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 (E(2)) and 1 ,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] enhanced alkaline phosphatas e (ALP) activity in human osteosarcoma SaOS-2 cells which had been gro wn in the presence of 10 nmol/L dexamethasone (SaOS + DEX calls). To d etermine whether this increase in ALP activity was associated with cha nges in receptor protein levels for E(2) (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. We stern and Northern blot analyses were used to determine whether E(2) a nd 1,25(OH)(2)D-3 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)(2)D-3, E(2), or E(2) + 1,2 5(OH)(2)D-3 increased the levels of both ER and ALP activity, as visua lized by enhanced cellular staining. Western analyses showed that 1,25 (OH)(2)D-3 and E(2), separately and in combination, significantly incr eased ER protein levels. 1,25(OH)(2)D-3 enhanced ER levels in a dose-d ependent manner [analysis of variance (ANOVA), F = 3.91, p < 0.05]; th is effect was augmented by E(2) (ANOVA, F = 5.98, p < 0.005). In compa rison, 17 alpha-E(2) + 1,25(OH)(2)D-3 and tamoxifen + 17 beta-E(2) + 1 ,25(OH)(2)D-3 did not increase ER levels compared with those obtained with 17 beta-E(2) + 1,25(OH)(2)D-3. ER mRNA levels were not significan tly increased by E(2), 1,25(OH)(2)D-3, or E(2) + 1,25(OH)(2)D-3 togeth er. In contrast, in a population of SaOS cells which had been in cultu re longer (similar to 40 passages more) than the previous cells, E(2) + 1,25(OH)(2)D-3 did not enhance ALP activity or ER levels above those obtained with 1,25(OH)(2)D-3 alone. These results showed that in resp onsive SaOS cells, E(2) enhanced both the stimulatory effects of 1,25( OH)(2)D-3 on ALP activity and the activation of ER. Thus changes in AL P activity are associated with changes in ER levels in SaOS + DEX cell s.