help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0537
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ray, S.
Right arrow Articles by Das, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ray, S.
Right arrow Articles by Das, S. K.
Endocrinology Vol. 148, No. 10 4774-4785
Copyright © 2007 by The Endocrine Society

Increased Level of Cellular Bip Critically Determines Estrogenic Potency for a Xenoestrogen Kepone in the Mouse Uterus

Sanhita Ray, Fuhua Xu, Ping Li, Nora S. Sanchez, Haibin Wang and Sanjoy K. Das

Division of Reproductive and Developmental Biology, Departments of Pediatrics (S.R., F.X., P.L., H.W., N.S.S., S.K.D.) and Cancer Biology (S.R., F.X., P.L., S.K.D.), Vanderbilt University Medical Center, Nashville, Tennessee 37232-2678

Address all correspondence and requests for reprints to: Sanjoy K. Das, Department of Pediatrics, Division of Reproductive and Developmental Biology, Vanderbilt University Medical Center, MCN-D4100, Nashville, Tennessee 37232-2678. E-mail: sanjoy.das{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Xenoestrogen mimics estrogen-like activities primarily based on alterations of gene expression and interactions with estrogen receptor (ER)-{alpha} and -ß. However, the requirement for large concentrations to induce estrogenic phenotypes and low affinity for ERs has challenged the notion that prevailing xenoestrogens are significant health hazards. Here in this study, we show that under certain conditions, exposure of xenoestrogen could be potentially harmful in respect to enhanced uterine estrogenicity. Previously, we have demonstrated that estradiol-17ß up-regulates uterine Bip, a stress-related endoplasmic reticulum protein, via an ER-independent mechanism in mice. Moreover, this protein essentially involves in estradiol-17ß-mediated uterine growth response and ER{alpha}-dependent gene transcription. Here, we demonstrate that among three tested xenoestrogens, only kepone (>15–30 mg/kg) exerts sustained inductive response for uterine Bip expression. Interestingly, this kepone-induced Bip strongly correlates with ER{alpha}-dependent growth and gene expressional responses in the mouse uterus. Furthermore, these effects were strongly suppressed after knockdown of uterine Bip, via the adenovirus approach. Although kepone at 7.5 mg/kg was not effective, it was strongly stimulatory by the adenovirus-driven forced expression of uterine Bip. In contrast, the control green fluorescence protein virus was not effective in the aforementioned responses. Furthermore, the induction of uterine Bip by stress-related signals also revealed the onset of uterine growth in mice when exposed to a sublethal dose of kepone. Collectively, studies provide novel molecular evidence that Bip acts as a critical regulator to amplify estrogenic potency for a weak xenoestrogen kepone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH BANNED IN the industrial nations, organochlorine compounds, such as pesticides and polychlorinated biphenyls, are still used in the third world countries (1, 2, 3) and are highly persistent organic pollutants that have been identified in diverse environmental conditions worldwide (4, 5, 6, 7, 8). This has been a major issue about the potential health and environmental risks associated with the exposure to human and wildlife (9, 10), by disrupting the endocrine system and affecting normal functions of hormone-responsive target tissues. In particular, the harmful effects of many organochlorine compounds in reproductive organs have been suspect for many years, but the mode and extent of their actions are not clearly defined (11, 12, 13).

In general, they have been attributed to their ability to mimic certain effects of primary estrogens, thus cataloging them as xenoestrogens (14, 15). In particular, they have drawn attention because of their ability to interact with nuclear estrogen receptor (ER)-{alpha} and -ß. Nonetheless, it has been argued that the extent of their effects is minimal because of low binding affinities for ERs (16, 17, 18) and the need for larger molar concentrations to produce a phenotypic effect (19, 20, 21, 22). However, there is also evidence that response to an "estrogen" in a target tissue is not necessarily related to its affinity for the receptor (23, 24). For example, doisynolic acid shows uterotrophic activity in the rat similar to that induced by estradiol-17ß (E2), yet shows little affinity for ERs (25, 26). Moreover, estrogenicity of xenobiotics may involve various steroid-binding protein pathways; they may interact with ER or other binding proteins that may not result in the similar kind of transactivation that normally occurs with natural ligands. The metabolic conversion of xenobiotics may also alter their estrogenic activity. For example, the pesticide methoxychlor is a more potent reproductive toxicant when given orally than when injected (27, 28). Which of the many metabolites that are important has yet to be established, but a recent comparison of the affinity of some metabolites for the ER indicates a span of four orders of magnitude (16). Furthermore, xenobiotics can be effective at very low doses comparable to their levels of exposure in humans and wildlife (29, 30). In addition, xenoestrogens exhibit significant differences in respect to coactivator recruitment and transcriptional activation, compared with the natural estrogens (31, 32, 33, 34).

