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Molecular Endocrinology, doi:10.1210/me.2004-0164
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Molecular Endocrinology 18 (11): 2603-2615
Copyright © 2004 by The Endocrine Society

Inhibiting Proteasomal Proteolysis Sustains Estrogen Receptor-{alpha} Activation

Meiyun Fan, Harikrishna Nakshatri and Kenneth P. Nephew

Medical Sciences (M.F., K.P.N.), Indiana University School of Medicine, Bloomington, Indiana 47405; Department of Surgery (H.N.), Department of Biochemistry and Molecular Biology, Walther Oncology Center; Indiana University Cancer Center (H.N., K.P.N.); and Department of Cellular and Integrative Physiology (K.P.N.), Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to:Kenneth P. Nephew, Ph.D., Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, Indiana 47405-4401. E-mail: knephew{at}indiana.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor-{alpha} (ER{alpha}) is a ligand-dependent transcription factor that mediates physiological responses to 17ß-estradiol (E2). Ligand binding rapidly down-regulates ER{alpha} levels through proteasomal proteolysis, but the functional impact of receptor degradation on cellular responses to E2 has not been fully established. In this study, we investigated the effect of blocking the ubiquitin-proteasome pathway on ER{alpha}-mediated transcriptional responses. In HeLa cells transfected with ER{alpha}, blocking either ubiquitination or proteasomal degradation markedly increased E2-induced expression of an ER-responsive reporter. Time course studies further demonstrated that blocking ligand-induced degradation of ER{alpha} resulted in prolonged stimulation of ER-responsive gene transcription. In breast cancer MCF7 cells containing endogenous ER{alpha}, proteasome inhibition enhanced E2-induced expression of endogenous pS2 and cathepsin D. However, inhibiting the proteasome decreased expression of progesterone receptor (PR), presumably due to the heterogeneity of the PR promoter, which contains multiple regulatory elements. In addition, in endometrial cancer Ishikawa cells overexpressing steroid receptor coactivator 1, 4-hydroxytamoxifen displayed full agonist activity and stimulated ER{alpha}-mediated transcription without inducing receptor degradation. Collectively, these results demonstrate that proteasomal degradation is not essential for ER{alpha} transcriptional activity and functions to limit E2-induced transcriptional output. The results further indicate that promoter context must be considered when evaluating the relationship between ER{alpha} transcription and proteasome inhibition. We suggest that the transcription of a gene driven predominantly by an estrogen-responsive element, such as pS2, is a more reliable indicator of ER{alpha} transcription activity than a gene like PR, which contains a complex promoter requiring cooperation between ER{alpha} and other transcription factors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ACTIONS OF estrogens are mediated primarily through estrogen receptors (ER{alpha} and ERß) (1), ligand-dependent transcription factors that interact directly with estrogen response elements (EREs) in the promoters of target genes (1). Cellular levels of ER{alpha} (2), along with a large number of receptor coregulator complexes (3), play key roles in controlling appropriate physiological responses in estrogen target tissues, such as breast and uterus. Levels of ER{alpha} mRNA and protein are regulated primarily by its cognate ligand, 17ß-estradiol (E2) (4, 5, 6). E2 binding results in rapid turnover of ER{alpha} protein through the ubiquitin (Ub)-proteasome pathway (7, 8, 9, 10, 11), which has been implicated in both the overall control of gene transcription (12, 13, 14, 15, 16) and transactivation function of ER{alpha} and other nuclear receptors (7, 17, 18, 19, 20, 21, 22, 23, 24).

The Ub-proteasome system consists of the 26S proteasome, a complex composed of a 20S catalytic core for protein proteolysis and two ATPase-containing 19S regulatory particles that recognize polyubiquitin-tagged substrates (25). Like many other transcription factors, stimulation of ER{alpha} transcriptional activation appears to be associated with receptor ubiquitination and proteasomal degradation (11, 26). Several proteins possessing Ub ligase activity (e.g. E6AP, p300, BRCA1, and MDM2), as well as SUG1, a component of the 19S proteasome, have been shown to associate with ER{alpha} and modulate receptor signaling (27, 28, 29, 30, 31, 32, 33, 34). These observations suggest that proteasome-mediated receptor degradation is important for ER function.

Recent studies have demonstrated that inhibiting proteasomal degradation increases transcriptional activity of many, but not all, nuclear receptors, indicating a receptor-specific effect of proteasome inhibition (17, 18, 19, 20, 21, 22, 23, 24). Blocking ER{alpha} turnover by a proteasome-specific inhibitor, MG132, results in decreased expression of an ER{alpha}-responsive luciferase reporter, implicating that proteasomal degradation of ER{alpha} is required for its transactivation function (7, 35). However, MG132, and other proteasome inhibitors, have recently been shown to deleteriously affect production of a functional firefly luciferase enzyme (36), complicating the assessment of studies utilizing only ER{alpha}-responsive reporters expressing luciferase, in combination with 20S proteasome inhibitors. In addition, several studies have recently suggested that receptor degradation may not be required for ER{alpha}-mediated transcription. Frasor et al. (11, 37) reported that the partial agonist/antagonist 4-hydroxytamoxifen (4-OHT), which protects ER{alpha} from proteasomal degradation, stimulates ER-mediated transcription of a group of genes in MCF7 cells (38). Dissociation of ER{alpha} activation from degradation has also been reported in pituitary tumor cells (39, 40).

