Myostatin negatively regulates the expression of the steroid receptor co-factor ARA70.
Myostatin is a transforming growth factor-beta (TGF-beta) superfamily member and a key negative regulator of embryonic and postnatal muscle growth. In order to identify downstream target genes regulated by Myostatin, we performed suppressive subtraction hybridization (SSH) on cDNA generated from the biceps femoris muscle of wild-type and myostatin-null mice. Sequence analysis identified several known and unknown genes as Myostatin downstream target genes. Here, we have investigated the regulation of gene expression of an androgen receptor (AR) binding co-factor, androgen receptor associated protein-70 (ARA70), by Myostatin. We show that in mouse there are two isoforms of ARA70 with high homology (79%) to human ARA70; an alpha-isoform which is a canonical ARA70 and a beta-isoform which has a 9 consecutive amino acid deletion and 6 amino acid substitutions in the carboxyl-terminal portion. Reverse Northern analysis on the differentially expressed cDNA library indicated that there is increased expression of ARA70 in the muscles of myostatin-null mice. In addition, Northern blot, together with semi-quantitative PCR analysis, confirmed that there is increased expression of ARA70 in myostatin-null biceps femoris muscle when compared to wild-type muscle. In corroboration of these results, addition of exogenous Myostatin results in down-regulation of ARA70 expression confirming that Myostatin is a negative regulator of ARA70 gene expression. Expression analysis further confirmed that ARA70 is up-regulated during myogenesis and that peak expression of ARA70 is observed following the peak expression of MyoD in differentiating myoblasts. Given that lack of Myostatin and increased expression of AR leads to hypertrophy, we propose that absence of Myostatin, at least in part, induces the hypertrophy phenotype by increasing the activity of AR by up-regulating the expression of ARA70, a known stimulating co-factor of AR.
Siriett
Victoria
V
School of Biological Sciences, University of Waikato, Hamilton, New Zealand.
Nicholas
Gina
G
Berry
Carole
C
Watson
Trevor
T
Hennebry
Alex
A
Thomas
Mark
M
Ling
Nicholas
N
Sharma
Mridula
M
Kambadur
Ravi
R
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D055435:Myostatin; D056918:Nuclear Receptor Coactivators; D009693:Nucleic Acid Hybridization; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
C525911:MSTN protein, human; C525912:Mstn protein, mouse; D055435:Myostatin; C111382:NCOA4 protein, human; C508724:NcoA4 protein, mouse; D056918:Nuclear Receptor Coactivators; D015513:Oncogene Proteins; D020033:Protein Isoforms; D014157:Transcription Factors; D016212:Transforming Growth Factor beta