Given their physicochemical differences and distinct biological effects, it is not surprising that a variety of mechanisms are used by endocrine disrupting chemicals. Therefore, understanding molecular mechanisms of estrogen and xenoestrogen action will be of considerable use in many areas. Because uterine sensitivity to estrogenic substances is reflected in well-characterized biological responses that culminate to increased uterine tissue weight and epithelial cell proliferation, xenoestrogen-mediated uterotrophic assays have been used to assess the potency of estrogenic actions. Although xenoestrogen may use both ER{alpha} and ERß, in the mouse uterus ER{alpha} has been a major receptor for the control of estrogen-dependent growth response (35, 36, 37). However, little is known regarding the mechanism of actions of xenoestrogen at the molecular level, although accumulated evidence suggest that xenoestrogens induce estrogenic phenotypes with or without involving ER (20, 21, 22, 38).

We have previously shown that Bip (also known as grp78 encoded by Hspa5), a member of the heat shock protein 70 family, is induced early by natural estrogens in the mouse uterus via the ER-independent mechanism (39). Furthermore, this protein plays an essential role in the regulation of estrogen-dependent ER{alpha}-mediated gene transcription and growth responses in the uteri of mice (40). Bip is a resident protein in the endoplasmic reticulum, where it binds with newly synthesized peptide chains during the processes of secretion and translocation of proteins (41, 42). Here, we examined the regulation and functional activities of Bip in xenoestrogen-mediated effects in the uteri of mice. Studies provide evidence that kepone (≥15 mg/kg), among three xenoestrogens, specifically induces sustainable levels of uterine Bip without involving ER but facilitates complex formation between Bip and ER{alpha} to control ER{alpha}-dependent uterine gene expression. These results are consistent with the increase in uterine growth by kepone in the wild-type but not in ER{alpha} null mice. Remarkably, heightened accumulation of uterine Bip, either by adenovirus-driven strategy or stress response, causes simulation of uterine cell proliferation by a suboptimal dose of kepone in mice, suggesting that Bip acts as a critical regulator to determine estrogenicity for a weak xenoestrogen kepone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Adult CD-1 (Charles River Laboratories, Wilmington, MA) mice were housed in our institutional animal care facility according to National Institutes of Health and institutional guidelines for laboratory animals. In general, 8- to 10-wk-old adult mice were ovariectomized and rested for 7 d before they received any injections. They received a single sc injection (0.1 ml/mouse) with sesame seed oil (control), E2 (4 µg/kg) (positive control), kepone (7.5, 15, or 30 mg/kg), methoxychlor (7.5, 15, or 30 mg/kg), or 1-(o-chlorophenyl)-1-(p-chlorophenyl) 2,2,2-trichloroethane (o,p’-DDT) (7.5, 15, or 30 mg/kg), and were killed at indicated time points. In addition, one group of mice was subjected to daily single injection of kepone (7.5 mg/kg) for 3 consecutive days and killed 24 h after the last injection. All compounds were dissolved in absolute ethanol and diluted to the desired concentrations in sesame seed oil. Tissues were rapidly flash frozen and kept at –70 C, or fixed in 10% formaldehyde before paraffin embedding for subsequent studies.

In some studies, wild-type and ER{alpha}(–/–) littermates (43) were analyzed in parallel. Both mice (C57BL/6J/129/J) were produced by the crossing of heterozygous females and males in our animal facility. These mice were also given a single injection (sc) of oil, kepone (15 or 30 mg/kg), ICI-182,780 (ICI) [7(9-4,4,5,5,5-pentafluoropenylsulfinyl) nonyl-estra-1,3,4(10-triene-3,17-diol)] (20 mg/kg), or the same dose of ICI 30 min before kepone. Mice were killed 24 h after the last injection. Tissues were collected as described previously.

