Long-term culture in dexamethasone unmasks an abnormal phenotype in osteoblasts isolated from osteoporotic subjects.

We have shown that osteoblastic cells derived from trabecular bone explants of osteoporotic subjects (OP cells) exhibited an altered alkaline phosphatase (ALP) response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] compared to control (CON) cells. Our hypothesis that OP cells have other intrinsic abnormalities was investigated using our cell models representing two different stages of differentiation. OP and CON cells were cultured in the absence (-DEX) or presence (+DEX) of 10 nM dexamethasone (DEX) in 10% fetal calf serum (FCS) prior to exposure to serum-free medium containing 1 nM of PTH and/or 17-beta estradiol (E2). Both OP and CON cells responded to DEX with a two-fold increase in basal ALP activity. While E2 or PTH+E2 had no effect on OP cells, both treatments inhibited ALP activity in CON cells (p<0.05). OP and CON cells grown in DEX also expressed PTH-stimulated adenylate cyclase (AC) activities higher than those of (-DEX) cells. OP+DEX cells, however, exhibited activities which were 8-fold higher than those of CON+DEX cells (p<0.001). In OP+DEX cells, E2 stimulated basal AC activity (p<0.05) but did not affect PTH-stimulated activity. In contrast, in CON+DEX cells, E2 had no effect on basal activity but inhibited PTH-stimulated AC activity (p<0.001). Osteocalcin production was 4-fold lower in OP+DEX cells compared to OP-DEX and CON cells (p<0.05) while osteocalcin mRNA levels were significantly lower in OP+DEX and CON+/-DEX cells compared to OP-DEX cells (p<0.05). E2 did not affect osteocalcin protein or mRNA levels in either OP or CON cells. No differences in mRNA levels were found for estrogen receptor-alpha (ER-a) in OP+/-DEX cells whereas these levels were significantly higher in CON+DEX compared to CON-DEX cells (p<0.05). These results indicate that DEX amplified the differences between OP and CON cells and confirm the presence of intrinsic osteoblastic abnormalities in patients with osteoporosis that persist in culture.

Immunohistochemical localization of the estrogen receptor in human osteoblastic SaOS-2 cells: association of receptor levels with alkaline phosphatase activity.

We have previously shown that the combination of estrogen (E2) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] enhanced alkaline phosphatase (ALP) activity in human osteosarcoma SaOS-2 cells which had been grown in the presence of 10 nmol/L dexamethasone (SaOS + DEX cells). To determine whether this increase in ALP activity was associated with changes in receptor protein levels for E2 (ER) in individual SaOS + DEX cells, a monoclonal antibody to ER and a histochemical stain for ALP were used localize the expression of these proteins in fixed cells. Western and Northern blot analyses were used to determine whether E2 and 1,25(OH)2D3 affected immunoreactive ER protein and mRNA levels, respectively. Our results showed that immunohistochemical staining for ER was primarily nuclear, whereas histochemical staining for ALP was cytosolic. Treatment of cells with 1,25(OH)2D3, E2, or E2 + 1,25(OH)2D3 increased the levels of both ER and ALP activity, as visualized by enhanced cellular staining. Western analyses showed that 1,25(OH)2D3 and E2, separately and in combination, significantly increased ER protein levels. 1,25(OH)2D3 enhanced ER levels in a dose-dependent manner [analysis of variance (ANOVA), F = 3.91, p < 0.05]; this effect was augmented by E2 (ANOVA, F = 5.98, p < 0.005). In comparison, 17 alpha-E2 + 1,25(OH)2D3 and tamoxifen + 17 beta-E2 + 1,25(OH)2D3 did not increase ER levels compared with those obtained with 17 beta-E2 + 1,25(OH)2D3. ER mRNA levels were not significantly increased by E2, 1,25(OH)2D3, or E2 + 1,25(OH)2D3 together. In contrast, in a population of SaOS cells which had been in culture longer (approximately 40 passages more) than the previous cells, E2 + 1,25(OH)2D3 did not enhance ALP activity or ER levels above those obtained with 1,25(OH)2D3 alone. These results showed that in responsive SaOS cells, E2 enhanced both the stimulatory effects of 1,25(OH)2D3 on ALP activity and the activation of ER. Thus changes in ALP activity are associated with changes in ER levels in SaOS + DEX cells.

17 beta-oestradiol enhances the stimulatory effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity in human osteosarcoma SaOS-2 cells in a differentiation-dependent manner.

