Modulation of Estrogen Receptor Levels in Mouse Uterus by Protein ...

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 11 Printed in U.S.A.

Modulation of Estrogen Receptor Levels in Mouse Uterus by Protein Kinase C Isoenzymes* SILVIA MIGLIACCIO, TODD F. WASHBURN, SILVIA FILLO, HECTOR RIVERA, ANNA TETI, KENNETH S. KORACH, AND WILLIAM C. WETSEL Department of Histology and Medical Embryology Institute (S.M.), University of Rome La Sapienza, Rome 00161, Italy; Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology (S.M., T.F.W., K.S.K.), and Hormone Action Group, Laboratory of Signal Transduction (S.F., H.R., W.C.W.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; the Department of Psychiatry and Behavioral Sciences, Duke University Medical Center (W.C.W.), Durham, North Carolina 27710; and the Department of Experimental Medicine, University of L’Aquila (A.T.), Rome, Italy 67100 ABSTRACT We have recently shown that protein kinase C (PKC) modifies estrogen receptor (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 additional estrogen target organs, such as the uterus. When uteri were incubated 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 ester reduced PKC activity levels, but increased ER binding. Interestingly, the changes in binding appeared to be due primarily to alterations in cytosolic ER levels, as binding in the nuclear fraction was minimally enhanced. When levels of ER messenger RNA were evaluated by Northern blot analysis, no differences were observed among the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that levels of ER cytosolic pro-

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HE UTERUS is recognized as one of the major targets of estrogen action, and its physiological homeostasis is controlled primarily by circulating levels of estradiol (E2) and progesterone (P) during the menstrual cycle (1– 4). E2 exerts its biological effects on the uterus as well as on other estrogen target organs by binding to the estrogen receptor (ER) (5, 6). Once the hormone is bound, the ER activates or represses the transcription of target genes by binding to highly specific DNA sequences, termed the estrogen-responsive elements (6, 7). This mechanism of action is not unique to the ER, as it is also shared by other members of steroid receptor superfamily (5, 6, 8 –11). Although ER levels can be controlled directly by estrogen, other agents can also affect the number of ERs, and they can influence its biological actions (3, 4, 7). Certain growth factors have been reported to modulate or mimic, in an autocrine and/or paracrine manner, the biological actions of E2 (12–15). Received February 17, 1998. Address all correspondence and requests for reprints to: Silvia Migliaccio, M.D., Ph.D., Histology and Medical Embryology Department, University of Rome La Sapienza, Via Antonio Scarpa 14, Rome 00161, Italy. E-mail: [email protected]. * This work was supported by funds from the NIEHS Intramural Program (to K.S.K. and W.C.W.) and the Department of Psychiatry and Behavioral Sciences at Duke University Medical Center (to W.C.W.).

tein in the H7-, TPA-, and staurosporine-treated groups were increased relative to those in the untreated controls. When uteri were incubated with diethylstilbestrol in the presence of either 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 PKCa, -bI, -bII, -d, and -z are expressed in the uterus, only PKCa and -bI are translocated from the soluble to the particulate fraction and then degraded after phorbol ester stimulation. Hence, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an important 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 important in the regulation of the ER activity in a variety of estrogen-responsive tissues. (Endocrinology 139: 4598 – 4606, 1998)

These effects can occur at a variety of different levels within the cell, and they may include changes in the transcription of ER-responsive genes, alterations in ER messenger RNA (mRNA) stability, perturbations in ER binding, and changes in ER activation and translocation (12–15). These effects are not confined to the ER, as similar effects have been reported for other members of the steroid receptor superfamily (16 –18). One signal transduction system that appears to influence glucocorticoid, progesterone, vitamin D, and ER responsiveness is protein kinase C (PKC) (19 –22). This family of enzymes controls a variety of diverse functions within the cell (23, 24) through their abilities to phosphorylate intracellular substrates and certain types of receptors, including the steroid receptors (19 –22, 25). In this respect, several investigators have reported that alterations in PKC activity can modulate ER responsiveness in different cells in vitro. For instance, exogenous activation of PKC by phorbol esters can down-regulate the ER in MCF-7 cells (26 –28) and thereby influence the responsiveness of these cells to E2 stimulation. Results from our own laboratory have shown that alterations in PKC activity, as evidenced by the proliferation or differentiation status of osteoblast cells, can regulate ER levels and estrogen responsiveness in these cells in vitro (25). To date, however, there is no evidence that this interaction can occur