In the present study, we investigated the role of the Ub-proteasome pathway in ER{alpha}-mediated transcriptional responses. Genetic and pharmacological approaches were used to disrupt ER{alpha} ubiquitination, proteasome-mediated proteolysis, and thus ER{alpha} degradation, including the 20S proteasome inhibitor MG132, a dominant-negative mutant of the NEDD8 conjugation enzyme (Ubc12C111S) (41, 42), a Ub mutant with all of its lysines mutated to arginine (UbK0) (43), and the partial agonist/antagonist 4-OHT. To determine the effect of blocking ER{alpha} degradation on E2-induced transcriptional responses, ER-responsive reporter assays and expression of endogenous ER-target genes were used. The results demonstrate that proteasomal degradation is not essential for transcriptional activity of ER{alpha} and indicate that the Ub-proteasome system functions to limit E2-induced transcriptional output.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inhibiting the Proteasome Increases ER{alpha} Transcriptional Output
The enzymatic activity of chloramphenicol acetyltransferase (CAT), luciferase (Luc) or ß-galactosidase (Gal) reporter proteins is commonly used for assessing transcriptional activity of nuclear receptors in the presence of proteasome inhibitors. Recent studies with breast cancer T47D cells revealed that proteasome inhibitors (MG132, lactacystin, and proteasome inhibitor I) interfere with the production of luciferase and galactosidase proteins by a posttranscriptional mechanism, whereas the enzymatic activity of CAT remains unaffected (36). To verify these observations in our experimental systems, we examined the effect of MG132 on expression of these reporter enzymes from constitutively active constructs in cervical carcinoma HeLa and breast cancer MCF-7 cells. Cells were transfected with Rous sarcoma virus (RSV)-CAT, simian virus 40 (SV40)-Luc, or cytomegalovirus (pCMV)-ß-gal and then treated with vehicle [dimethylsulfoxide (DMSO)] or MG132 (1 µM) for 24 h. Reporter enzyme activity was determined using standard assays for luciferase, CAT, and galactosidase. Treatment of HeLa cells with MG132 had no effect on CAT activity but decreased luciferase and galactosidase activity by 80% and 30%, respectively (Fig. 1AGo, left panel). Essentially similar results were obtained using MCF7 cells (Fig. 1AGo, right panel). These results agree with a previous report demonstrating that proteasome inhibitors have deleterious effects on the enzymatic activities of luciferase and galactosidase reporter proteins (36).



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  		Fig. 1. Proteasome Inhibition Enhances E2-Induced CAT Reporter Gene Expression in HeLa Cells Transfected with ER{alpha}

A, Effect of proteasome inhibition by MG132 on expression of reporter enzymes from constitutively active promoters. HeLa cells (left panel) were plated on 12-well dishes at a density of 1 x 105 cells per well and cultured in hormone-free medium for 3 d. The cells were transfected with 100 ng RSV-CAT, 100 ng SV40-Luc, or 5 ng pCMV-ß-gal using LipofectAMINE Plus Reagent. The DNA/LipofectAMINE mixture was removed 5 h later and cells were placed in hormone free medium containing either 0.1% vehicle (DMSO) or 1 µM MG132 for 24 h. Similarly, MCF7 cells (right panel) were plated at a density of 1.2 x 105 cells per well, transfected with 250 ng RSV-CAT, 250 ng SV40-Luc, or 10 ng pCMV-ß-gal and then treated with DMSO or MG132 for 24 h. Reporter enzyme activities were normalized against total cellular protein and expressed as the mean ± SD from three independent experiments, each in triplicate. B, Effect of MG132 on ER{alpha}-mediated CAT expression. HeLa cells were plated in 12-well dishes at a density of 1 x 105 cells per well and cultured in hormone-free medium for 2 d. The cells were transfected with 100 ng ERE-Vit-CAT and the indicated amount of pSG5-ER{alpha} using LipofectAMINE Plus reagent. The DNA/LipofectAMINE mixture was removed 5 h later and cells were placed in hormone-free medium for 24 h. Transfected cells were treated with DMSO or MG132 (1 µM) for 1 h and then treated with 10 nM E2 for 24 h. CAT activity was determined using the colorimetric CAT ELISA kit and normalized against total cellular protein. CAT activity is expressed as the mean ± SD of three independent experiments, each performed in triplicate. Fold increases in ERE-CAT in the presence of E2 ± MG132 are presented in the table. C, Effect of MG132 on E2-induced down-regulation of ER{alpha}. HeLa cells were plated in 60-mm dishes at a density of 3 x 105 cells per dish and cultured in hormone-free medium for 2 d. Cells were transfected with 100 ng pSG5-ER{alpha} using LipofectAMINE Plus reagent. The DNA/LipofectAMINE mixture was removed 5 h later, and cells were placed in hormone-free medium for 24 h. The transfected cells were treated with DMSO or MG132 (1 µM) for 1 h and then treated with 10 nM E2 for 8 h. Whole-cell lysates were prepared and subjected to immunoblotting analysis using an anti-ER{alpha} antibody (Chemicon, Temecula, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

 
Previously, we and others showed that E2 induces ER{alpha} degradation in transiently transfected HeLa cells and MG132 abolishes such degradation (8, 9, 42). Based on the above results, we further investigated the relationship between ER{alpha} turnover and E2induced transcriptional response using an E2-responsive CAT reporter. HeLa cells were transiently transfected with ERE-vitellogenin (Vit)-CAT and different doses of ER{alpha}-expressing construct (0.1–5 ng pSG5-ER{alpha}/105 cells). Cells were treated with vehicle (DMSO) or MG132 (1 µM) for 1 h followed by E2 (10 nM). CAT activity was measured 24 h after E2 treatment. Basal CAT activity increased, proportional to the amount of pSG5-ER{alpha} (Fig. 1BGo; open bars). As expected, E2 markedly induced CAT activity (Fig. 1BGo; gray bars); however, treatment with MG132 plus E2 resulted in greater CAT activity, compared with E2 alone (Fig. 1BGo; black vs. gray bars). Cells treated with MG132 alone exhibited slightly higher CAT activity than the DMSO control (Fig. 1BGo, hatched bars). A synergistic effect of MG132 plus E2 was observed in cells transfected with lower levels of ER{alpha} (0.1–0.3 ng pSG5-ER{alpha}/105 cells). For example, the combined treatment of MG132 and E2 increased ERE-CAT activity by about 7.4-fold in cells transfected with 0.1 ng pSG5-ER{alpha}/105 cells, whereas MG132 or E2 alone increased ERE-CAT activity by 1.82- or 3.10-fold, respectively (table in Fig. 2BGo). Immunoblot analysis showed that pretreatment with MG132 effectively blocked E2-induced ER{alpha} down-regulation in HeLa cells (Fig. 1CGo). Taken together, these observations demonstrate that ER{alpha} retains the capacity to activate transcription in the absence of proteasomal degradation, and blocking ER{alpha} turnover increases E2-induced transcriptional output. The results further suggest that, in cells containing low levels of ER{alpha}, proteasome-mediated receptor degradation plays a role in limiting E2-induced transcriptional responsiveness.