We tested the effect of osteoblastic differentiation on the interactive effects of 17 beta-oestradiol (E2) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on alkaline phosphatase activity. As cell models we utilized the more differentiated human osteosarcoma (SaOS) cells that had been cultured for 6 days in medium containing 10 nM dexamethasone (Dex) (SaOS+Dex cells) and the less differentiated cells cultured in the absence of Dex (SaOS-Dex cells). The cells were challenged with 1,25(OH)2D3 in the presence or absence of Dex for 24 h and then with E2 for an additional 24 h. In SaOS-Dex cells, alkaline phosphatase activity remained constant over the 48-h period and was not significantly affected by E2, 1,25(OH)2D3 or 1,25(OH)2D3+E2 treatment. On the other hand, in SaOS+Dex cells, 1,25(OH)2D3 and E2+1,25(OH)2D3 stimulated alkaline phosphatase activity (ANOVA, F = 154.2, P < 0.0001) with the maximal response at 48 h (P < 0.01). In SaOS+Dex cells, 1,25(OH)2D3 had dose-dependent stimulatory effects which were strongly enhanced by 10 nM E2 (ANOVA, F = 46.0, P < 0.001). Studies on dose-dependent effects of E2, in the presence or absence of 100 nM 1,25(OH)2D3, revealed that in the presence of 1,25(OH)2D3, the E2 dose-response curve was biphasic in SaOS+Dex cells (ANOVA, F = 3.40, P < 0.005), with maximum stimulation at 10 nM E2 (P < 0.01). The specificity of E2 was verified using the inactive 17 alpha-oestradiol and the oestrogen antagonist, tamoxifen. These data indicate that E2 and 1,25(OH)2D3 have positive interactive effects on alkaline phosphatase activity in human osteoblasts, and suggest that the expression of this interaction is dependent on the stage of differentiation of the cells.

Positive interaction between 17 beta-Estradiol and parathyroid hormone in normal human osteoblasts cultured long term in the presence of dexamethasone.

We previously developed two models of human osteoblasts with distinct differentiation stages using cells derived from iliac crest trabecular bone explants cultured long term in the presence (HOB + DEX) and absence (HOB - DEX) of 10 nM dexamethasone (DEX) (Wong et al., J Bone Miner Res 1990;5:803). Using these models from 36 subjects aged 41-80 years, we examined the effects of 17 beta-estradiol (E2) on cell proliferation, osteocalcin (OC) production, alkaline phosphatase (ALP) and basal and parathyroid hormone (PTH)-stimulated adenylate cyclase activities, as well as the steady-state mRNA levels of ALP, collagen type I(COLL), OC, and receptors for E2 (ER) and PTH (PTHr). E2 alone had no effect on [3H]thymidine uptake in (HOB - DEX) cells but appeared to stimulate the uptake in (HOB + DEX) cells in a dose-dependent manner, with maximum effect at 10(-10)M (p < 0.05). However, in the presence of 10(-6)M PTH, E2 inhibited the uptake in (HOB - DEX) cells (ANOVA, KW = 18.95, p < 0.005) but stimulated the uptake in (HOB + DEX) cells (KW = 13.52, p < 0.025). E2 decreased the amount of osteocalcin in culture media from both (HOB - DEX) and (HOB + DEX) cells (p < 0.05). PTH alone or E2, alone or in combination with 10(-9)M PTH, had no effect on ALP activity in (HOB - DEX) cells. In contrast, in (HOB + DEX) cells, E2 + PTH but not E2 alone, had biphasic effects on ALP activity, with maximum stimulation observed at 10(-11) and 10(-10)M E2, and a return to basal levels at 10(-9)M E2. E2 decreased basal adenylate cyclase activities in a dose-dependent manner in (HOB + DEX) but not (HOB - DEX) cells (KW = 13.48, p < 0.05). In (HOB + DEX) cells, E2 had biphasic effects on PTH-stimulated adenylate cyclase activity, with significant stimulation observed at 10(-10)M (p < 0.05). While E2 had no significant effect on osteoblastic marker mRNA levels in (HOB - DEX) cells, it decreased osteocalcin and stimulated PTHr mRNA levels in (HOB + DEX) cells. Thus, in our human osteoblastic cell models, estrogen regulated metabolic function largely in the more differentiated cells, by modifying the effects of PTH.

Age-dependent expression of osteoblastic phenotypic markers in normal human osteoblasts cultured long-term in the presence of dexamethasone.

We have previously shown that osteoblasts derived from trabecular bone explants and cultured long term in 10 nM dexamethasone ((HOB + DEX) cells) exhibited properties consistent with a more differentiated phenotype compared with those grown in the absence of dexamethasone ((HOB-DEX) cells). To characterize these two cell models further, we measured the steady-state mRNA levels of the phenotypic markers alkaline phosphatase (ALP), collagen type I (COLL) and osteocalcin (OC), OC production, and the activities of ALP and parathyroid hormone (PTH)-stimulated adenylate cyclase. These findings were then correlated with the age and sex of the bone donors. Long-term culture in dexamethasone significantly increased ALP and OC mRNA levels and the activities of ALP and PTH-stimulated adenylate cyclase but not OC production, in (HOB + DEX) compared with (HOB-DEX) cells (p < 0.05). When the data were examined with respect to the age of the bone donor, age-dependent differences in the expression and responses to dexamethasone were apparent. ALP and PTH-stimulated adenylate cyclase activities decreased with increasing age of the bone donor in (HOB-DEX) and (HOB + DEX) cells (p < 0.05). There were no significant correlations between phenotypic marker mRNA levels and bone donor age in (HOB-DEX) and ((HOB + DEX) cells. All age-dependent decreases in ALP and PTH-stimulated cyclase activities were enhanced in the (HOB + DEX) cells. However, when the data were examined according to the sex of the bone donor, there were no differences in mRNA levels, OC production, or ALP and cyclase activities between cells from male and female donors. These results indicate an age dependence in the expression of osteoblastic markers in human bone cells at different stages of differentiation: thus osteoblastic cultures derived from older donors are likely to contain fewer osteoprogenitor cells, lower levels of glucocorticoid receptors or represent more differentiated osteoblasts compared with those derived from younger donors.