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in E2-responsive whole tissues or organs. The present studies were conducted in whole uteri to evaluate whether ER levels can be modulated by alterations in PKC activity. Our results show that although changes in PKC activity modulate ER protein levels and binding, ER mRNA levels are unaffected. These data indicate that PKC affects the ER at some posttranscriptional or posttranslational step, and they suggest that this effect may be biologically relevant in modulating the responsiveness of E2 target organs such as the uterus. Materials and Methods Reagents and materials All electrophoresis reagents were obtained from Bio-Rad Laboratories, Inc. (Rockville Center, NY), whereas E2 was purchased from Research Plus (Denville, NJ). DMEM-Ham’s F-12 medium (phenol red, phosphate, and calcium free) and Coomassie brilliant blue-prestained mol wt markers were purchased from Life Technologies (Gaithersburg, MD). GeneScreen filters, [3H]E2, [g-32P]ATP, [32P]deoxy-CTP, and the Renaissance Western blot reagents were purchased from DuPont-New England Nuclear (Boston, MA). The BA-S 85 nitrocellulose membrane was from Schleichler & Schuell (Keene, NH), and the XAR-5 film was purchased from Eastman Kodak Co. (Rochester, NY). The nick translation kit, 14C-methylated protein standards, and 125I-labeled antirat IgG (Fab9)2 of sheep IgG were purchased from Amersham Corp. (Arlington Heights, IL). Agarose was obtained from FMC Bioproducts (Rockland, ME), the leupeptin was purchased from Boehringer Mannheim (Indianapolis, IN), and the peroxidase-labeled goat antirabbit IgG was obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). The ER monoclonal antibody H222 was a gift from Dr. Chris Nolan of Abbott Laboratories (North Chicago, IL). The kinase inhibitor H7 was purchased from Seikagaku Corp. America, Inc. (Rockville, MD); all other reagent grade chemicals were obtained from Sigma Chemical Co., Inc. (St. Louis, MO). The PKC antisera were obtained commercially from Oxford Biomedical Research (Oxford, MI), and protein A-Sepharose was obtained from Pharmacia Biotech (Piscataway, NJ).

Animals Female CD-1(ICR) Br female mice (Charles River Laboratories, Raleigh, NC) that had been ovariectomized 2 weeks before the study were used in these experiments. All mice were used in the experiments 14 days after ovariectomy to eliminate any potential contribution of endogenous estrogens (2). Animals were killed by cervical dislocation in accordance with the Guidelines for the Care and Use of Experimental Animals and under an approved protocol from the NIEHS animal care and use committee.

In vitro incubation of uteri Uteri were rapidly removed and cut longitudinally to expose the luminal surface. The uteri were placed in 2.5 ml DMEM-Ham’s F-12 medium containing 20 mm sodium molybdate. Different agents were added, and the uteri were incubated under 95% oxygen-5% carbon dioxide for 2 h at 37 C unless otherwise noted. The incubation period was terminated by transferring the uteri to 5 ml TEM buffer [10 mm Tris (pH 7.4), 1 mm EDTA, and 20 mm sodium molybdate] containing 2% SDS and 1 mm dithiothreitol at 25 C.

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Western blotting ER protein in the ERc and ERn fractions were first acetone precipitated (4). Samples (250 mg protein) were submitted to SDS-PAGE under denaturing conditions according to the method of O’Farrell (31), except that a 3% acrylamide-bis-acrylamide stacking gel was used. Acrylamide was substituted for bis-acrylamide as the cross-linking agent in the 10% running gel (30). The acrylamide cross-linker provides better separation and resolution of the nuclear doublet forms of the ER. Separation was achieved using a 32-cm model SE-620 Tall Boy gel (Hoeffer Scientific, San Francisco, CA) to resolve the ERn doublet. 14C-Methylated protein standards were used as mol wt markers for the gels that were analyzed by immunodetection. Coomassie brilliant blue, prestained, mol wt markers were employed as visual markers. Samples were electrophoretically transferred to nitrocellulose membranes using an LKB Multiphor II Nova Blot transfer unit (Pharmacia Biotech). Immunochemical detection of mouse uterine ER was performed using an indirect labeling technique (4). The ER was bound with the H222 primary antibody and an 125I-labeled secondary antibody. The epitope specificity of the H222 antibody resides near the steroid-binding region (32). Detection of the ER protein was performed by direct autoradiography with Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY). The data were quantified using a PhosphorImager scanner (Molecular Dynamics, Inc., Foster City, CA). For detection of the various PKC isoenzymes, uteri were incubated in medium alone or in the presence of 25 mm 12-O-tetradecanoylphorbol13-acetate (TPA) for 10, 30, 60, or 120 min. Additional groups were treated with 100 nm H7 for 30 or 120 min. Soluble and particulate fractions were prepared as described below for PKC activity assays. A small aliquot of each was saved for later protein analyses (33). Approximately 120 mg protein were loaded onto a 10% SDS-PAGE gel, and the separated materials were transferred to nitrocellulose (34). The membranes were treated as outlined previously (34). Blots were developed using the Renaissance Western blot reagents according to the manufacturer’s recommendations. The blots were analyzed with a AlphaImager Densitometer (Alpha Innotech Corp., San Leandro, CA). Although immunoreactive PKCz was present in the uterus, its levels were very low. Four hundred and fifty micrograms of uterine protein were incubated with the primary antiserum for 24 h at 4 C. Protein A-Sepharose was added to the mixture and incubated at 4 C overnight. All subsequent centrifugation and washing steps were performed according to Pharmacia’s suggestions. Samples were subjected to Western blot analysis as described above.

RNA extraction and Northern blot analysis At the end of the 2-h incubation period, uteri were immediately immersed in liquid nitrogen. Samples were pulverized to a fine powder using a mortar and pestle cooled with dry ice to avoid degradation of the RNA. The samples were homogenized in guanidine isothiocyanate, and total cellular RNA was isolated using the guanidine isothiocyanate/ cesium chloride gradient method (25). After formaldehyde denaturation, total RNA was separated using a 1% agarose gel, transferred to GeneScreen filters, processed according to the manufacturer’s instructions, and baked for 1 h at 80 C. A 829-bp ER probe (directed against the hormone-binding domain of the mouse ER) and a probe for ribosomal PL-7 were labeled with [32P]deoxy-CTP by nick translation. Blots were hybridized overnight at 42 C as previously described (25). At the end of the hybridization period, membranes were washed in 2 3 SSC solution (1 3 5 1.5 m sodium chloride and 0.15 m sodium citrate, pH 7.0) with 0.1% SDS at room temperature followed by 0.1 3 SSC with 0.1% SDS at 52 C. The blots were exposed to Kodak XAR-5 film at 280 C. The films for ER and PL-7 expression were scanned by densitometry, and expression of the ER was normalized against expression of PL-7.