In another set of experiments, ovariectomized wild-type mice were given injections of kepone (2.5 and 7.5 mg/kg) or oil (as vehicle control) and immediately subjected to physiologic stress using an ultrasonic sound repellant device (Victor Sonic PestChaser, Woodstream, PA) or direct application of endoplasmic reticulum stress inducer, tunicamycin (1.0 mg/kg, sc). Mice were killed after 24 h, and uterine tissues were collected for analysis.

Probes and Northern blot hybridization
c-RNA probes were generated from mouse-specific cDNA clones. The cDNA clones of Bip, secreted frizzled related protein 2 (SFRP-2), Wnt4, Wnt5a, and ribosomal protein L7 (rpL7) have previously been described (39, 44). For Northern blot hybridization, 32P-labeled antisense c-RNA probes were generated using appropriate RNA polymerases. The probes had specific activities of 2 x 109 dpm/µg. The Northern blot hybridization technique was the same as previously described (45). Stripping of the hybridized probe before subsequent rehybridization was achieved as described. Transcripts were detected by autoradiography. The abundance of mRNAs for each gene expression was quantitated by analysis of band intensities on the autoradiogram using densitometric scanning and was corrected against rpL7.

Antibodies and other reagents
The affinity purified polyclonal antibodies for Bip (catalog no. sc-1050), ER{alpha} (catalog no. sc-542), actin (catalog no. sc-1615), progesterone receptor (catalog no. sc-539), and green fluorescence protein (GFP) (catalog no. sc-1050) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Ki-67 antibody (catalog no. NCL-Ki67 paraffin) was purchased from Novocastra Laboratories Ltd., (Newcastle upon Tyne, UK). Bromodeoxyuridine (BrdU) (catalog no. B9285), E2, and methoxychlor [dimethoxydiphenyltrichloro ethane (DMDT)]; 1,1,1,-Trichloro-2,2,-bis-[p-methoxyphenyl]ethane) were purchased from Sigma Chemical Co. (St. Louis, MO). Kepone (chlordecone) [1,1a,3,3a,4,5,5a,5b,c-decachlorooctoahydro-1,3,4-metheno-2H-cyclobuta pentalen-2-one] and o,p’-DDT were purchased from Cerilliant Corporation (Austin, TX) and were at least 99% pure. ICI-182,780 (ICI) [7 (9-4,4,5,5,5-pentafluoropenylsulfinyl) nonyl-estra-1,3,4(10-triene-3,17 -diol)] was a gift of Zeneca Pharmaceuticals (Cheshire, UK). Chemical structures of kepone, o,p’-DDT, methoxychlor, E2, and ICI are shown in Fig. 1Go.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 1. Chemical structures of the natural estrogen E2, antiestrogen ICI-182,780, and the organochlorine pesticides kepone, methoxychlor, and o,p-DDT used in the present study.

 
Immunohistochemistry, immunoprecipitation, and Western blotting
These procedures were the same as previously described (46). For studies with immunoprecipitation and Western blotting, proteins were extracted in Tissue-PE LB lyses buffer (GenoTech, St. Louis, MO). For studies with BrdU incorporation into DNA, mice were injected with BrdU (50 mg/kg, ip), 2 h before they were killed. Formaldehyde-fixed paraffin-embedded tissue sections were stained for BrdU incorporation, using biotinylated antibody according to the manufacturer’s instruction (catalog no. 93–3944; Zymed Laboratories Inc., San Francisco, CA).

Recombinant adenoviral plasmids and generation of viral particles
The generation of recombinant adenoviral plasmids for the antisense Bip (rAdBipAs) and GFP control (rAdGFP) was previously described (40). The recombinant Bip-sense cDNA construct (rAdBipS) was generated as follows. The full-length coding region of mouse Bip cDNA was generated by RT-PCR using primers carrying linkers for EcoRV at 5'-ends of both: sense, 5'-GCCCCGGGATGATGAAGTTCACTGTG-3'; and antisense, 5'-GCCCCGGGCTACAACTCATCTTTTTC-3'. The amplified DNA fragment was inserted into a shuttle vector pAd-track cytomegalovirus (CMV) at the EcoRV site, in a direction of sense with respect to the cytomegalovirus (CMV) promoter. The identity of the clone was confirmed by sequencing. This clone DNA was linearized with PmeI and subsequently cotransfected with pAdEasy-1 into Escherichia coli BJ5183 to obtain the recombinant clone. The recombinant plasmid clones harboring either Bip-S or Bip-As DNA possess an additional CMV promoter that drives GFP independently. The viral packaging of these plasmids was performed by transfection into 293 cells, as described (47). Viral particles were purified through CsCl density gradient centrifugation and stored at –70 C.