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  		Fig. 2. Effect of MG132 on E2 Dose-Dependent Induction of Reporter Gene Expression in HeLa Cells

HeLa cells were plated in 12-well dishes at a density of 1 x 105 cells per well and cultured in hormone-free medium for 2 d. The cells were transfected with 100 ng ERE-Vit-CAT and 0.3 ng (A) or 1 ng (B) of pSG5-ER{alpha} using LipofectAMINE Plus reagent. The DNA/LipofectAMINE mixture was removed 5 h later and cells were placed in hormone-free medium for 24 h. The transfected cells were treated with DMSO or MG132 (1 µM) for 1 h and then treated with the indicated concentration of E2 for 24 h. CAT activities were normalized against total cellular protein and expressed as mean ± SD of three independent experiments, each performed in triplicate. EC50 range was calculated with a 95% confidence.

 
Effect of Inhibiting the Proteasome on E2 Sensitivity
Based on the observation that preventing receptor protein turnover increases ER{alpha}-mediated transcription, we examined the effect of inhibiting the proteasome on hormone sensitivity. HeLa cells were transfected with ERE-Vit-CAT and pSG5-ER{alpha}, treated with DMSO or MG132 for 1 h, and then treated with various doses of E2 (1 x 10–15 to 1 x 10–8 M). CAT activity was determined 24 h after the addition of ligand. In cells transfected with 0.3 ng (Fig. 2AGo) or 1 ng pSG5-ER{alpha} (Fig. 2BGo), a hyperbolic dose response to E2 was observed; the lowest dose of hormone that induced CAT activity was 1 x 10–11 M E2. Increasing ER{alpha} expression (0.3 ng vs. 1 ng pSG5-ER{alpha}) and pretreatment with MG132 augmented maximal CAT induction by E2, but no effect on E2 sensitivity was observed. The minimal dose of E2 required to induce CAT was 1 x 10–11 M under all experiment conditions, and the EC50 was not different (Fig. 2Go). These results demonstrate that blocking ER{alpha} degradation increases the magnitude of E2-induced gene transcription but has no effect on hormone sensitivity.

Inhibiting the Proteasome Extends the Duration of E2-Induced Gene Transcription
The results of the above experiments suggest that inhibiting the proteasome may extend the half-life of ligand-activated ER{alpha} and thus increase receptor transcriptional output. To test the possibility that MG132 treatment would subsequently extend the duration of an E2induced transcriptional response, we performed a time course analysis using luciferase as a reporter protein. The half-life of CAT in mammalian cells is about 50 h (44); in contrast, luciferase has an intracellular half-life of about 3 h (44), making it well suited for performing a dynamic analysis of promoter activation. Thus, we used HeLa cells transfected with ER{alpha} and ERE-pS2-Luc to study the effect of proteasome inhibition on E2-induced transcription in a time-dependent manner. In transfected HeLa cells, E2 induced a transient induction of luciferase activity, maximal at 6 h (Fig. 3AGo, solid circles). Pretreatment with MG132 decreased E2-induced luciferase expression at the early time points (1.5–6 h), but markedly increased E2-induced luciferase expression from 9–20 h (Fig. 3AGo, solid triangles).



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  		Fig. 3. Effect of Blocking ER{alpha} Turnover on Time-Dependent Induction of Reporter Gene Expression by E2 in HeLa Cells

A, Effect of MG132 on E2-induced expression of reporter gene. HeLa cells were plated in 12-well dishes at a density of 1 x 105 cells per well and cultured in hormone-free medium for 2 d. The cells were transfected with 250 ng ERE-pS2-Luc and 1 ng of pSG5-ER{alpha} using LipofectAMINE Plus reagent. The DNA/LipofectAMINE mixture was removed 5 h later, and cells were placed in hormone-free medium for 24 h. The transfected cells were treated with DMSO or MG132 (5 µM) for 1 h and then treated with 10 nM E2 for the indicated time period. Luciferase activity was determined using the Luciferase Assay System, normalized against total cellular protein. B, Effect of MG132 on SV40-Luc expression. HeLa cells were transfected with 100 ng SV40-Luc. The DNA/LipofectAMINE mixture was removed 5 h later and cells were placed in hormone-free medium containing either 0.1% vehicle (DMSO) or MG132 (5 µM) for the indicated time period. Luciferase activity was determined and normalized against total cellular protein. C, Normalized ERE-Luc activities. ER{alpha}-mediated luciferase activity in the presence of MG132 was normalized to luciferase activity from the SV40-Luc construct [Normalized ERE-Luc activity in the presence of MG132 = ERE-Luc activity in the presence of MG132 x (SV40-Luc activity/SV40-Luc activity in the presence of MG132)]. D, Effect of overexpressing Ubc12C111S on E2-induced reporter gene expression. HeLa cells were transfected with 250 ng ERE-pS2-Luc, 1 ng pSG5-ER{alpha}, along with 100 ng pcDNA or pcDNA-Ubc12C111S, and treated with 10 nM E2 for the indicated period of time. Luc activities were normalized against total cellular protein. E, Effect of overexpressing Ubc12C111S on SV40-Luc expression. HeLa cells were transfected with 100 ng SV40-Luc, along with 100 ng pcDNA-Ubc12C111S or control vector pcDNA. The DNA/LipofectAMINE mixture was removed 5 h later, and cells were placed in hormone-free medium for the indicated time period. Luc activities were normalized against total cellular protein. For all assays, Luc activities are expressed as mean ± SD from three independent experiments, each performed in triplicate.