ER binding assay Uteri were initially homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) for 10 sec at a setting of 6.5 and then centrifuged at 105,000 3 g for 45 min at 30 C. Cytosol (ERc) and nuclear (ERn) ER fractions were isolated as previously outlined (2). An ER exchange binding assay (29), as modified by Golding and Korach (30), was used to measure both ERc and ERn levels. Measurements obtained for each fraction were normalized to 100 mg DNA (30).

Extraction and measurement of PKC activity At the end of the 2-h incubation period, uteri were washed with PBS, minced, and homogenized in a buffer containing 20 mm Tris-HCl (pH 7.4), 2 mm EDTA, 10 mm EGTA, 250 mm sucrose, 5 mm dithiothreitol, 1 mm phenylmethylsulfonylfluoride, and 0.24 mm leupeptin. Samples were centrifuged at 100,000 3 g for 45 min at 4 C (34). After centrifugation, the supernatant (or soluble fraction) was saved. The pellet was

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homogenized in the same buffer, except it was made 0.1% Triton X-100. The samples were incubated and mixed continuously for 1 h at 4 C, then centrifuged as described above. This supernatant (or particulate fraction) was saved. Samples were loaded onto columns packed with diethylaminoethyl-Sephacel resin (Pharmacia Biotech) and were washed extensively with a solution of 20 mm Tris-HCl (pH 7.4), 20 mm sodium chloride, 0.5 mm EDTA, 0.5 mm EGTA, and 10 mm 2-mercaptoethanol. PKC was eluted with the same buffer, except that the sodium chloride concentration was increased to 100 mm. Aliquots of partially purified PKC from soluble and particulate fractions were taken and assayed for protein contents (33). PKC activity was assessed using a reaction mixture containing 20 mm Tris-HCl (pH 7.4), 8 mm magnesium chloride, 16 mm ATP, [g-32P]ATP, and 200 mg/ml histone III-S at 30 C for 3 min. Activity was determined in the presence or absence of 100 mm calcium chloride, 8 mg/ml phosphatidyl serine, and 2 mg/ml diolein. Samples were filtered with Whatman GF/C filters, and 32P incorporation into lysine-rich histone was quantified by liquid scintillation counting.

Statistics All data are expressed as the mean and sem. The data for the TPAstimulated changes in ER binding, down-regulation of PKC activity, and ERc and ERn binding in fresh uteri were analyzed by Student’s t tests. All other data were subjected to ANOVA, where a posteriori comparisons were made by Newman-Keuls tests (35).

Results

Our early studies demonstrated that alterations of PKC levels in an osteoblast-like cell line can modify ER binding (25). To determine whether an interaction between the ER and the PKC pathways is present in a classical estrogen target tissue such as the uterus, murine uteri were collected from adult animals and incubated in the presence of increasing concentrations of the selective PKC inhibitor H7 (36). After a 2-h incubation period, increasing concentrations of H7 (50 – 500 nm) enhanced total ER binding in a dose-dependent manner (Fig. 1A). Maximal binding was achieved at concentrations between 100 –500 nm. Interestingly, both ERc and ERn binding were differentially affected by the H7 treatment (Fig. 1B). Although H7 enhanced the number of binding sites for ERn, these effects were minimal and not dose dependent. By contrast, total binding in the ERc was significantly augmented by H7 in a dose-dependent manner. These data indicate that the PKC inhibitor H7 can enhance ER binding. To further delineate the role of PKC in ER binding, uteri were treated with phorbol ester to down-regulate the kinase. When uteri were incubated in the presence of 25 mm TPA, the number of ER-binding sites was augmented by approximately 185% [untreated, 140 6 4.87 fmol/mg DNA (n 5 6); treated, 259 6 9.06 fmol/mg DNA (n 5 6)]. These data are consistent with our previous results with the PKC inhibitor H7, indicating that PKC can regulate the number of ERbinding sites in the uterus. To demonstrate that PKC activity levels in the uterus were directly affected by phorbol ester treatment, activity was measured after 2 h of exposure to TPA. As expected, TPA stimulates translocation of PKC from the soluble to the particulate fraction (Fig. 2). Here, the level of PKC activity in this latter fraction was augmented by TPA exposure. In addition to translocation, TPA enhanced the loss of PKC activity, as the total activity (soluble plus particulate fractions) was less than that in the unstimulated control (Fig. 2, inset). These data indicate that down-regulation of PKC in the mouse uterus may lead to a loss of the enzyme.

FIG. 1. Effects of increasing concentrations of H7 on ER binding levels. Uteri were incubated for 2 h in the absence or presence of the selective PKC inhibitor H7. At the end of the incubation, uteri were rinsed with PBS, and ER levels were evaluated by binding assay. A, Data are presented as total ER binding levels. Open bar, Control or untreated group; filled bars, H7-treated groups. B, Results are depicted as binding in the ERc and ERn fractions, respectively. Crosshatched bars, ERc binding; horizontal bars, ERn binding. The results shown in the figures are representative of one of the two experiments conducted in duplicate where there are three observations per treatment group. *, P , 0.05 vs. the zero dose.

