A direct in vitro effect of 17p-estradiol Although gonadal steroids have a profound effect on skeletal tissues (1-3), and estrogen deficiency has been established as a major etiologic factor in postmenopausal osteoporosis (1, 2), their influence has been considered to be indirect. This prevailing opinion was due to a lack of evidence for either specific estradiol receptors in bone (4) or direct biologic effects of sex steroids on bone cells (5, 6). The situation has dramatically reversed since recent studies have demonstrated significant, albeit low, concentrations of 17/3-estradiol (E2) receptors (refs. 7-9; 1) as well as androgen receptors ( ¶) in bone cells. The brain type (BB) isoenzyme of creatine kinase (CK; ATP:creatine N-phosphotransferase, EC 2.7.3.2; ref. 10) involved in the "energy buffer" system, which regulates cellular concentrations of ATP and ADP, is the major component (11) of the E2-induced protein of the rat uterus (12). E2-induced protein synthesis, and more recently, modulation of CK activity, has been a useful marker for studies on the mechanism ofaction of E2 in uterus and in other tissues that contain E2 receptors (13), because of its rapid response to E2 in vivo and in vitro (14). Moreover, it is a convenient marker for estrogen-modulated gene expression, since E2 treatment increases the steady-state level of mRNA for CK BB in the rat uterus (15). The speed and sensitivity of the assay for CK activity makes CK stimulation an efficient response marker to detect the action of E2 and other hormones (16) in skeletal tissues. In this report, we present evidence that E2 acts directly on cultured osteoblasts and epiphyseal cartilage cells, leading to increased CK activity as well as increased [3H]thymidine incorporation into DNA. Moreover, we report a rapid and sex-specific action of E2 and testosterone (T) on these markers in bones of prepubertal rats. Rat epiphyseal cells were obtained from vitamin Ddeficient 16-to 18-day-old rats, which have a wider epiphyseal cartilage zone than normally fed rats. Epiphyseal cartilage plates were isolated under a binocular dissecting microscope. Cells released by digestion with 0.25% collagenase (Worthington) in phosphate-buffered saline for 60 min at 370C were cultured as described above for calvaria cells (in 2 mM Ca2+). tTo whom reprint requests should be addressed. ¶Spelsberg, T.
MATERIALS AND METHODS