Rapid membrane responses to dihydrotestosterone are sex dependent in growth plate chondrocytes.
Sex steroids are important regulators for longitudinal growth, bone mass accrual, and sexual dimorphism of the skeleton. 17β-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17β-estradiol, male chondrocytes produce 5α-dihydrotestosterone (DHT). Previously DHT was found to mediate sex-specific effects of testosterone in male cells, but it is not known if a membrane-signaling pathway is involved. In this study, we hypothesized that DHT can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10(-10)-10(-7)M testosterone or DHT for 3, 9, 90 and 270min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10(-7)M DHT for 9min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPβS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. DHT induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10(-7)M DHT (p<0.05), whereas testosterone had no effect. PKC activity was augmented at 9 and 90 min, and then decreased to baseline at 270min. Neither testosterone nor DHT affected PKC in female cells. U73122, quinacrine, and AACOCF3 inhibited DHT-induced activation of PKC. DHT treatment for 9 min had no effect in [(3)H]-thymidine incorporation in quiescent confluent cultures but caused a dose dependent increase in alkaline phosphatase specific activity. Inhibition of PLC reduced the response of to DHT in a dose dependent manner, indicating that PLC is involved. In conclusion, our study indicates that DHT, but not testosterone, has sex-specific rapid membrane effects in male growth plate chondrocytes involving PLC and PLA2-mediated PKC signaling pathways. Together with previous observations showing that male cells convert testosterone to DHT, these results suggest that DHT might act in the membrane through an autocrine/paracrine mechanism.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, United States.
Khairat
K
ElBaradie
Yun
Y
Wang
Barbara D
BD
Boyan
Zvi
Z
Schwartz
D000469:Alkaline Phosphatase / Q000378:metabolism; D000728:Androgens / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013196:Dihydrotestosterone / Q000494:pharmacology*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D006132:Growth Plate / Q000166:cytology; D008297:Male; D054467:Phospholipases A2 / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011944:Receptors, Androgen / Q000378:metabolism; D012727:Sex Characteristics; D013739:Testosterone / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism
D000728:Androgens; D011944:Receptors, Androgen; D013196:Dihydrotestosterone; D013739:Testosterone; D011493:Protein Kinase C; D054467:Phospholipases A2; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium