IMMUNOHISTOCHEMICAL LOCALIZATION OF THE ESTROGEN-RECEPTOR IN HUMAN OSTEOBLASTIC SAOS-2 CELLS - ASSOCIATION OF RECEPTOR LEVELS WITH ALKALINE-PHOSPHATASE ACTIVITY
Mk. Sutherland et al., IMMUNOHISTOCHEMICAL LOCALIZATION OF THE ESTROGEN-RECEPTOR IN HUMAN OSTEOBLASTIC SAOS-2 CELLS - ASSOCIATION OF RECEPTOR LEVELS WITH ALKALINE-PHOSPHATASE ACTIVITY, Bone, 18(4), 1996, pp. 361-369
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.