The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.

Estrogens have been shown to be essential for maintaining a sufficiently high bone mineral density and ER alpha expression has been demonstrated in bone cells. Recently, a novel estrogen receptor, estrogen receptor beta (ERbeta) has been identified. Here we demonstrate that also ERbeta is expressed in human osteoblasts, and that ER alpha and ERbeta are differentially expressed during human osteoblast differentiation. ERbeta mRNA expression increased gradually during osteoblast culture, resulting in an average increase of 9.9+/-5.3 fold (mean+/-S.D., n=3) at day 21 (mineralization phase) as compared to day 6 (proliferation phase). In contrast, ER alpha mRNA expression levels increased only slightly until day 10 (2.3+/-1.7 fold) and then remained constant. The observed differential regulation of ER alpha and beta is suggestive for an additional functional role of ERbeta to ER alpha in bone metabolism.

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