Northern blot analysis was performed to determine whether alterations in PKC activity influenced steady state mRNA levels of the ER. Uteri were exposed to vehicle alone, 100 nm H7, or 25 mm TPA. Two hours later, RNA was extracted and run in a Northern blot. Densitometric analyses of the ratio of ER to PL-7 expression revealed that ER mRNA levels were not influenced by either the H7 or TPA treatment (Fig. 3). Although H7 and TPA exerted no effect on uterine ER mRNA levels, ER binding was enhanced by these same treatments. Taken together, these mRNA and binding data in the uterus are similar to those previously reported by us in osteoblast cells (25), and they suggest that PKC regulates ER


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FIG. 2. Uterine PKC activity after treatment with phorbol ester or vehicle. Uteri were exposed to vehicle alone (dimethylsulfoxide) or 25 mM TPA for 2 h. PKC activity was measured in the soluble and particulate fractions as outlined in Materials and Methods. The data are expressed as picomoles of 32P incorporated into histone per min/mg protein. The inset shows a loss in total PKC activity (soluble plus particulate activity) after 2 h of continuous TPA exposure. The data were replicated three times. *, P , 0.05 vs. the control (CONT).

FIG. 3. Effects of H7 and TPA treatment on ER mRNA levels in the uterus. Northern blots were run on uteri that had been exposed to vehicle alone, 100 nM H7, or 25 mM TPA for 2 h. The locations of ER and PL-7 mRNAs are denoted in the figure. These data were replicated twice.

binding through a posttranscriptional or posttranslational mechanism. Western blot analysis of the uterine ER protein was performed to determine whether ER protein levels can be affected by agents that influence PKC. Uteri were incubated for 2 h in the presence or absence of increasing concentrations of H7. In concert with results from the ER binding studies, exposure to increasing amounts of H7 led to an augmenta-

tion of the amount of ER protein in the uterus (Fig. 4A). Densitometric analysis revealed that this augmentation of ER protein was greater than 4-fold over that of the unstimulated control. In additional experiments, PKC activity was manipulated by inhibiting the kinase with staurosporine (37) or by subjecting the kinase to down-regulation with phorbol ester. In both cases, the uterine ERc protein contents were enhanced by approximately 3-fold (Fig. 4B, left). Notably, no augmentation of ER protein was found in the nuclear fraction (Fig. 4B, right). These findings are consistent with our idea that PKC is regulating the ER at some posttranscriptional and/or posttranslational level. Although we conducted our studies in ovariectomized mice that presumably had very low or unmeasurable levels of circulating estrogens, we wanted to determine whether ER occupancy by an estrogen could influence PKC modulation of this receptor. As the synthetic estrogen, diethylstilbestrol (DES), is a potent ER agonist and because we have extensive experience with this agent, we selected it for use in this experiment. Uteri were removed immediately at the time of death and subjected to an ER binding assay, or they were incubated for 2 h with vehicle, DES, or DES in the presence of either H7 or TPA. In freshly dissected uteri, approximately 84% of the ER was located in the cytosolic fraction (Fig. 5, left). After 2 h of incubation, ER levels were reduced in the vehicle control group, particularly in the cytosolic fraction (Fig. 5, right). Exposure of the uterus to DES served to stimulate the localization of the ER from the cytosolic to the nuclear fraction. Additionally, DES somewhat retarded the loss of the total ER content from the uterus (e.g. ERc and ERn contents

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FIG. 4. Western blot analyses of ER protein after treatment with H7, TPA, or staurosporine. A, Uteri were treated for 2 h in the presence of increasing concentrations (0 –300 nM) of H7. Western blots were run as described in Materials and Methods. Total immunoreactive ER is depicted. B, To further verify that PKC could influence ER protein levels, uteri were treated with another PKC inhibitor, staurosporine (50 mM), or were down-regulated by TPA (2.5 or 25 mM) for 2 h. Note that ER protein in the cytosolic (left) and nuclear fractions (right) were examined. ER protein levels were quantified by densitometry. The data in these two panels were repeated three times.

with DES stimulation were greater than those with vehicle alone). Neither H7 nor TPA influenced ERc or ERn levels over those with DES treatment alone. These results suggest that DES may antagonize the PKC-induced modulation of ER binding in the cytosol by activating the ER and stimulating its localization from this compartment to the nucleus. Through its localization in the nucleus, the ERn may no longer be accessible to PKC. As tamoxifen and triphenylethylene derivatives have been reported to inhibit PKC activity in vitro (38), we extracted PKC from murine uteri and determined whether DES could affect PKC activity directly. When uteri were incubated for 2 h with DES, no effects on PKC activity were observed (soluble: control, 522.1 6 56.1; DES, 553.6 6 33.8; particulate: control, 29.6 6 6.1; DES, 21.3 6 9.4 pmol 32P incorporated into histone/minzmg protein; n 5 3 for each of the group conditions). These results suggest that DES does not influence uterine PKC activity directly. Collectively, our data indicate that PKC modulation of the ER only occurs for nonactivated receptors in the cytosol. Our results demonstrate that PKC can regulate ERc protein and binding levels in the uterus. As PKC is known to represent a family of isoenzymes, we sought to determine which PKC isoforms were expressed in the mouse uterus in an attempt to identify those isoforms that may be contributing to these changes in uterine ERc contents. Western blots were