 
As mentioned above, MG132 can inhibit luciferase production. To determine the effect of MG132 on luciferase synthesis in general, we transfected HeLa cells with a constitutively active luciferase construct (SV40-Luc). In contrast to what we observed using ERE-pS2-Luc, MG132 consistently decreased the expression of SV40-Luc during the 20-h period (Fig. 4BGo), excluding the possibility that MG132 enhances ERE-luc activity by stabilizing luciferase protein. To subtract the general inhibitory effect of MG132 on luciferase synthesis, at each time point shown in Fig. 3CGo, ER{alpha}-mediated luciferase expression in the presence of MG132 was normalized to luciferase activity from the SV40-Luc construct [normalized ERE-Luc activity in the presence of MG132 = ERE-Luc activity in the presence of MG132 x (SV40-Luc activity/SV40-Luc activity in the presence of MG132)]. The adjusted results clearly demonstrate that blocking receptor degradation with MG132 increases both the magnitude and duration of E2-induced gene transcription, suggesting that the duration of gene transcription induced by E2 is limited by ER{alpha} degradation through the 26S proteasome.



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  		Fig. 4. Ub Mutant Blocks ER Degradation and Sustained E2-Induced Gene Expression

A, Overexpression of UbK0 blocks E2-induced ER{alpha} degradation. HeLa cells were plated in 60-mm dishes at a density of 3 x 105 cells per dish and cultured in hormone-free medium for 2 d. The cells were transfected with 150 ng pSG5-ER{alpha}, along with 150 ng pcDNA-Ub or pCS2-UbK0, using LipofectAMINE Plus reagent. The DNA/LipofectAMINE mixture was removed 5 h later, and cells were placed in hormone-free medium for 24 h before treatment with DMSO or 10 nM E2 for 8 h. Whole-cell lysates were prepared and subjected to immunoblotting analysis using an anti-ER{alpha} antibody. The Coomasie-stained SDS-PAGE gels show that equal amounts of cell lysates were loaded. B, Effect of UbK0 on ER{alpha}-mediated luciferase expression. HeLa cells stably transfected with ER{alpha} were plated in 12-well dishes at a density of 1 x 105 cells per well and cultured in hormone-free medium for 2 d. The cells were transfected with 250 ng ERE-pS2-Luc, along with 100 ng pcDNA-Ub or pCS2-UbK0 as indicated, using LipofectAMINE Plus reagent. The DNA/LipofectAMINE mixture was removed 5 h later, and cells were placed in hormone-free medium for 24 h before treatment with DMSO or 10 nM E2 for the indicated time period. C, Effect of UbK0 on luciferase expression from SV40-Luc. HeLa cells stably transfected with ER{alpha} were transfected with 100 ng SV40-Luc, along with 100 ng pcDNA-Ub or pCS2-UbK0. Five hours later, the DNA/LipofectAMINE mixture was removed, and cells were placed in hormone-free medium for the indicated time period. Luciferase activity was normalized against total cellular protein and expressed as the mean ± SD from three independent experiments, each performed in triplicate.

 
Inhibiting ER{alpha} Ubiquitination Prolongs E2-Induced Gene Transcription
In a previous study, we used a dominant-negative mutant of the NEDD8 conjugation enzyme, Ubc12C111S, to inhibit ER{alpha} ubiquitination and degradation (42). Here we used Ubc12C111S as a means to investigate the role of ER{alpha} turnover in ER{alpha} transactivation function and to corroborate our observations using MG132. The impact of Ubc12C111S on the time-dependent induction of a reporter gene by ER{alpha} was investigated. HeLa cells were transfected with pSG5-ER{alpha} and ERE-pS2-Luc, along with a control vector (pcDNA) or a construct expressing the mutant Ubc12 (pcDNA-Ubc12C111S). In cells transfected with pcDNA, E2 transiently induced luciferase expression, and maximal induction was observed at 5 h (Fig. 3DGo, solid circles). However, in cells transfected with pcDNA-Ubc12C111S, a delay in peak expression of E2-induced luciferase activity was observed (9 h; Fig. 3DGo, solid triangles), and luciferase expression remained elevated, even 20 h after E2 treatment. No effect of Ubc12C111S on maximal E2-induced luciferase activity was observed (Fig. 3DGo, solid circles vs. solid triangles). To confirm that the observed effect of Ubc12C111S on ER{alpha}-mediated luciferase expression was specific, luciferase activity in cells cotransfected with SV40-Luc and Ubc12C111S was assessed over time. No effect of Ubc12C111S on SV40-Luc expression was seen at 6 and 12 h after transfection; a slight increase in luciferase expression was observed at 20 h (1.3-fold; Fig. 3EGo). Overall, these results demonstrate that inhibiting ER{alpha} ubiquitination prolongs ER{alpha}-mediated transcription, supporting the hypothesis that proteasome-mediated degradation of ER{alpha} serves as a means to limit the duration of E2 signaling.

Blocking Polyubiquitination Sustains E2-Induced Gene Expression
To determine the effect of blocking polyubiquitination on ER{alpha}-mediated transcription, we used a Ub mutant, UbK0, which has all of its lysines replaced by arginine. This mutant competes with endogenous ubiquitin and terminates ubiquitin chains, resulting in the accumulation of short ubiquitin conjugates that cannot be degraded efficiently by the proteasome (43). First, we examined the effect of overexpressing UbK0 on E2-induced ER{alpha} degradation. In HeLa cells cotransfected with wild-type Ub and ER{alpha}, the level of receptor protein decreased markedly after E2 treatment (Fig. 4AGo), accompanied by transient E2-induced expression of an ER-responsive luciferase reporter gene (Fig. 4BGo, 8 h vs. 24 h). In contrast, cells transfected with UbK0 showed sustained E2-induced luciferase expression (Fig. 4BGo), and no decrease in ER{alpha} protein levels was observed (Fig. 4AGo). Furthermore, the effect of UbK0 on ER{alpha}-induced luciferase was specific, as UbK0 showed no effect on expression of the SV40-Luc construct (Fig. 4CGo). These results demonstrate that blocking polyubiquitination of ER{alpha} stabilizes the receptor, resulting in the prolonged expression of an ER{alpha}responsive gene.

