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Expression of androgen receptor target genes in skeletal muscle.

Rana K, Lee NK, Zajac JD, MacLean HE - Asian J. Androl. (2014 Sep-Oct)

Bottom Line: The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR(∆ZF2) muscle.The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57(Kip2), Igf2 and calcineurin Aa, was increased in AR(∆ZF2) muscle, and the expression of all but p57(Kip2) was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle.We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Austin Health, University of Melbourne, Heidelberg, Victoria, Australia.

ABSTRACT
We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR)-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (AR(ΔZF2)) versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR(∆ZF2) muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57(Kip2), Igf2 and calcineurin Aa, was increased in AR(∆ZF2) muscle, and the expression of all but p57(Kip2) was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

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Relative AR gene expression. AR gene expression in (a) SkMC cultured as myoblasts in proliferation media (n = 9) or as myotubes in differentiation media (n = 10) for 24 h, normalized to myoblasts, *P < 0.05 vs SkMC myoblasts; (b) SkMC cultured in proliferation (myoblasts) or differentiation (myotubes) media and treated with 10 nmol l-1 DHT or vehicle (n ≥ 5 per group) for 12 and 24 h, normalized to myoblast 12 h vehicle; (c) GAST muscle of 18- to 19-week-old male mice following orchidectomy with control implants (orx control) at 8–9 weeks or orchidectomy with testosterone implants (orx + T) (n = 9 per group), normalized to orx control (model previously described26), *P < 0.05 vs orx control; (d) GAST muscle of 9-week-old ARΔZF2 male mice and WT male littermates (n = 6 per group), normalized to WT (model previously described24), *P < 0.05 vs WT; (e) WT male and female mice at E18.5 (all hind-limb muscles) (n ³ 9 per group), 4 weeks (GAST muscle) (n = 6 per group) and 12 weeks (GAST muscle) (n = 5 per group), normalized to 12 week males, #P < 0.05 vs E18.5 sex-matched control, §P < 0.05 vs 4 week sex-matched control and (f) 12-week-old male muscles (GAST, EDL, SOL, LA) (n=5 per group), normalized to GAST. **P < 0.001 vs GAST, EDL, SOL. All data are mean ± s.e.m. and one-way ANOVA, Tukey's post hoc test (age) and Student's t-test (sex) or Student's t-test (muscle) applied. AR: androgen receptor; DHT: dihydrotestosterone; EDL: extensor digitorum longus; GAST: gastrocnemius; s.e.m.: standard error of the mean; LA: levator ani; orx: orchidectomy; SOL: soleus; SkMC: skeletal muscle cells; WT: wildtype.
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Figure 1: Relative AR gene expression. AR gene expression in (a) SkMC cultured as myoblasts in proliferation media (n = 9) or as myotubes in differentiation media (n = 10) for 24 h, normalized to myoblasts, *P < 0.05 vs SkMC myoblasts; (b) SkMC cultured in proliferation (myoblasts) or differentiation (myotubes) media and treated with 10 nmol l-1 DHT or vehicle (n ≥ 5 per group) for 12 and 24 h, normalized to myoblast 12 h vehicle; (c) GAST muscle of 18- to 19-week-old male mice following orchidectomy with control implants (orx control) at 8–9 weeks or orchidectomy with testosterone implants (orx + T) (n = 9 per group), normalized to orx control (model previously described26), *P < 0.05 vs orx control; (d) GAST muscle of 9-week-old ARΔZF2 male mice and WT male littermates (n = 6 per group), normalized to WT (model previously described24), *P < 0.05 vs WT; (e) WT male and female mice at E18.5 (all hind-limb muscles) (n ³ 9 per group), 4 weeks (GAST muscle) (n = 6 per group) and 12 weeks (GAST muscle) (n = 5 per group), normalized to 12 week males, #P < 0.05 vs E18.5 sex-matched control, §P < 0.05 vs 4 week sex-matched control and (f) 12-week-old male muscles (GAST, EDL, SOL, LA) (n=5 per group), normalized to GAST. **P < 0.001 vs GAST, EDL, SOL. All data are mean ± s.e.m. and one-way ANOVA, Tukey's post hoc test (age) and Student's t-test (sex) or Student's t-test (muscle) applied. AR: androgen receptor; DHT: dihydrotestosterone; EDL: extensor digitorum longus; GAST: gastrocnemius; s.e.m.: standard error of the mean; LA: levator ani; orx: orchidectomy; SOL: soleus; SkMC: skeletal muscle cells; WT: wildtype.

Mentions: To determine if the AR gene is autologously regulated by androgens in muscle, we first examined regulation in vitro using the human myocyte cell line, SkMC, which we have previously shown expresses AR levels similar to human muscle.27AR mRNA was up-regulated in myotubes compared to myoblasts (Figure 1a). However, AR gene expression was not regulated by 12 or 24 h DHT treatment of either SkMC myoblasts or myotubes in vitro (Figure 1b).