run for PKCa, -bI, -bII, -g, -d, -e, and -z with isoenzymespecific antisera (34). Using mouse brain extract as a positive control, all of these PKC isoforms, except PKCg and -e, were found to be expressed in mouse uterus (data not shown). As activation of PKC is related to changes in ER binding, we examined the effects of TPA and H7 on activation, translocation, and down-regulation/degradation of PKC. Uteri were incubated with medium (control) or TPA for 10, 30, 60, and 120 min or with H7 for 30 or 120 min. While PKCa, -bI, -bII, -d, and -z were present in both soluble and particulate fractions of the uterus; in most cases, the soluble content far exceeded that of the particulate in the unstimulated uterus (Fig. 6). As expected, H7 had no effect on activation, translocation, or down-regulation of any of the PKC isoforms. This result is consistent with its role as an inhibitor of PKC (36). By contrast, administration of TPA stimulates a translocation of PKCa, -bI, and -bII from the soluble to the particulate fractions, relative to the effect of H7 and no stimulation. This translocation by phorbol ester can be seen within the first 10 min of stimulation and is still evident at 120 min. By comparison, translocation of PKCd and -z was unaffected by TPA treatment. Besides influencing translocation, TPA also stimulated degradation of certain of the PKC isoforms (Fig. 6). For instance, compared with the 30 and 60 min points, there was a loss in PKCa and -bI immunoreactivity in the particulate fractions after 120 min of stimulation. In addition, lower mol wt immunoreactive forms of PKCa were present in these particulate fractions after 10, 30, 60, and 120 min of TPA stimulation. The appearance of these immunoreactive species could be blocked with the immunizing peptide (data not shown). As TPA down-regulation of PKC led to an enhancement of ERc protein and binding in the uterus and because TPA treatment caused a selective degradation/down-regulation of PKCa and -bI in this tissue, either one or both of these two PKC isoenzymes may regulate ER levels in the cytosol of the uterus. Discussion

The present study extends our previous observations in osteoblast-like cells (25) on the interaction between the effects of ER and PKC in the uterus. In both cases, inhibition of PKC by H7 or down-regulation of the kinase by phorbol ester enhances ER binding. In the uterus, this potentiation in binding is accompanied by an increase in ER protein. The augmentation in these ER parameters appears to be due to a selective increase in ERc content, as there is a minimal enhancement in ERn binding, and these effects are not dose dependent. In addition, no effects on steady state ER mRNA levels are discerned by treatment with either H7 or TPA. Interestingly, preexposure of the uterus to DES localized the ER to the nuclear fraction and served to block the ability of PKC to influence ERc content. Analyses of the PKC isoforms by Western blot reveals that the uterus contains PKCa, -bI, -bII, -d, and -z. However, only PKCa, -bI, and -bII are translocated from the soluble to the particulate fraction in response to TPA administration. Moreover, at the end of a 1-h stimulation period, PKCa and -bI are clearly down-regulated in response to phorbol ester. Taken in concert, our data


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FIG. 5. Analyses of ER binding in uterus immediately after death or after 2 h of exposure to vehicle, DES, DES and H7, or DES and TPA. Left, Uteri were removed immediately from mice and assayed for ERc or ERn binding. Open bar, ERc binding; solid bar, ERn binding. *, P , 0.05, ERc vs. ERn binding. Right, Uteri were treated with vehicle alone, 100 nM DES alone, or DES in combination with 100 nM H7 or 25 mM TPA. At the end of 2 h, cytosol and nuclear fractions were made, and ER binding was assessed. Cross-hatched bars, ERc binding; horizontal bars, ERn binding. Each panel is representative of two independent experiments. *, P , 0.05 vs. the vehicle control.

demonstrate that PKC can regulate the ER in whole organs such as the uterus and that this interaction may be important in modulating the responsiveness and physiology of these tissues. In many tissues, estrogen can stimulate cell division and proliferation. The sensitivity of a tissue to estrogen usually depends upon the levels of the ER within that tissue. This latter relationship may be evident in some cases of breast or uterine cancer where the tumor looses its responsiveness to estrogens. Interestingly, some ER-negative cell lines contain higher levels of PKC activity than ER-positive ones (28). Although PKC has been reported to affect ER mRNA levels in some cell lines (39), the results of the present study and our experiments with osteoblast-like cells (25) indicate that PKC may regulate ER protein at some posttranscriptional level. In both preparations, a selective inhibitor of PKC (e.g. H7) was found to augment the number of ER-binding sites in a dosedependent manner. Further evidence that PKC is involved in this response derives from the phorbol ester experiments performed in the uterus. In this case, TPA depressed PKC activity or down-regulated the enzyme while enhancing ER binding. Taken together, these results suggest that the PKC-ER interaction may be relevant under a variety of physiological and pathological circumstances. The PKC-mediated enhancement of ER binding could be attributed to several different mechanisms. For instance, in MCF-7 cells, phorbol ester has been reported to alter the levels of ER mRNA and to exert effects on the levels of ER protein and binding (39). These effects were attributed primarily to changes in the stability of the ER mRNA and were observed to be maximal after 24 h of continuous exposure to TPA. In our uterine tissue experiments, although changes in

