Osteoblast and osteocyte apoptosis associated with androgen action in bone: requirement of increased Bax/Bcl-2 ratio

Both the number and the activity of osteoblasts are critical for normal bone growth and maintenance. Although a potential role for estrogen in protection of bone mass through inhibition of osteoblast apoptosis has been proposed, a function for androgen is much less clear. The aim of this study was to establish a direct role for androgen to influence osteoblast apoptosis both in vitro and in vivo. AR-MC3T3-E1 cells, with androgen receptor (AR) overexpression controlled by the type I collagen promoter, were treated with the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT). Apoptosis was assessed by three different techniques including DNA fragmentation, caspase-3 activation, and changes in mitochondrial membrane potential. Transactivation of AR by DHT enhanced apoptosis while 17beta-estradiol (E(2)) treatment reduced apoptosis in both proliferating preosteoblasts and mature osteocyte-like cells. To explore mechanism, the apoptosis regulators Bcl-2 (antiapoptotic) and Bax (proapoptotic) were evaluated. Western analysis revealed that DHT decreased Bcl-2 resulting in a significantly increased Bax/Bcl-2 ratio. Regulation of Bcl-2 was post-transcriptional since bcl-2 mRNA levels were unaffected by DHT treatment. Furthermore, ubiquitination of Bcl-2 was increased and serine phosphorylation was reduced, consistent with inhibition of MAP kinase signaling by DHT. Increased Bax/Bcl-2 ratio was essential since either Bcl-2 overexpression or Bax downregulation by RNA interference (RNAi) partially abrogated or reversed DHT-enhanced osteoblastic apoptosis. In order to establish physiologic significance in vivo, AR-transgenic mice with AR overexpression in the osteoblast lineage and thus enhanced androgen sensitivity were characterized. In male AR-transgenic mice, increased osteoblast apoptosis was observed in vivo even in association with new bone formation. Thus, although estrogen can be antiapoptotic, androgen stimulates osteoblast and osteocyte apoptosis through an increased Bax/Bcl-2 ratio even in anabolic settings. These results identify a new mechanism for androgen regulation of osteoblast activity distinct from estrogen, and suggest that enhanced apoptosis can be associated with anabolic stimulation of new bone growth. Androgens thus play a distinct role in skeletal homeostasis.