TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition.

Although recent clinical studies have suggested a possible role for sclerostin, a secreted Wnt antagonist, in the pathogenesis of postmenopausal osteoporosis, the detailed mechanisms how estrogen deficiency regulates sclerostin expression have not been well-elucidated. Bilateral ovariectomy or a sham operation in female C57BL/6 mice and BALB/c nude mice was performed when they were seven weeks of age. The C57BL/6 mice were intraperitoneally injected with phosphate-buffered serum (PBS), 5 µg/kg ß-estradiol five times per week for three weeks, or 10 mg/kg TNF-α blocker three times per week for three weeks. Bony sclerostin expression was assessed by immunohistochemistry staining in their femurs. The activity and expression of myocyte enhancer factors 2 (MEF2), which is essential for the transcriptional activation of sclerostin, in rat UMR-106 osteosarcoma cells were determined by luciferase reporter assay and western blot analysis, respectively. Bony sclerostin expression was stimulated by estrogen deficiency and it was reversed by estradiol supplementation. When the UMR-106 cells were treated with well-known, estrogen-regulated cytokines, only TNF-α, but not IL-1 and IL-6, increased the MEF2 activity. Consistently, TNF-α also increased the nuclear MEF2 expression. Furthermore, the TNF-α blocker prevented the stimulation of bony sclerostin expression by ovariectomy. We also found that there was no difference in sclerostin expression between ovariectomized nude mice and sham-operated nude mice. In conclusion, these results suggest that TNF-α originating from T cells may be at least in part responsible for stimulating the sclerostin expression observed in an estrogen-deficient condition.

Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TRalpha transcriptional factors.

Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states. In muscles, glucose uptake is performed by the GLUT-4 protein, which is encoded by the SLC2A4 gene. SLC2A4 gene expression increases in response to conditions that improve insulin sensitivity, including chronic exercise. However, since chronic exercise improves insulin sensitivity, the increased SLC2A4 gene expression could not be clearly attributed to the muscle contractile activity per se and/or to the improved insulin sensitivity. The present study was designed to investigate the role of contractile activity per se in the regulation of SLC2A4 gene expression as well as in the participation of the transcriptional factors myocyte enhancer factor 2D (MEF2D), hypoxia inducible factor 1a (HIF-1a), and thyroid hormone receptor-alpha (TRalpha). The performed in vitro protocol excluded the interference of metabolic, hormonal, and neural effects. The results showed that, in response to 10 min of electrically induced contraction of soleus muscle, an early 40% increase in GLUT-4 mRNA (30 min) occurred, with a subsequent 65% increase (120 min) in GLUT-4 protein content. EMSA and supershift assays revealed that the stimulus rapidly increased the binding activity of MEF2D, HIF-1a, and TRalpha into the SLC2A4 gene promoter. Furthermore, chromatin immunoprecipitation assay confirmed, in native nucleosome, that contraction induced an approximate fourfold (P < 0.01) increase in MEF2D and HIF-1a-binding activity. In conclusion, muscle contraction per se enhances SLC2A4 gene expression and that involves MEF2D, HIF-1a, and TRalpha transcription factor activation. This finding reinforces the importance of physical activity to improve glycemic homeostasis independently of other additional insulin sensitizer approaches.

Influence of growth factors on poultry myogenic satellite cells.

Skeletal muscle development in avian and mammalian embryos depends on the proliferation, differentiation, and fusion of embryonic myoblasts. During the late fetal period and following birth or hatching, myogenic satellite cells are responsible for this developmental function. Satellite cells, which are found adjacent to existing skeletal muscle fibers fuse with these fibers and their nuclei direct the synthesis of new protein and function in the maturation of muscle. These events are controlled by specific growth factors that are produced locally by satellite cells and other cells in the muscle. Progress in our understanding of the early events in myogenesis has been made possible by the development of satellite cell cultures and media formulations that allow the assessment of the role of growth factors in skeletal muscle growth and development. Because of the key role that satellite cells play in skeletal muscle growth, development, and regeneration, many scientists in both the agricultural and medical communities have focused their research on understanding the physiology of this cell. From an agricultural perspective, a better understanding of the mechanisms regulating satellite cell activity may lead to procedures to increase the deposition and efficiency of lean muscle (meat) accretion and, perhaps, improve the nutrient composition of meat products.

Requirement of a novel gene, Xin, in cardiac morphogenesis.