both ER binding and protein occurred, no changes in steady state ER mRNA levels were noted. A similar effect was observed by us in an osteoblast-like cell line (25). This absence of an effect on ER mRNA could be due to several factors. For instance, experiments that report changes in transcript levels typically expose cells to TPA for more than 24 h. As we only treated the uterus with TPA for 2 h, this period may be too short to observe any alterations in ER mRNA. Alternatively, the nature of the interactions between PKC and the ER may vary among different estrogen-responsive tissues and cell lines. In either case, acute exposure to TPA appears to affect uterine ER binding and protein levels at some posttranscriptional or posttranslational step that may include enhanced translational efficiency of the ER mRNA and/or inhibition of degradation of the ER. PKC has been observed to phosphorylate the progesterone (40) and vitamin D receptors (20). More recently, TPA treatment has been shown to lead to the in vitro phosphorylation of Ser118 on the human ER (41). At the present time, it is unclear whether this phosphorylation event represents a direct interaction between PKC and the ER or whether it is mediated by some other kinase that may be activated by PKC (42). Presently, the effects of Ser118 phosphorylation on ER function are controversial. Ali et al. (43) reported that mutation of Ser118 depresses ER activity by 75% compared with that in wild-type cells. On the other hand, LeGoff et al. (44) noted that the mutation only marginally affected activity. Regardless, it is conceivable that PKC could phosphorylate the ER in our own experiments. Phosphorylation of the ER could alter its conformational properties and thereby affect the ability of the ER to bind to estradiol, to its DNA response

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FIG. 6. Western blot analyses of PKC isoforms in mouse uterus. Western blots were run for PKCa, -bI, -bII, -d, and -z using the soluble and particulate fractions from uterus. The approximate size of each of these PKC isoforms is depicted on the right. Uteri were incubated in medium alone; with TPA for 10, 30, 60, or 120 min; or with H7 for 30 or 120 min. Changes in the levels of the PKC isoforms were evaluated by densitometry. Each experiment was replicated three times.

elements, and/or to the coactivators or corepressors that control the transcription of estrogen-responsive genes. In both osteoblast-like cells (25) and the uterus, we have found that inhibition or down-regulation of PKC increased the levels of ER-binding sites. Exposure to the PKC inhibitor H7 greatly enhanced uterine ERc binding in a dose-dependent manner. Although the levels of nuclear binding sites were increased by this treatment, the effect was small and was not dose dependent. The effects of PKC agents on the ER was further confirmed by our Western blot analyses, where administration of TPA or the PKC inhibitor, staurosporin, served to augment levels of the ERc protein. Negligible effects on ERn levels were observed. One characteristic of unoccupied glucocorticoid (45), progesterone (46), and estrogen (47) receptors is that they shuttle back and forth between the cytosol and the nucleus. Under this scenario, PKC could preferentially affect the unoccupied ERc. The small alterations in ERn binding could be due to some of the ERc traversing to the nuclear compartment. Regardless, these data

do not permit clear discrimination of whether PKC-ER interactions occur in cytosolic and/or nuclear compartments. In an effort to more clearly identify the location of the PKC-ER interaction, uterine tissues were treated with the synthetic estrogen DES in the presence of H7 or TPA. Parenthetically, ER agonists have been shown to bind to the ER and to localize it to the nucleus (47). In our experiment, the agonist DES had several effects. Compared with those in the vehicle control group, total ER levels were enhanced. In addition, DES localized the ER to the nucleus. Neither H7 nor TPA exerted any effect on ER binding over and above that of this DES treatment alone. In this situation, DES may abrogate the effect of PKC on the ER either by inhibiting the enzyme or, most likely, by removing the ER from a location accessible to PKC. In the former situation, tamoxifen and other estrogenic-like compounds have been reported to inhibit PKC activity in vitro (38). In our uterine experiments, however, DES had no effect on PKC activity. Thus, as DES stimulates a localization of the ER to the nucleus, this event


may serve to remove the ER from a location where PKC can interact with or phosphorylate the ER. Phosphorylation of the ER could not only affect the conformation of the receptor (as discussed above), but it could also serve to target the ER to the ubiquitin-proteosome pathway for degradation. Indeed, inhibition of PKC by H7 or down-regulation of the enzyme by TPA may serve to block this targeting process and thereby lead to enhanced ER binding and protein. Our experiments in osteoblast-like cells (25) and in the uterus clearly establish a role for PKC in regulating ER levels. PKC, however, is not a single entity; rather, it represents a family of enzymes that is composed of at least 11 different members (48). Our Western blot analyses reveal that the uterus contains 5 different PKC isoforms: PKCa, -bI, -bII, -d, and -z. These isoforms include members of the Ca21-dependent, Ca21-independent, and atypical groups. Interestingly, these same PKC isoforms are also expressed in the uterine HEC-1-B and SKUT-1-B cell lines (49). In our own uterine experiments, activation of PKC by phorbol ester was associated with translocation of only the Ca21-dependent isoforms: PKCa, -bI, and -bII. No translocation of either PKCd or -z was noted. While phorbol ester neither binds nor activates PKCz, this compound has been reported to activate PKCd in vitro (50). Despite this fact, TPA does not stimulate the translocation of PKCd in all tissues. Thus, it is unclear in our own experiments whether PKCd contributes to any of the changes that we observed with the ER in the uterus. In striking contrast, TPA administration was associated with the loss or degradation of PKCa and -bI. As inhibition or down-regulation of PKC leads to an enhancement of ER binding and protein levels in the uterus, the TPA-stimulated loss of these two Ca21-dependent isoforms suggests that they may be intimately related to these biochemical changes in the ERc. Our results in the uterus demonstrate that PKC can regulate the numbers of ERc. In osteoblast-like cells, we have shown that PKC-induced alterations in ER binding can have important functional consequences (25). The interactions between the PKC and ER systems are probably complex because they may occur at multiple levels within the cell, and they may be tissue or cell specific. For example, PKC has been reported to affect ER binding, protein, and mRNA levels in some cells (39), but not in others (25, 51). PKC has also been reported to affect ER activity beyond that of the receptor. For instance, in many tissues activation of PKC is associated with rapid changes in some of the members of the activating protein-1 family of transcription factors (52). Overexpression of c-Fos, c-Jun, or Jun-B has been reported to suppress estrogen-dependent transcription of estrogen response element-containing reporter genes (53). Thus, when these results are considered within the context of our own experiments in the uterus and in osteoblast-like cells (25), they suggest that PKC may be able to affect not only the ER itself, but also its ability to signal. Although our results in osteoblast-like cells (25) and in the uterus demonstrate that TPA can enhance ER binding, other investigators have observed phorbol esters to have no effect (51) or to depress ER binding (39). In these cases, the discrepancy in results may be due to the concentration of TPA used, the duration of treatment, or the physiological or dif-