Proteasome Inhibition Enhances ER{alpha}-Mediated Transcription in MCF7 Breast Cancer Cells
To further investigate the role of ER{alpha} degradation in receptor transactivation ability under physiologically relevant conditions, we examined the effect of inhibiting the proteasome in MCF7 breast cancer cells, which endogenously express ER{alpha}. First, we examined the effect of MG132 on ERE-Vit-CAT expression in MCF7 cells. MCF7 cells were transiently transfected with ERE-Vit-CAT and then treated with DMSO or MG132 (1 µM) for 1 h before E2 (10 nM) treatment. CAT activity was determined 24 h after E2 treatment. A 17.8 ± 1.7 fold increase in CAT expression was seen in MCF7 cells treated with E2, compared with the control; treatment with MG132 further increased E2-induced CAT activity to 25.6 ± 2.5 fold. Therefore, inhibiting the proteasome enhanced ER{alpha} transcriptional activity in MCF7 cells, indicating that ER{alpha} degradation plays a key role in limiting E2-induced transcriptional responses in breast cancer cells.

To determine the effect of proteasome inhibition on transcription of ER{alpha}-target genes in breast cancer cells, we pretreated MCF7 cells with MG132 and examined E2-induced pS2 gene expression. ER{alpha} regulates pS2 transcription through an imperfect palindromic ERE at position –405 to –393 of its promoter region (45); pS2 expression is considered a reliable indicator of ER{alpha} transcriptional activity (46). Timedependent effects of MG132 on heterogeneous nuclear pS2 RNA (pS2 hnRNA) levels, which reflect the rates of pS2 gene transcription (47, 48, 49, 50), were examined. Primers amplifying the conjoining sequence between the first intron and second exon of the pS2 gene were used, and expression of pS2 hnRNA was assessed by real-time quantitative RT-PCR (Q-PCR). After administration of E2, levels of pS2 hnRNA increased by 3 h, peaked at 12 h, and then declined by 70% during the next 8 h (Fig. 5AGo, gray bars). However, at all time points examined, E2-induced expression of pS2 hnRNA was markedly enhanced by pretreatment with MG132 (Fig. 5AGo, black vs. gray bars), and pS2 hnRNA levels declined only by 15% from 12–20 h after the combined treatment (Fig. 5AGo, black bars). MG132 alone showed no effect on basal pS2 hnRNA expression (Fig. 5AGo, hatched bars). In agreement with what we observed with pS2 hnRNA, the combined treatment of MG132 plus E2 resulted in greater expression of pS2 mRNA after 6 h, compared with E2 treatment alone (Fig. 5BGo, black vs. gray bars); pS2 mRNA levels remained markedly elevated up to 20 h, the last time point examined (Fig. 5BGo, black bars). The coordinate increase in E2-induced expression of both pS2 hnRNA and pS2 mRNA by MG132 excludes the possibility that MG132 inhibits the hnRNA splicing process or stabilizes pS2 mRNA. Therefore, it seems reasonable to conclude that blocking the proteasome with MG132 enhances E2-induced pS2 transcription initiation. Together, these results demonstrate that inhibiting the proteasome increases both the magnitude and duration of E2-induced expression of the endogenous pS2 gene in breast cancer cells.



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  		Fig. 5. Effects of MG132 on ER{alpha}-Mediated Transcription of Endogenous Target Genes in MCF7 Cells

MCF7 cells were plated at a density of 3 x 106 per 10-cm dish and allowed to grow in hormone-free medium for 3 d. The cells were pretreated with MG132 (5 µM) for 1 h and then treated with 10 nM E2 for the indicated time periods. Total RNA was prepared and subjected to Q-PCR analysis to determine the expression levels of pS2 hnRNA (A), pS2 mRNA (B), cathepsin D mRNA (C), and PR mRNA (D). For all Q-PCR assays, the relative levels of mRNA were normalized with ß-actin mRNA and standardized such that values obtained in cells treated with vehicle (DMSO) only were set to 1. The results were expressed as mean ± SD from two independent experiments, each in duplicate. To determine the effect of MG132 on E2-induced ER degradation, MCF7 cells were treated as in panel A and subjected to whole-cell lysate preparation and immunoblotting with an anti-ER antibody (E). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

 
We also examined the effect of MG132 on mRNA expression of cathepsin D and progesterone receptor (PR), two well-known E2-regulated genes, in MCF7 cells. As shown in Fig. 5CGo, a transient increase in cathepsin D mRNA expression was observed after treatment with E2. Pretreatment with MG132 enhanced both basal and E2-induced cathepsin D expression at 3 and 6 h (Fig. 5CGo, black vs. gray bars); however, at 12 and 24 h, the effect of MG132 was no longer apparent. Treatment of MCF7 cells with E2 increased PR mRNA levels 7-fold by 3 h, and PR mRNA levels remained elevated throughout the experiment period (Fig. 5DGo, gray bars). MG132 pretreatment decreased E2-induced expression of PR mRNA by more than 50% at all time points examined (Fig. 5DGo, black vs. gray bars), which agrees with a recent report that MG132 inhibits ER{alpha}-induced increase in PR protein levels (7). The differential effects of MG132 on these ER{alpha}-target genes demonstrate that promoter context must be considered when evaluating MG132 regulation of ER{alpha}-mediated transcription. Immunoblotting analysis showed that pretreatment with MG132 efficiently blocked E2-induced ER{alpha} down-regulation in MCF7 cells (Fig. 5EGo).

4-OHT Stimulates ER{alpha}-Mediated Transcription without Inducing ER{alpha} Degradation
The antiestrogen 4-OHT has been shown to up-regulate ER{alpha} levels by blocking ER{alpha} degradation (37), and previous studies have shown that 4-OHT functions as an ER{alpha} agonist in Ishikawa endometrial cancer cells (51, 52). To further examine the relationship between receptor stability and ER{alpha}-mediated transcription, we stably transfected ER{alpha}-negative Ishikawa cells with ER{alpha}. The ER{alpha}(+) Ishikawa cells were then transfected with a luciferase reporter construct containing the human C3 promoter (C3T1-Luc) and then treated with either E2 (10 nM) or 4-OHT (1 µM) for 16 h. After E2 administration, a 2-fold increase in luciferase activity was observed (Fig. 6AGo), accompanied by a marked decrease in ER{alpha} protein level (Fig. 6BGo). Treatment with 4-OHT also stimulated expression of luciferase (80% of E2-stimulated luciferase expression) (Fig. 6AGo), but the antiestrogen did not down-regulate ER{alpha} (Fig. 6BGo). Thus, these results demonstrate that the partial agonist activity of 4-OHT and ER{alpha} degradation are not coupled in endometrial cancer cells. It has been reported that steroid receptor coactivator 1 (SRC-1), by stimulating transcription activity of 4-OHT liganded ER{alpha} (53), can convert 4-OHT to a full agonist. We reasoned that if receptor degradation is essential for ER{alpha} to initiate transcription, SRC1 should enhance 4-OHT-stimulated ER{alpha} transactivation activity and, in parallel, induce proteasomal degradation of 4-OHT-liganded ER{alpha}. To test this reasoning, the ER{alpha}(+)Ishikawa cells were cotransfected with a construct expressing SRC1 and C3T1-Luc and then treated with either E2 (10 nM) or 4-OHT (1 µM) for 16 h. As expected, overexpressing SRC1 resulted in similar 4-OHT- and E2-stimulated ER{alpha} activity (Fig. 6AGo); however, 4-OHT did not induce receptor down-regulation (Fig. 6BGo). Thus, under these experimental conditions, 4-OHT, even when behaving as a full agonist in the presence of an increased level of SRC-1, did not induce ER{alpha} degradation. Taken together, these results demonstrate that ER{alpha}-mediated gene transactivation can be uncoupled from receptor degradation.



