title: Androgen receptor polyglutamine repeat length affects receptor activity and C2C12 cell development.
abstract: Testosterone (T) has an anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. The effects of AR glutamine repeat length on skeletal muscle are unknown. In this study we examined the effect of AR CAG repeat length on AR function in C2C12 cells. AR expression vectors harboring 14, 24, and 33 CAG repeats were used to assess AR transcriptional activity. C2C12 cell proliferation, differentiation, gene expression, myotube formation, and myonuclear fusion index were assessed. Transcriptional activity increased with increasing repeat length and in response to testosterone (AR14 = 3.91 ± 0.26, AR24 = 25.21 ± 1.72, AR33 = 36.08 ± 3.22 relative light units; P < 0.001). Ligand activation was increased for AR33 (2.10 ± 0.04) compared with AR14 (1.54 ± 0.09) and AR24 (1.57 ± 0.05, P < 0.001). AR mRNA expression was elevated in each stably transfected line. AR33 cell proliferation (20,512.3 ± 1,024.0) was decreased vs. AR14 (27,604.17 ± 1,425.3; P < 0.001) after 72 h. Decreased CK activity in AR14 cells (54.9 ± 2.9 units/μg protein) in comparison to AR33 (70.8 ± 8.1) (P < 0.05) was noted. The myonuclear fusion index was lower for AR14 (15.21 ± 3.24%) and AR33 (9.97 ± 3.14%) in comparison to WT (35.07 ± 5.60%, P < 0.001). AR14 and AR33 cells also displayed atypical myotube morphology. RT-PCR revealed genotype differences in myostatin and myogenin expression. We conclude that AR polyglutamine repeat length is directly associated with transcriptional activity and alters the growth and development of C2C12 cells. This polymorphism may contribute to the heritability of muscle mass in humans.
authors.affiliation: Department of Kinesiology, School of Public Health, University of Maryland, College Park, Maryland 20742, USA.
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Sheppard
authors.forename: Espen E
authors.initials: EE
authors.lastname: Spangenburg
authors.forename: Eva R
authors.initials: ER
authors.lastname: Chin
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Roth
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013739:Testosterone / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
chemical_list: D003470:Culture Media; D010455:Peptides; D012333:RNA, Messenger; D011944:Receptors, Androgen; C097188:polyglutamine; D013739:Testosterone