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ferentiation status of the cells or tissue under study. Clearly, additional experiments are required to decide these issues. Although our present results demonstrate that PKC can affect ER levels in tissues such as the uterus, reports from other laboratories indicate that estrogen can also affect PKC. For instance, in the pituitary, estrogen has been reported to enhance levels of PKC activity (54). Furthermore, certain estrogenic compounds can directly influence PKC activity in vitro (38). Taken together, these data indicate that PKC and the ER may form a feedback system where each one is involved in regulating the other. This bidirectional communication may be important not only in controlling the proliferation and differentiation status of certain hormone target cells, but it may also be relevant in regulating their responsiveness and sensitivity to a myriad of different stimuli. Acknowledgment We thank Dr. Chris Nolan at Abbot Laboratories (North Chicago, IL) for his kind gift of the ER monoclonal antibody H222.

References 1. Gorski J, Toft D, Shyamala G, Smith D, Notides A 1968 Hormone receptors: studies on the interaction of estrogen with the uterus. Recent Prog Horm Res 24:45–50 2. Korach KS 1979 Estrogen action in the mouse uterus: characterization of the cytosol and nuclear receptor system. Endocrinology 104:1324 –1332 3. Quarmby VE, Korach KS 1984 The influence of 17b-estradiol on patterns of cell division in the uterus. Endocrinology 114:694 –702 4. Washburn TF, Holcutt A, Brautigan DL, Korach KS 1991 Uterine estrogen receptor in vitro: phosphorylation of nuclear specific forms on serine residues. Mol Endocrinol 5:235–242 5. Evans RM 1988 The steroid and thyroid hormone receptor family. Science 240:889 – 895 6. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Steroid receptor family: structure and function. Endocr Rev 11:201–220 7. Curtis SW, Korach KS 1990 Uterine estrogen receptor interaction with estrogen-responsive DNA sequences in vitro: effects of ligand binding on receptorDNA complexes. Mol Endocrinol 4:276 –286 8. Beato M 1989 Gene regulation by steroid hormones. Cell 58:335–344 9. Green S, Chambon P 1988 Nuclear receptor enhances our understanding of transcription regulation. Trends Genet 4:309 –319 10. Baniahmad A, Tsai MJ 1993 Mechanisms of transcriptional activation by steroid hormone receptors. J Cell Biochem 51:11151–11156 11. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/ thyroid receptor superfamily members. Annu Rev Biochem 63:451– 486 12. McLachlan JA, Nelson KG, Takahashi S, Bossert NL, Newbold RR, Korach KS 1991 Estrogen and growth factors in the development, growth, and function of the female reproductive tract. In: Schomberg DW (ed) Growth Factors in Reproduction. Springer-Verlag, New York, pp 197–203 13. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658 – 4662 14. Ma ZQ, Santagati S, Patrone P, Pollio G, Vegeto E, Maggi A 1994 Insulin-like growth factors activate estrogen receptor to control the growth and differentiation of the human neuroblastoma cell line SK-ER3. Mol Endocrinol 8:1465–1473 15. Katzenellebogen BS, Norman MJ 1990 Multihormonal regulation of the progesterone receptor in MCF-7 human breast cancer cells: interrelationships among insulin/insulin-like growth factors, serum and estrogen. Endocrinology 126:891– 891 16. Aronika SM, Katzenellenbogen BS 1991 progesterone receptor regulation in uterine cells: stimulation by estradiol, cyclic adenosine 39,59-monophosphate and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinology 128:2045–2052 17. Power RF, Lydon JP, Conneely OM, O’Malley BW 1991 Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 252:1546 –1547 18. Culig Z, Hobish A, Crnauer MV, Radmayr C, Trapman J, Hittmair A, Bartsch G, Klocker H 1994 Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor I, keratinocyte growth factor, and epidermal growth factor. Cancer Res 54:5474 –54781 19. Krishnan AV, Feldman D 1991 Activation of protein kinase C inhibits vitamin receptor D gene expression. Mol Endocrinol 5:605– 612

