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 phosphatase (ALP) activity in human osteosarcoma SaOS-2 cells which had been grown in the presence of 10 nmol/L dexamethasone (SaOS + DEX calls). To determine whether this increase in ALP activity was associated with changes 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. Western and Northern blot analyses were used to determine whether E(2) and 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) + 1,25(OH)(2)D-3 increased the levels of both ER and ALP activity, as visualized by enhanced cellular staining. Western analyses showed that 1,25(OH)(2)D-3 and E(2), separately and in combination, significantly increased ER protein levels. 1,25(OH)(2)D-3 enhanced ER levels in a dose-dependent manner [analysis of variance (ANOVA), F = 3.91, p < 0.05]; this effect was augmented by E(2) (ANOVA, F = 5.98, p < 0.005). In comparison, 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 significantly increased by E(2), 1,25(OH)(2)D-3, or E(2) + 1,25(OH)(2)D-3 together. In contrast, in a population of SaOS cells which had been in culture 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 responsive 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 ALP activity are associated with changes in ER levels in SaOS + DEX cells.