In vivo virus delivery
This was followed essentially the same as previously described (40). In brief, adenoviral particles were first inoculated directly into the uterine lumen of both horns (20 µl solutions in saline containing 1 x 1011 virus particles per horn) from the oviduct end just before ovariectomy. They were given rest for 7 d before they received the second inoculum (~100 µl solution in saline containing 1 x 1011 virus particles) through the tail vein. They were again rested for two more days before receiving injections of kepone or oil (as a vehicle control) for 24 h. Uterine tissues were appropriately collected for subsequent analysis.

RT-PCR
Procedures for the RT and comparative PCR followed the previously described methods (48, 49) with some modifications. In brief, total RNA was extracted from mouse uterus using Trizol (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instruction. RT with oligo-dT priming was performed to generate cDNAs from 4 µg total RNA using Superscript II following the instructions provided by the manufacturer. DNA amplification was performed with Taq DNA polymerase (Invitrogen, San Diego, CA) using the following primers: cyclin D1 (329 bp), 5'-GCGTACCCTGACACCAATCT-3' and 5'-CACAACTTCTCGGCAGTCAA-3'; Mad2 (182 bp), 5'-TCCCTACAGACACCCTCCAC-3' and 5'-TTCTTGCGCTTCTGGAAGAT-3'; and rpL7 (246 bp), 5'-TCAATGGAGTAAGCCCAAAG-3' and 5'-CAAGAGACCGAGCAATCAAG-3'. PCR conditions were 94 C for 4 min and then appropriate number of cycles (see Fig. 5EGo) for linear amplification using 94 C for 30 sec, 55 C for 30 sec, and 72 C for 45 sec, followed by incubation at 72 C for 10 min. Amplified fragments were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. The intensity of each band was measured by Scion Image (Scion Corp., Frederick, MD), and the abundance of mRNAs for each gene expression was corrected against rPL7.


Figure 5
View larger version (43K):
[in this window]
[in a new window]

  		FIG. 5. A, Analysis of adenovirus-mediated expression in the uterus. Uterine tissues were collected after administration of adenoviruses rAdBipS, rAdBipAs, or rAdGFP (control) in mice, as described in Materials and Methods, followed by injections of kepone as indicated for 24 h. Proteins were analyzed by Western blotting for the expression of Bip, GFP, and actin. This experiment was repeated at least three times with similar results. B, Analysis of uterine cell proliferation by BrdU or Ki-67. Adult ovariectomized wild-type mice were subjected to adenoviruses rAdBipS or rAdGFP (as control) as described in Materials and Methods, followed by injections (sc) of kepone (7.5 mg/kg) for 24 h. BrdU (50 mg/kg, ip) was injected 2 h before killing. Representative tissue sections are shown from the analysis of at least five different mice for each group. C and D, Quantitation of BrdU- or Ki-67-positive cells. The data presented here are after the analysis of at least five different mice from each group. *, Values are statistically different (P < 0.001) based on ANOVA (for Fig. 5CGo: F = 2368, df = 19 for BrdU; F = 1099, df = 19 for Ki-67; and for Fig. 5DGo: F = 1203, df = 19 for BrdU; F = 1532, df = 19 for Ki-67), followed by the Dunnett’s t test. E, Kepone regulates uterine expression of cyclin D1 and Mad2 in wild-type mice. Uterine tissues were collected after administration of adenoviruses rAdBipS, rAdBipAs, or rAdGFP (control) in mice, as described in Materials and Methods, followed by injections of kepone as indicated for 24 h. In addition, adult ovariectomized wild-type mice were subjected to oil (vehicle control) or E2 (100 ng/mouse, as positive control) and killed after 24 h. In general, three to five mice were used for each group analysis. Independent preparation of total RNAs (in triplicate) were analyzed by comparative RT-PCR as described in the Materials and Methods. The band intensities shown in the figure were measured by densitometric analysis, and the relative levels of mRNAs for gene specific expression were determined after correction with that of rpL7. *, Values are statistically different (P < 0.05, Student’s t test) compared with corresponding groups. ge, Glandular epithelium; le, luminal epithelium.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kepone selectively modulates Bip and maintains its sustained up-regulation in a dose-dependent manner in the mouse uterus
Previously, we demonstrated that Bip and Wnt-signaling genes are regulated by E2 in the mouse uterus (39, 44). Here, we examined whether xenoestrogens viz. kepone, methoxychlor, and o,p’-DDT also possess similar responses to these uterine genes. Based on previous reports with respect to the doses used for these compounds for studies with regulation of uterine genes (13, 20, 21, 22), we tested all three xenoestrogenic compounds in mice using doses 7.5, 15.0, and 30.0 mg/kg for 24 h. Northern blot hybridization analyses revealed that kepone (Fig. 2AGo), similar to E2 (Fig. 2DGo), was capable of inducing uterine Bip mRNA expression (3- to 4-fold) at or above 15 mg/kg over the control (oil). In contrast, o,p’-DDT (Fig. 2BGo) or methoxychlor (Fig. 2CGo) was unable to show such a response. In addition, none of these xenoestrogenic compounds (Fig. 2Go, A–C) had any effects on SFRP-2, a Wnt-signaling antagonist. Although consistent with previous results (39), this gene was suppressed by E2 (Fig. 2DGo). Similarly, other E2-responsive Wnt signaling genes (viz. Wnt4, Wnt5a, and ß-catenin) (44) were also not affected by these xenoestrogenic compounds at the aforementioned doses (data not shown). Overall, these results suggest that uterine Bip is specifically altered by kepone in an estrogen-responsive manner and provides a basis for further investigation of kepone-dependent regulations.