A novel gene, Xin, from chick (cXin) and mouse (mXin) embryonic hearts, may be required for cardiac morphogenesis and looping. Both cloned cDNAs have a single open reading frame, encoding proteins with 2,562 and 1,677 amino acids for cXin and mXin, respectively. The derived amino acid sequences share 46% similarity. The overall domain structures of the predicted cXin and mXin proteins, including proline-rich regions, 16 amino acid repeats, DNA-binding domains, SH3-binding motifs and nuclear localization signals, are highly conserved. Northern blot analyses detect a single message of 8.9 and 5.8 kilo base (kb) from both cardiac and skeletal muscle of chick and mouse, respectively. In situ hybridization reveals that the cXin gene is specifically expressed in cardiac progenitor cells of chick embryos as early as stage 8, prior to heart tube formation. cXin continues to be expressed in the myocardium of developing hearts. By stage 15, cXin expression is also detected in the myotomes of developing somites. Immunofluorescence microscopy reveals that the mXin protein is colocalized with N-cadherin and connexin-43 in the intercalated discs of adult mouse hearts. Incubation of stage 6 chick embryos with cXin antisense oligonucleotides results in abnormal cardiac morphogenesis and an alteration of cardiac looping. The myocardium of the affected hearts becomes thickened and tends to form multiple invaginations into the heart cavity. This abnormal cellular process may account in part for the abnormal looping. cXin expression can be induced by bone morphogenetic protein (BMP) in explants of anterior medial mesoendoderm from stage 6 chick embryos, a tissue that is normally non-cardiogenic. This induction occurs following the BMP-mediated induction of two cardiac-restricted transcription factors, Nkx2.5 and MEF2C. Furthermore, either MEF2C or Nkx2.5 can transactivate a luciferase reporter driven by the mXin promoter in mouse fibroblasts. These results suggest that Xin may participate in a BMP-Nkx2.5-MEF2C pathway to control cardiac morphogenesis and looping.

Proinflammatory cytokines regulate myogenic cell proliferation and fusion but have no impact on myotube protein metabolism or stress protein expression.

The objective of the present study was to evaluate the effect of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-alpha (IL-1a), on myoblast proliferation and fusion and on myocyte protein metabolism and stress protein expression. Proliferation was suppressed (p < 0.05) by both cytokines, alone and in combination, and at lower concentrations, the suppression was additive. Likewise, fusion was retarded (p < 0.05) by these cytokines alone and in combination. Myosin synthesis was not altered acutely or chronically by TNF-alpha alone or by the combination of this cytokine with IL-1alpha. Chronic exposure to TNF-alpha did not alter total cellular protein synthesis, but exposure to IL-1alpha and the cytokine combination resulted in an increase (14% to 19%, p < 0.05) in synthesis. Neither total cellular protein nor myosin degradation were influenced by either cytokine alone or by the combination. There was no detectable induction, acutely or chronically, of any of the stress proteins evaluated (HSC70, HSP70, or HSP60). These data suggest that cytokines may alter muscle growth and development prenatally and postnatally and that the changes in muscle protein metabolism during periods of immune challenge are not direct effects of TNF-alpha or IL-1alpha.

Alpha-tropomyosin gene expression in Xenopus laevis: differential promoter usage during development and controlled expression by myogenic factors.

Tropomyosins (TMs) constitute a group of contractile proteins encoded by a multigene family showing distinct cell-type-specific and developmental expression patterns. In mammals and birds, the alpha-TM gene is the most complex and can produce several muscle and non-muscle isoforms. We report here the characterization of the 5' region of the Xenopus laevis alpha-TM gene and its developmental expression. The 5' region of the gene is structurally related to the avian and mammalian cognates and presents two promoters flanking a pair of alternatively spliced exons, 2a/2b, where exon 2a is a smooth-muscle-specific exon. The internal promoter is used to generate a non-muscle low molecular weight TM whilst muscle TM isoforms originate from the distal promoter. RNase protection analysis shows that the two promoters have distinct temporal programs of activation. The internal promoter is activated early in oogenesis and non-muscle transcripts are found throughout oogenesis, embryogenesis and in adult tissues. Only low molecular weight non-muscle TM-encoding mRNAs are expressed in oogenesis. The distal promoter is silent during oogenesis, and the skeletal muscle alpha-TM transcripts accumulate from stage 15 in the embryo and are expressed in adult striated muscle tissues. In situ hybridization indicates that these transcripts are expressed in both the somites and heart of the embryo. Ectopic expression of myogenic factors, but not the MEF2 myocyte-specific enhancer factor 2 factors SL1 and SL2, can induce the expression of the alpha-TM gene suggesting that the gene is a direct target for myogenic but not for MEF2 factors. The amphibian alpha-TM gene constitutes a gene marker for studying the developmental control expression of muscle genes in the different myogenic lineages.