In previous studies we established that human bone and human osteoblast-like cells (hOB cells) cultured from bone express 5α-reductase (5α-R) activity, as demonstrated by the conversion of testosterone and androstenedione to their corresponding 5α-reduced metabolites, 5α-dihydrotestosterone (DHT) and 5α-androstanedione. Two 5α-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, 5α-reductase isozyme expression in genital skin fibroblasts cultured from foreskin of three males was determined. Pharmacological and biochemical studies using selective inhibitors of the 5α-R isozymes were performed, and gene expression was assessed by RT-PCR. In hOB cells, LY191704, a potent nonsteroidal selective inhibitor of 5α-R type 1, and the 4-azasteroid 17β-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5α-androstan-3-one (a dual inhibitor of 5α-R types 1 and 2) inhibited 5α-R activity with a 50% inhibitory concentration (IC50) of approximately 4 nM. Finasteride, a selective inhibitor of 5α-R type 2, blocked 5α-R activity with an IC50 of approximately 60 nM. The IC50 of progesterone, a physiological substrate for 5α-R, was approximately 200 nM. In genital skin fibroblasts, LY191704 inhibited 5α-R with an IC50 of more than 5000 nM, whereas finasteride and 17β-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5α-androstan-3-one effectively inhibited 5α-R with IC50 of approximately 4 nM. Experiments to determine 5α-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 5α-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 5α-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 5α-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.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Clinical Biochemistry
- Biochemistry, medical