Figure 2
View larger version (70K):
[in this window]
[in a new window]

  		FIG. 2. Uterine regulation of gene-specific mRNAs by xenoestrogenic compounds and E2. Dose-response studies were conducted with adult ovariectomized wild-type mice injected (sc) with kepone (7.5, 15, or 30 mg/kg) (A), o,p’-DDT (7.5, 15, or 30 mg/kg) (B), methoxychlor (7.5, 15, or 30 mg/kg) (C), or E2 (4 µg/mouse) (D) (as a positive control) for 24 h. Mice injected with oil and killed after 24 h served as vehicle controls. Total RNA (6 µg) was separated by formaldehyde-agarose gel electrophoresis, transferred and UV cross-linked to nylon membrane, and hybridized as described in Materials and Methods. Hybridization was performed using 32P-labeled cRNA probes sequentially to Bip, SFRP-2, and rpL7. Representative autoradiograms are shown with exposures at 5 h for Bip, 15 h for SFRP-2, and 3 h for rpL7. Temporal studies were performed with uterine total RNA (6 µg in each lane) analyzed at indicated times, using kepone at 7.5 mg/kg (E) or 30 mg/kg (F), or E2 (4 µg/mouse) (G). Hybridization was performed using 32P-labeled cRNA probes sequentially to Bip, and rpL7. Autoradiographic exposure times were similar as indicated previously. In general, three to five mice were used for each group analysis. These experiments (A–G) were repeated three times with independent RNA samples. In the bar plot, the abundance of mRNAs for each gene expression was quantitated by analysis of band intensities using densitometric scanning and was corrected against rpL7. *, Values are statistically different (P < 0.05, Student’s t test) from the oil-treated group.

 
We have previously demonstrated that E2 temporally regulates uterine Bip (39, 40), thus, we analyzed whether kepone-dependent regulation of Bip mRNAs occurs in a temporal fashion. Consistent with our previous studies, E2 was able to induce uterine Bip mRNAs early (within 2 h) and maintained high until 24 h (Fig. 2GGo). In case of kepone, as shown in Fig. 2EGo, our results indicate that kepone at its lowest dose (7.5 mg/kg) had an induction of Bip mRNAs by 2 h (~3-fold) over the control. However, this was declined to the basal level by 6 h and remained low until 48 h. In contrast, the applications of kepone using doses 15 (data not shown) and 30 mg/kg (Fig. 2FGo) were able to show the increase of Bip mRNAs by 2 h (~2-fold), followed by steady increases at 6 and 24 h (4- to 5-fold), and thereafter a slight decline by 48 h. Overall, these results suggest that kepone maintains sustained up-regulation of uterine Bip RNAs only at or above 15 mg/kg in mice.

