Abstract

Insulin-like growth factor-I (IGF-I) has been reported to mediate the effects of oestradiol-17β in the osteoblast-like osteosarcoma cell line ROS 17/2·8 and to stimulate directly cell proliferation in cell cultures derived from rat calvaria. Few data are available on the role of IGF-I in androgen stimulation of cultured skeletal cells and in oestrogen and androgen stimulation of bone and cartilage in vivo. The purpose of the present study was to compare the effect of IGF-I in rats in vivo with its effect in vitro on calvarial bone cells from females (responding only to oestrogens) and from males (responding only to androgens, such as testosterone and dihydrotestosterone). We found that IGF-I stimulated, in a dose- and time-dependent manner, the specific activity of creatine kinase (CK, a marker of skeletal cell division), in both female and male calvarial bone cells, in ROS 17/2·8 cells and in epiphyseal cartilage cell cultures. Maximal stimulation occurred at 30 or 100 nm within 1–2 h after stimulation. In ROS 17/2·8 cells, IGF-I stimulated [³H]thymidine incorporation, after 22 h of treatment, in parallel with CK activity. IGF-II, at higher doses than IGF-I (maximal stimulation at 300 nm), stimulated CK specific activity in female- and male-derived calvarial cell cultures. When IGF-I (50 nm) was applied together with oestradiol-17β (30 nm) or with dihydrotestosterone (300 nm) there was no additional response in the cultures. IGF-I injection (1·5 μg) into immature female or male rats resulted in a time-dependent stimulation of CK specific activity in epiphyses and diaphyses starting at 2 h. Injection of IGF-I (1·5 μg) together with either 5 μg oestradiol-17β to females or 50 μg dihydrotestosterone to males did not change the pattern or the extent of response to IGF-I. The response of both male- and female-derived bone cells to IGF-I suggests that it could play a role in the effects of androgens as well as oestrogens in bone and cartilage.

Journal of Endocrinology (1994) 143, 251–259

Abstract

We have previously demonstrated sex-specific stimulation of creatine kinase specific activity (CK) in bone cells both in vivo and in vitro, in primary culture cells derived from rat and human bone and in established human bone-derived cell lines. We found that the female-derived cell line, SaOS-2, responded to 17β-estradiol (E₂) by increased CK specific activity. The effects of E₂ on the CK activity in SaOS-2 cells was inhibited by 100-fold excess of 4-hydroxytamoxifen (Tam) as well as by the other anti-estrogen, ICI 164,384. Tam by itself had some stimulatory effect whereas ICI 164,384 showed no estrogenic activity. We also demonstrated the estrogenic-like effect of another anti-estrogen, raloxifene (Ral), which is agonist only in the SaOS-2 osteoblast-like cells but not in the human endometrial, Ishikawa cell line. Ishikawa cells respond to E₂ and to Tam by increased CK activity. In both osteoblasts and endometrial cell lines, Ral and Tam were inhibitory in the presence of E₂. The effects of E₂ on SaOS-2 cells are at least partially mediated by the estrogen receptor (ER) at the level of transcription as demonstrated by transient transfection experiments using the human creatine kinase promoter chloramphenicol acetyltransferase in these cells.

Pretreatment of SaOS-2 with calcitropic hormones, either 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) or human parathyroid hormone (1–34) (hPTH(1–34)) increased the stimulation of CK by E₂ by 40–60% relative to E₂ alone and significantly increased the sensitivity of the cells to E₂ by lowering the effective hormonal dose needed for stimulation of CK by E₂ by 100-fold. This stimulatory effect of pretreatment of the cells with 1,25(OH)₂D₃ was due to a 2·5-fold increase in the level of ER expression as measured directly by enzyme immunoassay in the SaOS-2/1 subline. The increase in the responsiveness to E₂ by hPTH(1–34) was not due to an increase in ER level in the cells. We can conclude that in cell cultures as in vivo, Ral shows different effects depending on the cell type, namely estrogenic-like activity in skeletal cells but not in uterine cells. We can also conclude that as with rat-derived cells, in bone cells derived from human bone 1,25(OH)₂D₃ increased the sensitivity to E₂ due to an increase in the number of ER in the cells, whereas PTH(1–34) augmented the response to E₂ without increasing ER, by another, as yet unknown, mechanism. These studies suggest that the treatment of pathological bone disorders may be improved by combined hormone therapy.

Journal of Endocrinology (1996) 150, 275–285

Department of Biodynamics, Weizmann Institute of Science, Rehovot, Israel

(Received 4 June 1974)

Luteinizing hormone (LH) stimulates ovarian ornithine decarboxylase (ODC), the first enzyme in the pathway of polyamine synthesis, when injected into mature (Kobayashi, Kupelian & Maudsley, 1971; Jänne & Williams-Ashman, 1971) or immature rats (Kaye, Icekson, Lamprecht, Gruss, Tsafriri & Lindner, 1973). Kobayashi et al. (1971) observed a rise in ovarian ODC activity on the afternoon of prooestrus, which may be related to the ovulatory process (Kobayashi et al. 1971) and/ or the early stages of corpus luteum formation (Williams-Ashman, Jänne, Coppoc, Geroch & Schenone, 1972). We show here that ovarian ODC activity is stimulated by LH preferentially in the Graafian follicle.

Groups of three to five Wistar-derived female rats (approximately 100 days old) selected after at least two successive regular 4-day oestrous cycles, were used for surgical isolation of Graafian follicles and corpora lutea. Only corpora lutea