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  		Fig. 6. Uncoupling of 4-OHT-Induced ER{alpha} Activation and ER{alpha} Degradation

A, 4-OHT stimulates ER{alpha}-mediated gene expression in Ishikawa cells. Ishikawa cells stably transfected with ER{alpha} were plated in 12-well dishes at a density of 1 x 105 cells per well and cultured in hormone-free medium for 2 d. The cells were transfected with 250 ng C3T1-Luc, along with 100 ng pcDNA or pcDNA-SRC1, using LipofectAMINE Plus reagent. The DNA/LipofectAMINE mixture was removed 5 h later, and cells were placed in hormone-free medium for 24 h before treatment with 10 nM E2 or 1 µM 4-OHT for 16 h. Luciferase activity was normalized against total cellular protein and expressed as mean ± SD from three independent experiments, each performed in triplicate. B, Effect of 4-OHT on ER{alpha} protein level. Ishikawa cells stably transfected with ER{alpha} were plated in 60-mm dishes at a density of 3 x 105 cells per dish and cultured in hormone-free medium for 3 d before treatment with 10 nM E2 or 1 µM 4-OHT for 16 h. Whole-cell lysates were prepared and subjected to immunoblotting analysis using an anti-ER antibody. GAPDH was used as a loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Like other rapidly turned over transcription factors, engagement of ER{alpha} in transactivation is coupled to ER{alpha} degradation by the Ub-proteasome pathway (7, 8, 9, 10, 11, 35). However, the functional impact of ER{alpha} degradation on cellular responses to E2 has not been well established. In this study, we analyzed the effect of blocking ER{alpha} degradation on E2-induced transcriptional output. We demonstrate that blocking ER{alpha} turnover prolongs the ability of ER{alpha} to transactivate target genes and increases the output of E2-induced gene transcription. We also show that 4-OHT can act as a full agonist in Ishikawa cells overexpressing SRC-1 to stimulate ER{alpha} transcriptional activity, without inducing receptor degradation. Furthermore, proteasome inhibition by MG132 increases ER{alpha}-mediated reporter gene expression, as well as expression of endogenous ER{alpha}-target genes (pS2 and cathepsin D), in MCF7 breast cancer cells. These data demonstrate that proteasomal degradation is not essential for ER{alpha} transcriptional activity; ER{alpha} remains functional after escaping ubiquitination and proteasomal proteolysis. An important implication of this study is that the E2induced transcriptional response is limited by receptor degradation through the Ub-proteasome system, and defects in proteasome-mediated degradation of ER{alpha} could lead to an enhanced cellular response to E2.

In this study, several approaches targeting different steps in ubiquitination/proteasome proteolysis were used to block ER{alpha} degradation. MG132 was used to inhibit ER{alpha} proteolysis by specifically blocking activity of the 20S proteasome. A dominant-negative mutant (Ubc12C111S) of the NEDD8 conjugation enzyme was used to block ER{alpha} ubiquitination by inhibiting Ub ligase activity (41, 42). A Ub mutant with all of its lysines mutated to arginine (UbK0) was used to block ER{alpha} polyubiquitination by terminating polyubiquitin chains (43). One concern regarding the use of these approaches is a lack of specificity, such that the observed effect on enhanced E2-induced transcriptional output could be due to stabilization of multiple regulatory proteins, in addition to ER{alpha}. However, several observations suggest that this is not the case. MG132, Ubc12C111S, and UbK0 substantially enhance E2-induced, but not basal, expression of ERE reporter genes or the endogenous pS2 gene, suggesting that the effect of these inhibitors on ER{alpha} target gene expression is hormone dependent and thus receptor dependent. Furthermore, a time-dependent effect on E2-induced gene transcription was observed, which agrees with the ability of these inhibitors to block ligand-induced ER{alpha} degradation. Finally, no timedependent effect on SV40-Luc expression was observed, in contrast to ERE-Luc, suggesting that these inhibitors do not broadly affect gene transcription in a time-dependent manner. Therefore, we conclude that MG132, Ubc12C111S, and UbK0 enhance E2induced gene transcription primarily by extending the lifetime of functional ER{alpha}.

Consistent with our ER{alpha} findings, proteasome inhibition has been shown to enhance the transcriptional response mediated by other nuclear receptors, including the glucocorticoid receptor (GR) (17, 24), aryl hydrocarbon receptor (18), peroxisome proliferator-activated receptor {alpha} (19), retinoid receptors (20), and the vitamin D3 receptor (21). However, it has also been reported that MG132 decreases transcriptional activity of PR and androgen receptor (22, 23), indicating that the effect of proteasome inhibition on transcriptional activity could be receptor specific. This is presumably due to the involvement of mechanisms other than modulation of receptor levels; for example, MG132 inhibited androgen receptor activity by eliminating androgen-induced nuclear translocation and coactivator recruitment (22, 23).