To determine whether this effect of kepone is reflected at the protein levels, we also analyzed expression by Western blotting. Consistent with the aforementioned results, the analysis of dose-dependent effects of kepone by 24 h revealed a significant induction of uterine Bip levels (~4-fold) at doses 15 and 30 mg/kg (Fig. 3AGo). Although kepone at 7.5 mg/kg was not effective, however, the analysis of the same dose of kepone at different times exhibited an increase of Bip levels by 2 h (~3-fold) but failed to sustain during the observed periods for 24 h (Fig. 3BGo). These results are also consistent with the mRNA data. In contrast, the multiple injections (3x) of kepone (7.5 mg/kg) demonstrated a distinct up-regulation of Bip (~6-fold) by 24 h of the last injection (Fig. 3BGo), suggesting a cumulative effect on gene expression after the chronic exposure of kepone. Because Bip controls estrogen-regulated ER{alpha} functions (40), we also analyzed the expression of ER{alpha} protein levels in the aforementioned conditions. Concomitant changes were revealed by kepone between the ER{alpha} and Bip levels in the mouse uterus (Fig. 3Go, A and B).


Figure 3
View larger version (51K):
[in this window]
[in a new window]

  		FIG. 3. Regulation of Bip in the uteri of wild-type and ER{alpha}(–/–) mice by kepone. A, Dose-response studies. Adult ovariectomized wild-type mice were injected (sc) with kepone at 7.5, 15, or 30 mg/kg, and killed at 24 h. Uterine tissue extracts (30 µg protein) were analyzed by Western blotting for Bip, ER{alpha}, and actin. B, Temporal studies. Adult ovariectomized wild-type mice were given a single injection (sc) of kepone at 7.5 mg/kg and analyzed at indicated times. In addition, mice were injected with multiple (3x) injections of kepone at 7.5 mg/kg and killed 24 h after the last injection [24(M)]. Uterine tissue extracts (30 µg protein) were analyzed by Western blotting for Bip, ER{alpha}, and actin. C (i), Western blot analysis. Regulation of Bip by kepone in the uteri of wild-type and ER{alpha}(–/–) mice. Adult ovariectomized wild-type and ER{alpha}(–/–) mice were given injections (sc) of oil, kepone (15 mg/kg), ICI (20 mg/kg), or the same dose of ICI 30 min before kepone, and mice were killed at 24 h. Uterine tissue extracts (20 µg protein) were analyzed by Western blotting for Bip and actin. C (ii), Northern blot analysis. ICI effectively blocks uterine LF regulation by E2 in the uteri of wild-type mice. Adult ovariectomized wild-type mice were given injections (sc) of oil, E2 (4 µg/mouse), ICI (20 mg/kg), or the same dose of ICI 30 min before E2, and mice were killed at 24 h. In general, three to five mice were used for each group in aforementioned analyses (A–C). These experiments were repeated three times with independent samples. In the bar plot, the abundance of each protein expression was quantitated by analysis of band intensities using densitometric scanning and was corrected against actin. *, Values are statistically different (P < 0.05, Student’s t test) from the corresponding control group. **, Values are statistically different (P < 0.05, Student’s t test) from the E2-treated group. D, Analysis of protein-protein interaction. Uterine tissue extracts for the indicated groups were subjected to immunoprecipitation using Bip antibody, followed by Western blotting using specific antibodies to Bip (upper panel) or ER{alpha} (lower panel). The intense bands represent a heavy chain subunit of IgG, which also serves as an internal loading control. In our control experiments, immunoprecipitation using normal goat serum did not detect any specific bands for Bip or ER{alpha} by Western blotting (data not shown). These experiments were repeated two times with similar results.

