Wayne E. Taylor

Myostatin, a member of the transforming growth factor-beta superfamily, is a genetic determinant of skeletal muscle growth. Mice and cattle with inactivating mutations of myostatin have marked muscle hypertrophy. However, it is not known whether myostatin regulates skeletal muscle growth in adult men and whether increased myostatin expression contributes to wasting in chronic illness. We examined the hypothesis that myostatin expression correlates inversely with fat-free mass in humans and that increased expression of the myostatin gene is associated with weight loss in men with AIDS wasting syndrome. We therefore cloned the human myostatin gene and cDNA and examined the gene's expression in the skeletal muscle and serum of healthy and HIV-infected men. The myostatin gene comprises three exons and two introns, maps to chromosomal region 2q33.2, has three putative transcription initiation sites, and is transcribed as a 3.1-kb mRNA species that encodes a 375-aa precursor protein. Myostatin is expressed uniquely in the human skeletal muscle as a 26-kDa mature glycoprotein (myostatin-immunoreactive protein) and secreted into the plasma. Myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and 2 fibers. The serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in HIV-infected men with weight loss compared with healthy men and correlate inversely with fat-free mass index. These data support the hypothesis that myostatin is an attenuator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men.

Testosterone supplementation increases skeletal muscle mass and decreases fat mass; however, the underlying mechanisms are unknown. We hypothesized that testosterone regulates body composition by promoting the commitment of mesenchymal pluripotent cells into myogenic lineage and inhibiting their differentiation into adipogenic lineage. Mouse C3H 10T1/2 pluripotent cells were treated with testosterone (0-300 nM) or dihydrotestosterone (DHT, 0-30 nM) for 0-14 d, and myogenic conversion was evaluated by immunocytochemical staining for early (MyoD) and late (myosin heavy chain II; MHC) myogenic markers and by measurements of MyoD and MHC mRNA and protein. Adipogenic differentiation was assessed by adipocyte counting and by measurements of peroxisomal proliferator-activated receptor gamma 2 (PPAR gamma 2) mRNA and PPAR gamma 2 protein and CCAAT/enhancer binding protein alpha. The number of MyoD+ myogenic cells and MHC+ myotubes and MyoD and MHC mRNA and protein levels increased dose dependently in response to testosterone and DHT treatment. Both testosterone and DHT decreased the number of adipocytes and down-regulated the expression of PPAR gamma 2 mRNA and PPAR gamma 2 protein and CCAAT/enhancer binding protein alpha. Androgen receptor mRNA and protein levels were low at baseline but increased after testosterone or DHT treatment. The effects of testosterone and DHT on myogenesis and adipogenesis were blocked by bicalutamide. Therefore, testosterone and DHT regulate lineage determination in mesenchymal pluripotent cells by promoting their commitment to the myogenic lineage and inhibiting their differentiation into the adipogenic lineage through an androgen receptor-mediated pathway. The observation that differentiation of pluripotent cells is androgen dependent provides a unifying explanation for the reciprocal effects of androgens on muscle and fat mass in men.

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C 2 C 12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli(Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([ 3 H]thymidine incorporation), and protein synthesis ([1- 14 C]leucine incorporation) in C 2 C 12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C 2 C 12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

Testosterone supplementation in men decreases fat mass; however, the mechanisms by which it inhibits fat mass are unknown. We hypothesized that testosterone inhibits adipogenic differentiation of preadipocytes by activation of androgen receptor (AR)/beta-catenin interaction and subsequent translocation of this complex to the nucleus thereby bypassing canonical Wnt signaling. We tested this hypothesis in 3T3-L1 cells that differentiate to form fat cells in adipogenic medium. We found that these cells express AR and that testosterone and dihydrotestosterone dose-dependently inhibited adipogenic differentiation as analyzed by Oil Red O staining and down-regulation of CCAAT/enhancer binding protein-alpha and -delta and peroxisome proliferator-activated receptor-gamma2 protein and mRNA. These inhibitory effects of androgens were partially blocked by flutamide or bicalutamide. Androgen treatment was associated with nuclear translocation of beta-catenin and AR. Immunoprecipitation studies demonstrated association of beta-catenin with AR and T-cell factor 4 (TCF4) in the presence of androgens. Transfection of TCF4 cDNA inhibited adipogenic differentiation, whereas a dominant negative TCF4 cDNA construct induced adipogenesis and blocked testosterone's inhibitory effects. Our gene array analysis indicates that testosterone treatment led to activation of some Wnt target genes. Expression of constitutively activated AR fused with VP-16 did not inhibit the expression of CCAAT/enhancer binding protein-alpha in the absence of androgens. Testosterone and dihydrotestosterone inhibit adipocyte differentiation in vitro through an AR-mediated nuclear translocation of beta-catenin and activation of downstream Wnt signaling. These data provide evidence for a regulatory role for androgens in inhibiting adipogenic differentiation and a mechanistic explanation consistent with the observed reduction in fat mass in men treated with androgens.

In the proposed "zinc finger" DNA-binding motif, each repeat unit binds a zinc metal ion through invariant Cys and His residues and this drives the folding of each 30-residue unit into an independent nucleic acid-binding domain. To obtain structural information, we synthesized single and double zinc finger peptides from the yeast transcription activator ADR1, and assessed the metal-binding and DNA-binding properties of these peptides, as well as the solution structure of the metal-stabilized domains, with the use of a variety of spectroscopic techniques. A single zinc finger can exist as an independent structure sufficient for zinc-dependent DNA binding. An experimentally determined model of the single finger is proposed that is consistent with circular dichroism, one- and two-dimensional nuclear magnetic resonance, and visual spectroscopy of the single-finger peptide reconstituted in the presence of zinc.