RESUMO
In previous studies we established that human bone and human osteoblast-like cells (hOB cells) cultured from bone express 5alpha-reductase (5alpha-R) activity, as demonstrated by the conversion of testosterone and androstenedione to their corresponding 5alpha-reduced metabolites, 5alpha-dihydrotestosterone (DHT) and 5alpha-androstanedione. Two 5alpha-R isozymes (types 1 and 2) have been identified in various tissues. As their nature in bone is unknown, we investigated which isozymes were expressed in first passage hOB cells cultured from bone specimens obtained from six donors (five women and one man). For comparison, 5alpha-reductase isozyme expression in genital skin fibroblasts cultured from foreskin of three males was determined. Pharmacological and biochemical studies using selective inhibitors of the 5alpha-R isozymes were performed, and gene expression was assessed by RT-PCR. In hOB cells, LY191704, a potent nonsteroidal selective inhibitor of 5alpha-R type 1, and the 4-azasteroid 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one (a dual inhibitor of 5alpha-R types 1 and 2) inhibited 5alpha-R activity with a 50% inhibitory concentration (IC(50)) of approximately 4 nM. Finasteride, a selective inhibitor of 5alpha-R type 2, blocked 5alpha-R activity with an IC(50) of approximately 60 nM. The IC(50) of progesterone, a physiological substrate for 5alpha-R, was approximately 200 nM. In genital skin fibroblasts, LY191704 inhibited 5alpha-R with an IC(50) of more than 5000 nM, whereas finasteride and 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one effectively inhibited 5alpha-R with IC(50) of approximately 4 nM. Experiments to determine 5alpha-reductase activity in homogenates of hOB cells as a function of pH showed very low activity at pH 5.5, but a broad shoulder of activity from pH 6.0-9.0, which was not inhibited by finasteride, but was nearly completely blocked by LY191704. RT-PCR revealed that 5alpha-R type 1 and 2 mRNAs were expressed in both bone and genital skin fibroblasts. Based on our pharmacological and biochemical studies, it appears that 5alpha-R activity in hOB cells is catalyzed predominantly by the type 1 rather than the type 2 isozyme. This expression pattern is in contrast to that in genital skin fibroblasts, where the activity of the type 2 isozyme prevails. As in most androgen target tissues DHT is biologically more active as an androgen than testosterone, DHT is formed in bone by 5alpha-R type 1 action from circulating testosterone, and bone cells also express the androgen receptor, local DHT production may play a physiological role in human bone homeostasis.