 
Kepone regulates Bip via an ER-independent mechanism but directs interaction between Bip and ER{alpha} in the mouse uterus
Previously, we have shown that E2-dependent regulation of uterine Bip is mediated via an ER-independent mechanism (39), thus, we wanted to examine whether kepone-induced uterine Bip response follows a similar pathway of action. Based on the aforementioned results, ovariectomized wild-type and ER{alpha}(–/–) mice were similarly subjected to kepone (15 mg/kg) or oil, and analyzed uterine levels of Bip by Western blotting experiments. Our results revealed that up-regulation of uterine Bip (~3.5- to 4.0-fold) was indeed detected by kepone over the oil control in both mice [Fig. 3CGo (i)]. Furthermore, an injection of ER-antagonist ICI 182,780 (ICI, 20 mg/kg) given 30 min before kepone was not antagonistic to the aforementioned response, whereas ICI by itself had very little effects. Moreover, consistent with the previous report (20, 21), this dose of ICI was strongly inhibitory to E2-dependent regulation of uterine lactoferrin (LF) gene expression in ovariectomized wild-type mice [Fig. 3CGo (ii)]. Overall, these results suggest that kepone-dependent regulation of uterine Bip is mediated via ER-independent mechanism.

Because Bip’s role is primarily mediated through protein-protein interaction, and because Bip molecularly interacts with ER{alpha} to control ER{alpha}-dependent uterine estrogen signaling that includes uterine growth and gene expression (40), we wanted to examine whether kepone-induced uterine Bip also demonstrates a similar protein-protein interaction with ER{alpha}. Uterine protein extracts were subjected to coimmunoprecipitation analysis using either Bip (Fig. 3DGo) or ER{alpha} (data not shown) antibodies, followed by Western blotting for each of these proteins. Although a low level of interaction between Bip and ER{alpha} was detected in the oil group, a markedly induced interaction between these proteins was revealed by kepone treatments (Fig. 3DGo). Furthermore, this interaction appears to be specific because Bip-specific antibody was unable to pull down progesterone receptor (data not shown), a kepone-responsive gene in the mouse uterus (21). In addition, immunoprecipitation using preimmune serum did not detect any specific bands for Bip and ER{alpha} by Western blotting (data not shown). Collectively, these results suggest that kepone influences uterine Bip without involving ERs but directs facilitated interaction of Bip with ER{alpha}.

Uterotrophic effect of kepone strongly correlates with the uterine levels of Bip, and this effect is critically dependent on ER{alpha}
Because Bip essentially controls estrogen-dependent ER{alpha}-mediated uterine growth (40), we examined whether kepone-dependent up-regulation of uterine Bip correlates with the ER{alpha}-dependent uterotrophic changes in mice. Similar to the E2 effects, ovariectomized wild-type mice, given injections of kepone either a single at 15 or 30 mg/kg or multiple (3x) at 7.5 mg/kg and analyzed 24 h after the last injection, were responsive to significant increases in uterine wet weights, compared with oil (Fig. 4AGo). Although a single injection of kepone at 7.5 mg/kg was not effective. Furthermore, analysis of uterine cell proliferation, using BrdU incorporation (Fig. 4Go, B and C) and Ki-67 immunostaining (Fig. 4CGo), revealed a similar result. Interestingly, these observations were consistent with the detection of heightened levels of uterine Bip by kepone at higher doses (15–30 mg/kg) (for comparison, see Fig. 3Go, A and B).


Figure 4
View larger version (104K):
[in this window]
[in a new window]

  		FIG. 4. A, Analysis of uterine wet weights. Adult ovariectomized wild-type mice (10–12 mice in each group) were given single injections of oil, E2 (4 µg/kg), kepone (K) (at 7.5, 15, or 30 mg/kg), or multiple injections of kepone [3 x at 7.5 mg/kg, K-7.5 (M)], and killed 24 h after the last injection. In addition, ovariectomized ER{alpha}(–/–) mice (five to seven mice in each group) were subjected to kepone (30 mg/kg) and oil, and killed at 24 h. *, Values are statistically different (P < 0.001) based on ANOVA (F = 99.7; df = 29), followed by the Dunnett’s t test. B, BrdU incorporation into DNA. Similar to the aforementioned experiments in A, mice were injected with BrdU (50 mg/kg, ip) 2 h before killing. Formaldehyde-fixed paraffin-embedded tissue sections were stained for BrdU incorporation as described in Materials and Methods. Representative data are shown from the analysis of at least five different mice for each group. Tissue sections are shown. Reddish-brown nuclear deposits (as shown by dark black spots in the nuclei of epithelial cells) indicate the sites of positive immunostaining. C, Quantitation of BrdU- or Ki-67-positive cells. The data presented here are after the analysis of at least five different mice from each group. *, Values are statistically different (P < 0.001) against the oil (control), based on ANOVA (BrdU: F = 1782, df = 29; Ki-67: F = 1755, df = 29), followed by the Dunnett’s t test. D, Analysis of uterine cell proliferation by BrdU or Ki-67 in ER{alpha}(–/–) mice. Adult ovariectomized ER{alpha}(–/–) mice were given injections (sc) of oil or kepone (30 mg/kg) and analyzed after 24 h. Representative data are shown from the analysis of at least five different mice for each group. ge, Glandular epithelium; le, luminal epithelium.

