Work from our laboratory as well as others have established a role for ER in mediating lactotroph proliferation and PRL expression, both in the presence and absence of E2. We have previously shown that, even in the absence of E2, the “pure” anti-estrogen ICI caused a rapid and robust degradation of ERα, leading to inhibition of cell proliferation. We also demonstrated that although Tam and Ral failed to significantly inhibit lactotroph proliferation, like ICI, they are potent inhibitors of PRL release from GH3 cells 
. These studies lead us to hypothesize that differential biological outcomes can be expected, depending upon the ability of the anti-estrogen to degrade ERα.
Our results show that ICI induces proteasome-mediated degradation of ERα in GH3 cells which was reversed by the broad spectrum proteasome inhibitor MG132 (). The ability of ICI to degrade ER via
the proteasome is consistent with previous reports in a variety of cell types 
. We next questioned whether preventing ICI-induced ERα degradation limits its growth inhibitory effect on GH3 cells. A simple approach would have been to block the ICI-induced ERα degradation with MG132 (), and then assess its effect on cell proliferation. However, we (data not shown) and others have shown that prolonged use of proteasome inhibitors results in apoptosis in lactotrophs 
. We further explored this issue by comparing the effects of MPP, a highly specific ERα antagonist that does not degrade ERα 
. Unlike ICI, which has a rapid and robust effect on ERα degradation, MPP had no effect on ERα degradation. Compared to ICI, MPP by itself was ineffective at inhibiting GH3 cell proliferation. Supporting the above conclusion were the results of the anchorage-independent growth assay as well as clonogenic property of GH3 cells. While ICI completely blocked colony formation, MPP had no significant effect. In addition, preincubation of GH3 cells with 10-fold excess MPP, blocks ICI-induced inhibition of cell proliferation (). The inability of MPP to suppress cell proliferation as well as anchorage independent growth of GH3 cells was not due to its ineffectiveness, since both ICI and MPP were effective at blocking ERE activity in reporter gene assays (). Further, this differential effect of ICI and MPP were observed in two other rat lactotroph cell lines (data not shown). Taken together, we conclude that in the absence of E2 occupation of ERα by an antagonist is not sufficient to inhibit lactotroph proliferation. Instead, ERα degradation must follow upon antagonist occupation of the receptor.
While both MPP and ICI are competitive inhibitors of ligand binding to ERα, it could be hypothesized that their differential effects on cell proliferation are due to their interaction with different pools of ERα. Although both our work (unpublished observations) as well as work from other laboratories have demonstrated the presence of nuclear as well as extra-nuclear ERα in GH3 cells, it is unlikely that ICI and MPP target different pools of ERα. This is based on our observations that excess MPP effectively blocks the ICI induced inhibition of cell proliferation. An alternate mechanism which could explain the differential growth suppressive effects of ICI and MPP, is an involvement of non-genomic/non-classical signaling mechanisms activated by anti-estrogen-occupied ER. Indeed, ICI-mediated Erk1/2 activation appears to be critical for growth modulation in immature cerebellar neurons 
. Our preliminary results demonstrate that ICI induced growth suppression of pituitary lactotrophs is accompanied with decrease in cyclin D3 expression and phosphorylation of Rb (unpublished observations). These results are consistent with a previous study demonstrating a decrease in cyclin D3 levels in response to ICI treatment in PR1 pituitary lactotrophs 
. Future studies will examine whether ICI-mediated growth suppression is due to modulation of a non-genomic signaling pathway that leads to decrease cyclin D3 expression and decreased phosphorylation of Rb.
We next explored the role of ERα degradation on PRL production and release. The use of a reporter assay, western blotting for intracellular PRL levels, and Nb2 assay for secreted PRL we found that both ICI and MPP are equally effective at suppressing PRL gene expression (), decreased intracellular PRL (), as well as decreased release of PRL from GH3 cells into CM (). When GH3 cells were preincubated with a 10 -fold excess of MPP, MPP failed to block the decreased expression of prl, intracellular levels of PRL as well as the amount of PRL released into the CM. These results indicate that occupation, and not degradation of ERα, is sufficient to inhibit prl expression, and subsequently, its production and release from GH3 cells. Since both ICI and MPP were effective at suppressing prl expression, we examined whether either compound inhibits ERE-mediated transcriptional activity. At the 10 nM levels, both ICI and MPP inhibited ERE activity by more than 75%. However, at 10 nM levels, MPP did not have any significant effect on cell proliferation or ERα degradation. This suggests that suppression of ERE transactivation is not the underlying mechanism by which ICI inhibits cell proliferation.
Taken together our results indicate that in pituitary lactotrophs, an ERα degrading antagonist such as ICI, but not an ERα occupying antagonist such as MPP, initiates a signal cascade that inhibits cell proliferation. On the other hand, occupation of ERα by either type of antagonist is sufficient for inhibiting prl expression and release. Anti-estrogens have been proposed as a class of novel therapeutics for suppressing prolactionomas. Therefore, a complete understanding of their differential effects on the lactotrophs could help in the development of more effective therapeutics.