Abstract
In addition to reproductive tissue, sex hormones induce transcriptional events in many connective tissue cells, including osteoblasts. Some sex hormone receptor modulators with bone sparing effects selectively target estrogen or androgen receptors, whereas others appear more promiscuous, in part through enzymatic metabolism. Rat osteoblasts express significant oxidative 3alpha-hydroxysteroid dehydrogenase activity, which can convert precursor substrates to potent androgen receptor agonists. Here we show that they also express 3-ketosteroid reductase activity, exemplified by 7-methyl-17-ethynyl-19-norandrostan-5 (10)en-3-one (tibolone) conversion to potent estrogen receptor alpha agonists. Conversion was rapid and quantitative, with 3alpha-hydroxytibolone as the primary metabolite. Consistently, tibolone induced estrogen receptor alpha-dependent gene promoter activity through cis-acting estrogen response elements, increased the stimulatory effect of TGF-beta on Smad-dependent gene promoter activity, and enhanced prostaglandin E2-induced activity of transcription factor Runx2. Rat osteoblasts express the 3-ketosteroid reductase AKR1C9, an aldo-keto reductase gene family member. Exposure to prostaglandin E2 increased AKR1C9 gene promoter activity and mRNA expression. AKR1C9 promoter activity was also enhanced by overexpression of protein kinase A catalytic subunit or transcription factor C/EBPdelta, and the effect of PGE2 was reduced by dominant negative C/EBPdelta competition or C/EBPdelta antisense expression. Moreover, prostaglandin E2 increased the amount of functional endogenous nuclear C/EBPdelta that could bind specifically to a distinct domain approximately 1.8-kb upstream from the start site of AKR1C9 transcription. In summary, in addition to 3alpha-hydroxysteroid dehydrogenase, rat osteoblasts express significant and regulatable 3-ketosteroid reductase activity. Through these enzymes, they may selectively metabolize precursor compounds into potent steroid receptor agonists locally within bone.
Highlights
Sex steroids regulate gene expression in neural, cardiovascular, skin, and other connective tissue cells, and more recent evidence reveals an increase in breast and vascular disease with long term sex hormone replacement therapy (HRT) [51, 53,54,55]
We previously reported that osteoblasts possess potent oxidative 3␣-hydroxysteroid dehydrogenase activity [12], and show here that they exhibit significant reductive 3-ketosteroid reductase activity by which they rapidly convert the androgen receptor (AR) agonist tibolone to the estrogen receptor (ER)␣ agonists 3␣- and 3-hydroxytibolone. 3␣-hydroxytibolone is the sole tibolone metabolite produced by recombinant rat AKR1C9 in vitro [18], unlike the appearance of both 3␣- and 3-hydroxytibolone in intact rat osteoblasts
We found that the gene promoter for AKR1C9 is induced in osteoblasts by protein kinase A (PKA) activation and is driven by transcription factor CCAAT enhancerbinding protein (C/EBP)␦
Summary
Cells—Primary osteoblast-enriched cultures were isolated from parietal bones of 22-day-old Sprague-Dawley rat fetuses (Charles River Breeding Laboratories), as approved by the Yale Institutional Animal Care and Use Committee. Cells pooled from the last three digestions express features of differentiating osteoblasts, including high levels of runt homology domain nuclear factor 2 (Runx2), parathyroid hormone receptor, type I collagen synthesis, and alkaline phosphatase They increase osteocalcin expression in response to vitamin D3, exhibit differential sensitivity to transforming growth factor  (TGF-), bone morphogenetic protein 2, and various prostaglandins (PGs), and form mineralized nodules under conditions promoting long term differentiation in vitro. Runx activity was assessed with luciferase reporter plasmid 5XGAL4 driven by five GAL4 response elements in cells co-transfected with an expression plasmid encoding a Runx2-GAL4 DNA binding domain fusion protein (M1Runx2) [12, 29]. Smad-dependent gene expression was assessed with luciferase reporter plasmid SBE4 driven by four Smad response elements [29]. A significant difference was assumed by a p value of Ͻ0.05
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
