MODULATION OF ESTROGEN-RECEPTOR LEVELS IN MOUSE UTERUS BY PROTEIN-KINASE-C ISOENZYMES

We have recently shown that protein kinase C (PKC) modifies estrogen r eceptor (ER) binding and modulates the responsiveness to estrogens in a clonal osteoblast-like cell line stably transfected with the ER. The purpose of the present study was to determine whether the interaction observed between the ER and PKC signaling in these cells occurs in ad ditional estrogen target organs, such as the uterus. When uteri were i ncubated for 2 h with increasing concentrations of a kinase inhibitor (H7), ER binding was enhanced in a dose-dependent manner. Stimulation of PKC with phorbol eater reduced PKC activity levels, but increased E R binding. Interestingly, the changes in binding appeared to be due pr imarily to alterations in cytosolic ER levels, as binding in the nucle ar fraction was minimally enhanced. When levels of ER messenger RNA we re evaluated by Northern blot analysis, no differences were observed a mong the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that l evels of ER cytosolic protein in the H7-, TPA-, and staurosporine-heat ed groups were increased relative to those in the unheated controls. W hen uteri were incubated with diethylstilbestrol in the presence of ei ther H7 or TPA, no change in cytosolic ER levels was found, suggesting that only unoccupied ERs are responsive to modulation by PKC. Western blotting of the various PKC isoforms indicated that although PKC alph a, -beta(I), -beta(II), -delta, and -zeta are expressed in the uterus, only PKCa and -beta(I) are translocated from the soluble to the parti culate fraction and then degraded after phorbol ester stimulation. Hen ce, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an import ant role in the modulation of uterine ER levels and that PKC may exert its effect on the ER at some posttranscriptional or posttranslational step. Finally, our results show that an ER-PKC interaction occurs in a whole organ such as the uterus and that this interaction may be impo rtant in the regulation of the ER activity in a variety of estrogen-re sponsive tissues.