Progesterone inactivation in human osteoblasts

Although progesterone (P) has been implicated in the control of bone formation, P use in postmenopausal women appears to have little impact on bone. Functional P receptors are expressed in osteoblasts so the reason for these disappointing results is unclear. An important component of P action is 'pre-receptor' metabolism by intracellular enzymes. We have now investigated the potential role of P metabolising enzymes in mediating P action in human osteoblasts. Primary cultures of osteoblasts were generated by outgrowth from collagenase treated trabecular bone specimens obtained during elective knee surgery for osteoarthritis or rheumatoid arthritis (n=7). Sub-confluent monolayers were incubated in triplicates for 16 hr with C14-labeled-P and 50nM unlabeled P (approximate in vivo luteal phase P concentrations). Steroids were extracted into dichloromethane and identified by two-dimensional thin-layer chromatography (TLC). In parallel, RNA was extracted and RT-PCR analysis carried out to identify P metabolizing enzymes.

Extensive P metabolism was observed in all cultures (up to 40%) with the activity being of the order of 1pmol/mg protein/hr. The two major P metabolites were the inactive steroids 20alpha-DH-P and 5alpha-DH-P. High levels of AKR1C1 (also called 20alpha-HSD) and 5alpha-reductase type 1 mRNA which are the enzymes responsible for these conversions were detected in all cases. Several ring A reduced metabolites (3alpha,5alpha- and 3beta,5alpha-TH-P and their 20-reduced products) were detected in smaller amounts indicating expression of AKR1C2 and either 3beta-HSD type 1 or hydroxysteroid-epimerase. 72 hr treatment with dexamethasone or estradiol had no impact on P metabolism.

P appears to undergo extensive intracellular inactivation in human osteoblasts. At levels of P seen in vivo this inactivation is likely to substantially reduce the impact of this steroid on osteoblasts. Development of synthetic P derivatives which are not metabolized by these enzymes may improve bioactivity and improve their utility as bone active agents.