associate with C2C12 myoblast proliferation rate. Glucocorticoid treatment of C2C12 cells during differentiation reduced the differentiation capacity dose dependently, which is accompanied by a dose dependent reduction of myogenin mRNA level, and increased MyoD1, myf-5, and MRF4 mRNA levels compared to untreated control cells. Therefore, we conclude that glucocorticoid treatment reduces differentiation of C2C12 myoblasts probably through reduction of differentiation-specific myogenin mRNA level, while inducing higher mRNA levels of proliferation-associated MRF genes.