In MCF7 cells, we observed differential effects of MG132 on E2-induced transcription of endogenous pS2, cathepsin D, and PR gene, suggesting that proteasome inhibition can have promoter-specific effects on gene transcription. Although the reason for this is not clear, these observations raise the intriguing possibility of a differential requirement of ER{alpha} turnover in gene transcription, such that ER{alpha} degradation is required for PR transcription, but not for pS2 and cathepsin D. However, another attractive possibility is that multiple regulatory elements, other than an ERE, could be differentially regulated by proteasome inhibition; the different structures of the PR, pS2, and cathespin D promoters may favor this possibility. For endogenous genes, the effect of estrogen is usually mediated through cross-talk between the ERE and nearby regulatory elements, and there appears to be an inverse correlation between the influence of nearby elements and the strength of the ERE (54). The ERE sequence in pS2 promoter deviates from the consensus palindromic ERE by 1 bp and, when isolated from surrounding sequences, is able to mediate estrogen responsiveness (45); however, for the cathepsin D promoter, although the ERE-like sequence deviates from the consensus ERE by only 2 bp, it is unable to confer estrogen regulation alone and must cooperate with other regulatory elements (54). In the case of the PR promoter, only a half-site ERE is found, and estrogen induction of PR appears to require cooperation with nearby Sp1 and AP-1 sites (55). Based on the observation that ERE-Vit-CAT (Fig. 1BGo) and ERE-pS2-Luc (Fig. 2Go) activities correlate with cellular concentrations of ER{alpha}, we suggest that ER{alpha} levels are the determining factor for the transcription activity of genes controlled exclusively by ERE. We further suggest that transcriptional activity of endogenous genes driven predominantly by an ERE (e.g. pS2) may depend upon the availability of ER{alpha}. In contrast, the level of ER{alpha} is unlikely to be the sole determining factor for the transcription of genes without a consensus ERE in their complex promoters (e.g. PR). In support of this notion, it has been reported that E2-induced transcription of the PR gene does not parallel ER{alpha} occupancy (55). Therefore, it is possible that MG132 inhibits PR expression through other protein factors, either directly or indirectly. In this respect, when evaluating the transcriptional activity of ER{alpha}, after escaping proteasome degradation, promoter context must be considered. Based on our own and the results of others (50), it is plausible that the transcription rate of a gene driven predominantly by an ERE is a more reliable readout of ER{alpha} transcription activity than a gene containing a complex promoter requiring ER{alpha} plus other transcription factors.

Our results differ from a previous study by Reid et al. (35), showing that MG132 prevented recruitment of phosphorylated RNA pol II (p-Pol II) to the pS2 promoter. This is most likely due to different experimental conditions and endpoints used in the two studies. For example, in their study Reid et al. used a higher dose (10 µM) and longer pretreatment (7 h) with MG132. However, under that condition, it is not clear whether the drug had any effect on p-Pol II recruitment to non-estrogen-responsive promoters. In addition, although {alpha}-amantin was used to clean the pS2 promoter before p-Pol II recruitment analysis, it is not clear that gene transcription resumed immediately (within a 2 h period) after {alpha}-amantin treatment. Thus, whether the differential recruitment of p-Pol II, in the absence or presence of MG132 after {alpha}-amantin pretreatment, is correlated with pS2 gene transcription remains an open question. However, the observation by Reid et al. (35) that the 20S proteolytic subunit does not associate with the pS2 promoter in response to E2 stimulation, agrees with numerous studies showing that the 20S proteasome subunit is not required for transcription initiation and elongation (56, 57, 58, 59, 60). Our observation further shows that 20S proteasome activity is not essential for ER{alpha}-mediated gene transcription.

Although the mechanism(s) by which the proteasome modulates ER{alpha}-mediated transactivation remains to be fully elucidated, chromatin immunoprecipitation assays have demonstrated that both unliganded and liganded receptors constantly cycle on and off estrogen-responsive promoters (35). MG132 appears to halt this cyclic interaction, leading to prolonged occupancy of ER{alpha} on EREs (35). The cyclic turnover of ER{alpha} could be a mechanism used by cells to prevent multiple rounds of transcription initiation from a single promoter, thus ensuring an appropriate cellular response to changes in circulating concentrations of hormone. To support this explanation, recent studies of GR show that proteasome inhibition dramatically increases both the residence time of GR on its target promoter and transcriptional output (24). In addition to extending the half-life of ligand-activated ER{alpha}, other factors, such as increased cellular concentration of receptor coactivators, could contribute to the enhancement of transcription by proteasome inhibition. Several ER{alpha} coactivators, including the steroid receptor coactivator family members (SRC1, SRC2, and SRC3) and cAMP response element binding protein (CREB)-binding protein/p300, are substrates of proteasomal degradation; proteasome inhibition appears to increase cellular concentrations of these coactivators (61).

We found that blocking ER{alpha} degradation (using MG132, Ubc12C111S, or UbK0) decreases E2induced ERE-pS2-Luc expression at earlier time points (1.5–6 h) after E2 treatment (Figs. 3Go and 4Go). Although the reason for this is unknown, one possibility is that ubiquitination and 20S proteasome activity are required for optimal ER{alpha} activation, perhaps by facilitating the release of ER{alpha} from preexisting corepressor complexes. To fully elucidate the physiological role(s) of ubiquitination, identification of the primary Ub ligase(s) for ER{alpha}, as well as the ubiquitination site(s) in this receptor, will be necessary.

In target tissues where ER{alpha} levels are limiting, the magnitude of the response to E2 is correlated with cellular ER{alpha} concentrations (2, 62). The Ub-proteasome pathway, by modulating receptor protein turnover, could play an important role in determining cellular responses to circulating E2 levels. Our results indicate that both the magnitude and duration of E2-induced gene transcription are limited by proteasome-mediated degradation of ER{alpha}; therefore, it seems reasonable to speculate that defects in ER{alpha} degradation could lead to enhanced cellular responsiveness to estrogens. In support of this possibility, it has been demonstrated that thyroid hormone and insulin, by blocking ligand-induced ER{alpha} degradation, can augment E2-stimulated cell proliferation (39, 63). Therefore, our future studies will examine the functional impact of proteasome-mediated ER{alpha} degradation on complex biological responses to estrogens, such as mammary gland development. In addition, aberrant ER{alpha} expression and estrogen responsiveness have been linked to breast tumor pathogenesis and development (64, 65, 66). Our previous studies demonstrate that blocking ER{alpha} degradation render breast cancer cells insensitive to the growth-inhibitory effects of ICI 182,780, a potent ER{alpha} down-regulator (42). Whether defects in the ER{alpha} degradation pathway contribute to deregulated estrogen signaling in breast cancer cells and play a role in disease progression to antiestrogen resistance remains to be elucidated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
The construction of pSG5-ER{alpha}(HEGO), ERE2-pS2-Luc, pcDNA-HA-Ubc12C111S, C3T1-Luc, pcDNA-SRC1, pCS2-UbK0, and ERE-Vit-CAT has been described previously (43, 67, 68).