4606


20. Hsieh J-C, Jurutka PW, Galligan MA, Terpening CM, Haussler CA, Samuels DS, Shimizu N, Haussler MR 1991 Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. Proc Natl Acad Sci USA 88:9315–9319 21. Cho H, Katzenellebogen B 1993 Synergistic activation of estrogen receptormediated transcription by estradiol and protein kinase. Mol Endocrinol 7:441– 452 22. Weigel NL 1996 Steroid hormone receptors and their regulation by phosphorylation. Biochem J 319:657– 667 23. Nishizuka Y 1988 The molecular heterogeneity of protein kinase C and its implication for cellular regulation. Nature 334:661– 665 24. Anderson WB, Estival A, Tapiovaara H, Gopalakrishna R 1985 Altered subcellular distribution of protein kinase C (a phorbol ester receptor). A possible role in tumor promotion and regulation of cell growth: relationship to changes in adenylate cyclase activity. Adv Cyclic Nucleotide Res 19:287–306 25. Migliaccio S, Wetsel WC, Fox WM, Washburn TF, Korach KS 1993 Endogenous protein kinase C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels. Mol Endocrinol 7:1133–1143 26. Ree AH, Landmark BF, Walaas SI, Lahooti H, Eilvar L, Eskild W, Hansson V 1991 Down-regulation of messenger ribonucleic acid and protein levels for estrogen receptor by phorbol ester and calcium in MCF-7 cells. Endocrinology 129:339 –344 27. Tzukermann M, Zhang X-K, Pfahl M 1991 Inhibition of estrogen receptor activity by the tumor promoter 12– 0-tetradecanoylphorbol-13-acetate: a molecular analysis. Mol Endocrinol 5:1983–1992 28. Ha¨hnel R, Gschwedt M 1995 The interaction between protein kinase C (PKC) and estrogens. Int J Oncol 7:11–16 29. Anderson JN, Clark JH, Peck Jr EJ 1972 Oestrogen and nuclear receptor. Determination of specific sites by [3H]oestradiol exchange assay. Biochem J 526:561–567 30. Golding TS, Korach KS 1988 Nuclear estrogen receptor molecular heterogeneity in the mouse uterus. Proc Natl Acad Sci USA 85:69 –72 31. O’Farrel PH 1975 High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007– 4021 32. Greene GL, Sobel NB, King WJ, Jensen EV 1984 Immunochemical studies of estrogen receptor. J Steroid Biochem 20:51–56 33. Lowry O, Rosebrough M, Farr A, Randall R 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275 34. Wetsel WC, Khan WA, Merchenthaler I, Rivera H, Halpern AE, Phung HM, Negro-Vilar A, Hannun YA 1992 Tissue and cellular distribution of the extended family of protein kinase C isoenzymes. J Cell Biol 117:121–133 35. Winer BJ 1971 Statistical Principles in Experimental Design. McGraw-Hill, New York 36. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y 1984 Isoquinolinesulfoinamides, a novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036 –5041 37. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F 1986 Staurosporine, a potent inhibitor of phospholipid/Ca21 dependent protein kinase. Biochem Biophys Res Commun 135:397– 402 38. Bignon E, Kishimoto A, Pons M, de Paulet AC, Gilbert J, Miquel J-F, Nishi-

39.

40. 41. 42.

43. 44.

45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

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zuka Y 1990 Dual action of hydroxylated diphenylthylene estrogens on protein kinase C. Biochem Biophys Res Commun 166:1471–1478 Saceda M, Knabbe C, Dickson RB, Lippman ME, Bronzert D, Lindsey RK, Gottardis MM, Martin MB 1991 Post-transcriptional destabilization of estrogen receptor mRNA in MCF-7 cells by 12-O-tetradecanoylphorbol-13-acetate. J Biol Chem 266:17809 –17814 Boyle DM, van der Walt LA 1988 High performance affinity chromatography of human progesterone receptor. J Steroid Biochem 30:239 –244 Joel PB, Traish AM, Lannigan DA 1995 Estradiol and phorbol ester cause phosphorylation of serine 118 in the human estrogen receptor. Mol Endocrinol 9:1041–1052 Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Maushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494 Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:1153–1160 LeGoff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor: identification of hormone-regulated sites and examination of their influence on transcriptonal activity. J Biol Chem 269:4458 – 4466 Picard D, Yamamoto KR 1987 Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J 6:3333–3340 Guiochon-Mantel A, Lescop P, Christin-Maitre S, Loosfelt H, PerrotApplanat M, Milgrom E 1991 Nucleoplasmic shuttling of the progesterone receptor. EMBO J 10:3851–3859 Dauvois S, White R, Parker MG 1993 The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci 106:1377–1388 Newton AC 1995 Protein kinase C: structure, function, and regulation. J Biol Chem 270:28495–28498 Bamberger A-M, Bamberger CM, Wald M, Kratzmeier M, Schulte HM 1996 Protein kinase C (PKC) isoenzyme expression pattern as an indicator of proliferative activity I uterine tumor cells. Mol Cell Endocrinol 123:81– 88 Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Igarashi K, Nishizuka Y 1988 The structure, expression, and properties of additional members of the protein kinase C family. J Biol Chem 263:6927– 6932 Lake LM, Murai JT, Gerchenson LE 1982 Inhibition by 12-O-tetradecanoylphorbol-13-acetate of 17b-estradiol-induced initiation of DNA synthesis in rabbit endometrial cells in culture. Carcinogenesis 3:703–705 Karin M 1991 The AP-1 complex and its role in transcriptional control by protein kinase C. In: Cohen P, Foulkes JG (eds) The Hormonal Control Regulation of Gene Transcription. Elsevier, Amsterdam, pp 235–253 Doucas V, Spyrou G, Yaniv M 1991 Unregulated expression of c-Jun or c-Fos proteins but not Jun D inhibits oestrogen receptor activity in human breast cancer derived cells. EMBO J 10:2237–2245 Maeda T, Lloyd R 1993 Protein kinase C activity and messenger RNA modulation by estrogen in normal and neoplastic rat pituitary tissue. Lab Invest 68:472– 480