Expression of androgen receptor target genes in skeletal muscle.

Rana K, Lee NK, Zajac JD, MacLean HE - Asian J. Androl. (2014 Sep-Oct)

Relative AR gene expression. AR gene expression in (a) SkMC cultured as myoblasts in proliferation media (n = 9) or as myotubes in differentiation media (n = 10) for 24 h, normalized to myoblasts, *P < 0.05 vs SkMC myoblasts; (b) SkMC cultured in proliferation (myoblasts) or differentiation (myotubes) media and treated with 10 nmol l-1 DHT or vehicle (n ≥ 5 per group) for 12 and 24 h, normalized to myoblast 12 h vehicle; (c) GAST muscle of 18- to 19-week-old male mice following orchidectomy with control implants (orx control) at 8–9 weeks or orchidectomy with testosterone implants (orx + T) (n = 9 per group), normalized to orx control (model previously described26), *P < 0.05 vs orx control; (d) GAST muscle of 9-week-old ARΔZF2 male mice and WT male littermates (n = 6 per group), normalized to WT (model previously described24), *P < 0.05 vs WT; (e) WT male and female mice at E18.5 (all hind-limb muscles) (n ³ 9 per group), 4 weeks (GAST muscle) (n = 6 per group) and 12 weeks (GAST muscle) (n = 5 per group), normalized to 12 week males, #P < 0.05 vs E18.5 sex-matched control, §P < 0.05 vs 4 week sex-matched control and (f) 12-week-old male muscles (GAST, EDL, SOL, LA) (n=5 per group), normalized to GAST. **P < 0.001 vs GAST, EDL, SOL. All data are mean ± s.e.m. and one-way ANOVA, Tukey's post hoc test (age) and Student's t-test (sex) or Student's t-test (muscle) applied. AR: androgen receptor; DHT: dihydrotestosterone; EDL: extensor digitorum longus; GAST: gastrocnemius; s.e.m.: standard error of the mean; LA: levator ani; orx: orchidectomy; SOL: soleus; SkMC: skeletal muscle cells; WT: wildtype.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215656&req=5

Figure 1: Relative AR gene expression. AR gene expression in (a) SkMC cultured as myoblasts in proliferation media (n = 9) or as myotubes in differentiation media (n = 10) for 24 h, normalized to myoblasts, *P < 0.05 vs SkMC myoblasts; (b) SkMC cultured in proliferation (myoblasts) or differentiation (myotubes) media and treated with 10 nmol l-1 DHT or vehicle (n ≥ 5 per group) for 12 and 24 h, normalized to myoblast 12 h vehicle; (c) GAST muscle of 18- to 19-week-old male mice following orchidectomy with control implants (orx control) at 8–9 weeks or orchidectomy with testosterone implants (orx + T) (n = 9 per group), normalized to orx control (model previously described26), *P < 0.05 vs orx control; (d) GAST muscle of 9-week-old ARΔZF2 male mice and WT male littermates (n = 6 per group), normalized to WT (model previously described24), *P < 0.05 vs WT; (e) WT male and female mice at E18.5 (all hind-limb muscles) (n ³ 9 per group), 4 weeks (GAST muscle) (n = 6 per group) and 12 weeks (GAST muscle) (n = 5 per group), normalized to 12 week males, #P < 0.05 vs E18.5 sex-matched control, §P < 0.05 vs 4 week sex-matched control and (f) 12-week-old male muscles (GAST, EDL, SOL, LA) (n=5 per group), normalized to GAST. **P < 0.001 vs GAST, EDL, SOL. All data are mean ± s.e.m. and one-way ANOVA, Tukey's post hoc test (age) and Student's t-test (sex) or Student's t-test (muscle) applied. AR: androgen receptor; DHT: dihydrotestosterone; EDL: extensor digitorum longus; GAST: gastrocnemius; s.e.m.: standard error of the mean; LA: levator ani; orx: orchidectomy; SOL: soleus; SkMC: skeletal muscle cells; WT: wildtype.
Mentions: To determine if the AR gene is autologously regulated by androgens in muscle, we first examined regulation in vitro using the human myocyte cell line, SkMC, which we have previously shown expresses AR levels similar to human muscle.27AR mRNA was up-regulated in myotubes compared to myoblasts (Figure 1a). However, AR gene expression was not regulated by 12 or 24 h DHT treatment of either SkMC myoblasts or myotubes in vitro (Figure 1b).

Bottom Line: The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR(∆ZF2) muscle.The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57(Kip2), Igf2 and calcineurin Aa, was increased in AR(∆ZF2) muscle, and the expression of all but p57(Kip2) was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle.We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Austin Health, University of Melbourne, Heidelberg, Victoria, Australia.

ABSTRACT
We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR)-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (AR(ΔZF2)) versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR(∆ZF2) muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57(Kip2), Igf2 and calcineurin Aa, was increased in AR(∆ZF2) muscle, and the expression of all but p57(Kip2) was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

Show MeSH
Related in: MedlinePlus