Cell Lines
The human cervical carcinoma cell line HeLa and the breast cancer cell line MCF-7 were purchased from ATCC (Manassas, VA). The ER{alpha}-negative endometrial Ishikawa cell line was kindly provided by Dr. S. Hyder (University of Missouri, Columbia, MO). HeLa and Ishikawa cells were maintained in MEM with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum. MCF7 cells were maintained in the same medium with the addition of 6 ng/ml insulin. Before experiments involving hormone treatment, cells were cultured in hormone-free medium (phenol red-free MEM with 3% dextran-coated charcoal-stripped fetal bovine serum) for 3 d.

Transient Transfection and Reporter Enzyme Assays
Cells (80% confluence) were transfected with an equal amount of total plasmid DNA (adjusted by corresponding empty vectors) by using LipofectAMINE Plus Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s guidelines. The DNA/LipofectAMINE mixture was removed 5 h later and cells were placed in hormone-free medium. Unless stated otherwise, 24 h after transfection, cells were treated with vehicle (DMSO) or MG132 (Sigma Chemical Co., St. Louis, MO) for 1 h before E2 (Sigma) treatment. At the end of the experiment, cell lysates were prepared for reporter enzyme assays. Luciferase activity was determined using the Luciferase Assay System (Promega Corp., Madison, WI), Gal activity was determined using a chemiluminescent reporter assay (PE Applied Biosystems, Foster City, CA), and CAT activity was determined using the colorimetric CAT ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN). Total cellular protein was determined by using the Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA). Reporter activities were expressed as relative light units normalized to total cellular protein.

Q-PCR
MCF7 cells were plated at a density of 3 x 106 per 10-cm dish and allowed to grow in hormone-free medium for 3 d. The cells were pretreated with MG132 (5 µM) for 1 h before E2 (10 nM) treatment. Total RNA was prepared by a RNAeasy Mini Kit (QIAGEN, Valencia, CA), according to the manufacturer’s protocol. RNA (2 µg) was reverse transcribed in a total volume of 40 µl containing 400 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (New England Biolabs, Beverly, MA), 400 ng random hexamers (Promega), 80 U RNase Inhibitor, and 1 mM deoxynucleotide triphosphates. The resulting cDNA was used in subsequent Q-PCR reactions, performed in 1x iQ SYBR Green Supermix (Bio-Rad) with 5 pmol forward and reverse primers. The primers used in the Q-PCR were, for pS2 mRNA: forward primer, 5'-ATACCATCGACGTCCCTCCA-3'; and reverse primer, 5'-AAGCGTGTCTGAGGTGTCCG-3' (69); for pS2 hnRNA: forward primer, 5'-TTGGAGAAGGAAGCTGGATGG-3' (start position 3997, within the intron); reverse primer, 5'-ACCACAATTCTGTCTTTCACGG-3' (start position 4126, within the second exon); for PR: forward primer, 5'-TCAGTGGGCAGATGC TGTATTT-3'; and reverse primer, 5'-GCCACATGGTAAGGCATAATGA-3' (70); for cathepsin D: forward primer, 5'-GTACATGATCCCCTGTGAGAAGGT-3'; reverse primer, 5'-GGGACAGCTTGTAGCCTTTGC-3' (71); and for ß-actin: forward primer, 5'-TGCGTGACATTAAGGAGAAG-3'; and reverse primer, 5'-GCTCGTAGCT CTTCTCCA-3'. Q-PCR was performed in 96-well optical plates (Bio-Rad) using an iCycler system (Bio-Rad) for 40 cycles (94 C for 10 sec, 60 C for 40 sec), after an initial 3-min denaturation at 94 C. The relative concentration of RNA was calculated using the {Delta}{Delta}Ct method according to Relative Quantitation of Gene Expression (Applied Biosystems User Bulletin) with ß-actin mRNA as an internal control. Results were expressed as relative RNA levels standardized such that values obtained in cells treated with vehicle (DMSO) only were set to 1.


   ACKNOWLEDGMENTS
 
We thank Teresa Craft and Annie Park for excellent technical assistance; Dr. Curt Balch for critical review of this manuscript; Dr. Michele Pagano for providing the pCS2-UbK0 construct; and Dr. Wenlin Shao and Dr. Myles Brown for the Q-PCR primers of pS2 hnRNA.


   FOOTNOTES
 
This work was supported by the United States Army Medical Research Acquisition Activity, Award Nos. DAMD 17-02-1-0418 and DAMD17-02-1-0419 (to K.P.N.); American Cancer Society Research and Alaska Run for Woman Grant TBE-104125 (to K.P.N.); American Cancer Society Grant RPG-00-122-01-TBE and National Institutes of Health Grant CA89153 (to H.N.); and the Walther Cancer Institute (to M.F.).

Abbreviations: CAT, Chloramphenicol acetyltransferase; DMSO, dimethylsulfoxide; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response elements; GR, glucocorticoid receptor; hnRNA, heterogeneous nuclear RNA; Luc, firefly luciferase; 4-OHT, 4-hydroxytamoxifen; p-Pol II, phosphorylated RNA pol II; PR, progesterone receptor; Q-PCR, real-time quantitative reverse transcription-PCR; RSV, Rous sarcoma virus; SRC, steroid receptor coactivator; SV40, simian virus 40; Ub, ubiquitin; Ubc, ubiquitin-conjugation enzyme; Vit, vitellogenin.

Received for publication April 19, 2004. Accepted for publication July 21, 2004.


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 ABSTRACT
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 MATERIALS AND METHODS
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