Wayne E. Taylor

Exercise in Maintenance Hemodialysis Patients Induces Transcriptional Changes in Genes Favoring Anabolic Muscle

Exercise training increases exercise capacity of maintenance hemodialysis patients, but the cellular mechanisms responsible for this effect are unclear. We studied the effects of different forms of exercise training (endurance, strength, or a combination where patients underwent about one-half each

Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells

Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub‐cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT–PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC‐I and ‐II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co‐expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC‐II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose‐dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC‐II. Its predominant nuclear localization suggests that it may play a role in transcriptional regulation. J. Cell. Physiol. 190: 170–179, 2002.

Regulation of Myogenic Differentiation by Androgens: Cross Talk between Androgen Receptor/ β-Catenin and Follistatin/Transforming Growth Factor-β Signaling Pathways

Androgens are important regulators of body composition and promote myogenic differentiation and inhibit adipogenesis of mesenchymal, multipotent cells. Here, we investigated the mechanisms by which androgens induce myogenic differentiation of mesenchymal multipotent cells. Incubation of mesenchymal multipotent C3H 10T1/2 cells with testosterone and dihydrotestosterone promoted nuclear translocation of androgen receptor (AR)/beta-catenin complex and physical interaction of AR, beta-catenin, and T-cell factor-4 (TCF-4). Inhibition of beta-catenin by small inhibitory RNAs significantly decreased testosterone-induced stimulation of myogenic differentiation. Overexpression of TCF-4, a molecule downstream of beta-catenin in Wnt signaling cascade, in C3H 10T1/2 cells significantly up-regulated expression of myoD and myosin heavy chain II proteins and of follistatin (Fst), which binds and antagonizes native ligands of the TGF-beta/Smad pathway. Gene array analysis of C3H 10T1/2 cells treated with testosterone revealed that testosterone up-regulated the expression of Fst and modified the expression of several signaling molecules involved in the TGF-beta/Smad pathway, including Smad7. Lowering of testosterone levels in mice by orchidectomy led to a significant decrease in Fst and Smad7 expression; conversely, testosterone supplementation in castrated mice up-regulated Fst and Smad7 mRNA expression in androgen-responsive levator ani muscle. Testosterone-induced up-regulation of MyoD and myosin heavy chain II proteins in C3H 10T1/2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. In conclusion, our data suggest the involvement of AR, beta-catenin, and TCF-4 pathway during androgen action to activate a number of Wnt target genes, including Fst, and cross communication with the Smad signaling pathway.

THE GENETIC BASIS OF MALE INFERTILITY

Defective spermatogenesis can be the end result of a multitude of causes, such as systemic disease, malnutrition, endocrinologic disorder, genetic defects, anatomic obstruction of the passage of spermatozoa, infections, and environmental toxins. A genetic basis of infertility is thought to exist in a majority of infertile men currently classified as having idiopathic infertility. Despite advances in molecular technology, the pathophysiology of spermatogenic failure in a majority of infertile men remains unknown. Although a large number of genes and loci in experimental animals are associated with sterility, the human homologues of most of these genes have not been cloned yet. Infertility is a heterogeneous syndrome in men; therefore, it is likely that a multitude of genes and loci will be implicated in different infertility subsets.

Human KIT ligand promoter is positively regulated by HMGA1 in breast and ovarian cancer cells.

KIT ligand (KL) and its receptor, c-kit, are coexpressed in many types of cancer cells and have been implicated in tumor growth and angiogenesis. While Sertoli cell-specific regulation of the KL promoter has been well characterized, regulation in cancer cells remains to be elucidated. We recently reported microarray results demonstrating that increased high-mobility group (HMG) A1a protein expression correlates with increased KL transcription in MCF-7 human breast cancer cells. Sequence analysis indicates a potential for multiple HMGA1 binding sites within the human KL promoter. In order to better define the underlying molecular mechanisms that HMGA1 uses to facilitate malignant transformation of cancer cells, we have used a variety of methods to determine whether HMGA1a directly regulates the human KL promoter in breast and ovarian cancer cells. Our results indicate that: (i) KL promoter activity is significantly higher in MCF-7 cells overexpressing HMGA1a; (ii) HMGA1a protein binds to AT-rich regions of the KL promoter DNA both in vitro and in vivo; (iii) mutation of the AT-rich regions inhibits HMGA1a binding in vitro; and (iv) HMGA1a-specific inhibition significantly decreases transcription of KL in OCC1 human ovarian cancer cells. In addition, MCF-7 cells with transgenic HMGA1 overexpression stained positive for the KL protein by immunocytochemistry and immunohistochemistry, and were growth-inhibited by KL neutralization. The cumulative evidence indicates that HMGA1 positively regulates the human KL promoter in breast and ovarian cancer cells and implicates serum KL as a diagnostic marker for HMGA1-positive carcinomas.

419 TESTOSTERONE INHIBITION OF INSULIN SIGNALING PATHWAY GENES AND ADIPOGENIC GENES OBSERVED IN 3T3-L1 MOUSE PREADIPOCYTE CELLS BY EXPRESSION PROFILE.

The effect of testosterone administration on insulin sensitivity in patients with diabetes is poorly understood. Epidemiological studies in men indicate that type 2 diabetes mellitus and coronary artery disease are correlated with lower free testosterone concentrations in serum, while testosterone treatment in men is associated with increased insulin sensitivity. The purpose of this study is to analyze the gene expression profile in a preadipocyte cell line 3T3-L1, during growth in normal and adipogenic medium (AM), and subsequently study the effect of testosterone on expression of various genes involved in the insulin signaling pathway. We analyzed the effect of testosterone (100 nM) treatment on the expression pattern of 112 genes involved in insulin signaling pathway by GEArray (MM-030) from SuperArray. We observed that over 30 genes of this pathway are highly induced over five-fold during adipogenesis. Testosterone treatment led to significant change in at least 19 genes in this pathway. MEK 1, a component of MAP kinase pathway, was up-regulated after 48 hours of testosterone treatment, whereas we found significant inhibition in the expression of other MAP kinase pathway genes, Kras2, B-raf, MNK, and protein kinase C lambda. Inhibition was also observed for the primary target genes for insulin signaling (leptin and C/EBP-Beta) as well as PI-3 kinase pathway genes, eg, Frap 1, PIK3r2 (p85b subunit), and PAI-1, UCP-1, and Vegfa. We performed quantitative analysis by real-time reverse-transcription-PCR and Western blot analysis of various genes/proteins regulated by testosterone in this pathaway in order to have better understanding of the role of testosterone during insulin resistance, observing around three-fold inhibition of expression for leptin, C/EBP-Beta, PPAR-gamma2, and C/EBP-alpha. The information obtained from this study may provide the rationale for targeting key components responsible for insulin resistance in patients with type 2 diabetes.