Carboxyl-terminal parathyroid hormone peptide (53-84) elevates alkaline phosphatase and osteocalcin mRNA levels in SaOS-2 cells.

Previous findings in our laboratory have shown that hPTH-(53-84) stimulates alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells. In the present study, we examined the effects of hPTH-(53-84) and hPTH-(1-34) on the expressions of alkaline phosphatase, osteocalcin, and collagen type I mRNA levels in the human osteosarcoma cell line SaOS-2. The effect of hPTH-(53-84) on alkaline phosphatase and osteocalcin message levels was dose dependent (ANOVA, p < 0.005 and p < 0.001, respectively), with significant stimulation observed at 10 nM. Treatment with 10 nM hPTH-(53-84) for 24 h resulted in significant 2- and 1.4-fold increases in mRNA levels for alkaline phosphatase and osteocalcin, respectively (p < 0.05), but had no effect on collagen type I expression. The increased alkaline phosphatase mRNA levels was associated with a 1.5-fold increase in enzyme activity (p < 0.05). In contrast, under similar incubation conditions, hPTH-(1-34) had no significant effects on alkaline phosphatase or osteocalcin mRNA levels. On the other hand, hPTH-(1-34) had dose-dependent stimulatory effects on collagen type I mRNA levels (ANOVA, p < 0.001), 10 nM hPTH-(1-34) stimulating collagen type I expression 1.6-fold (p < 0.05). The results indicate that carboxyl-terminal hPTH-(53-84) has direct and unique biologic effects in human osteoblast-like cells in culture.

17 beta-estradiol and parathyroid hormone potentiate each other's stimulatory effects on alkaline phosphatase activity in SaOS-2 cells in a differentiation-dependent manner.

We studied the effects of 17 beta-estradiol (E2) and PTH on alkaline phosphatase activity in SaOS-2 cells that had been passaged and grown continually in the presence (SaOS + Dex) or absence (SaOS - Dex) of 10(-8) M dexamethasone (Dex). We showed that the more differentiated SaOS + Dex cells had higher alkaline phosphatase activity and PTH-responsive adenylate cyclase than the less differentiated SaOS - Dex cells. In SaOS - Dex cells, E2 or PTH (1 x 10(-11)-1 x 10(-6) M) had no effect on alkaline phosphatase activity. On the other hand, in SaOS + Dex cells, PTH and E2 each had small but very significant stimulatory effects on alkaline phosphatase activity. The combined effects of PTH and E2 resulted in potentiation which was dose dependent for PTH and E2. 17 alpha-estradiol and tamoxifen (1 x 10(-11)-1 x 10(-6) M) had no effect on PTH-stimulated alkaline phosphatase activity in SaOS + Dex cells, but the latter inhibited the E2 effect, clearly demonstrating its specificity. In conclusion, we have shown that in more differentiated osteoblastic cells, E2 and PTH have interactive effects on alkaline phosphatase activity.

Modulation of parathyroid hormone-sensitive adenylate cyclase in ROS 17/2.8 cells by dexamethasone 1,25-dihydroxyvitamin D3 and protein kinase C.

We tested whether the protein kinase C (PKC) modulation of PTH-sensitive adenylate cyclase in ROS 17/2.8 cells is affected by the glucocorticoid dexamethasone and the vitamin D hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Basal and PTH- and forskolin-stimulated adenylate cyclase activities were determined in the presence or absence of 100 nM phorbol 12-myristate 13-acetate (PMA), the activator of PKC, in ROS 17/2.8 cells that had been previously cultured with or without dexamethasone or 1,25(OH)2D3. Dexamethasone treatment increased the basal, PMA-, PTH-, (PTH + PMA)- and (forskolin + PMA)-sensitive adenylate cyclase while 1,25(OH)2D3 decreased these effects. The stimulatory and inhibitory effects were dose-dependent with respect to dexamethasone and 1,25(OH)2D3, respectively. Dexamethasone increased, while 1,25(OH)2D3 decreased the maximal activity of both PTH-sensitive and PKC-modulated PTH-sensitive adenylate cyclase without affecting the half-maximal concentration (ED50) of PTH required for the activation of the enzyme. Additionally, dexamethasone, 1,25(OH)2D3 and PKC did not affect each other's ED50. Our results suggest that the effects of dexamethasone, 1,25(OH)2D3 and PKC on PTH-sensitive adenylate cyclase in ROS 17/2.8 cells are independent of each other.