 
We also examined whether this kepone-dependent regulation of cell proliferation is critical to the ER{alpha}, using ER{alpha}(–/–) mice. Because the aforementioned studies demonstrated that kepone at 30 mg/kg is responsive to uterotrophic changes in wild-type mice, ER{alpha}(–/–) mice were subjected in a similar manner, and uterine wet weight and cell proliferation were analyzed. Our results revealed that ER{alpha}(–/–) mice were unable to support uterine growth responses by kepone (Fig. 4Go, A and D), although this kepone treatment did show distinct up-regulation of uterine Bip [Fig. 3CGo (i)]. Overall, these results, in conjunction with the aforementioned protein-protein interaction studies, suggest that kepone-dependent uterotrophic effect is critically dependent on the cooperation of Bip and ER{alpha}.

Adenovirus-driven manipulation of uterine Bip modifies estrogenic potency of kepone
The aforementioned results suggested that kepone-induced uterine cell proliferation associates with the heightened levels of endogenous Bip. Because Bip can regulate E2-depedent uterine cell proliferation (40), and because a single injection of kepone at 7.5 mg/kg failed to induce Bip, we speculated that forced expression of Bip by the adenovirus-driven approach will accentuate cell proliferation using this suboptimal dose of kepone. To address this possibility, we first analyzed the status of uterine Bip levels after administration of adenoviruses [rAdGFP (control), or rAdBipS or rAdBipAs]. As shown in Fig. 5AGo, Western blot analyses show that delivery of rAdBipS virus was indeed effective to maintain forced expression of Bip in the uterus, compared with the control virus. In contrast, rAdBipAs was able to suppress the kepone-induced uterine Bip level. Overall, these results suggest that adenovirus-driven manipulation of Bip expression in the mouse uterus is effective. Under this condition of Bip overexpression, we also analyzed the status of uterine cell proliferation in the wild-type mice given a suboptimal dose of kepone (7.5 mg/kg). As shown by BrdU incorporation and Ki-67 immunostaining (Fig. 5Go, B and C), our results demonstrate that forced expression of Bip (via rAdBipS) in conjunction with kepone (7.5 mg/kg) was indeed supportive of uterine cell proliferation (7-fold), whereas the control virus was not effective. Moreover, neither of these viral treatments showed any growth regulatory response in mice when injected with oil (Fig. 5CGo).

To examine further whether Bip is specifically involved in the regulation of kepone-induced uterine cell proliferation, we introduced rAdBipAs or rAdGFP (as a control) in mice before the administration of kepone (15 mg/kg) or oil (as a vehicle control). As shown before, kepone at this dose, compared with oil, was effective in inducing cell proliferation in presence of the control virus (Fig. 5DGo). Although knockdown of Bip via rAdBipAs showed strong suppression of this kepone-induced effect.

Because adenovirus-driven manipulation of Bip affects kepone-induced ER{alpha}-dependent uterine cell proliferation, we have further characterized the regulation of expression of several growth regulatory estrogen-responsive ER-mediated uterine genes (cyclin D1 and Mad2) in this context (50). Consistent with the aforementioned analysis, our results revealed that either forced expression or suppression of Bip caused a respective increase or decrease of ER{alpha}-dependent gene expression by kepone compared with appropriate controls (Fig. 5EGo). Collectively, these results suggest that overexpression or suppression of Bip strongly correlates with a respective increase or decrease of cell proliferation and gene expression in the uteri of mice, in conjunction with kepone.

Stress-regulated signals modulate estrogenicity of kepone in mice
Because Bip is known to be induced by a variety of stress signals (42), and because increased uterine levels of Bip enhance the estrogenicity of kepone, we speculate that stress-inducing signals might enhance growth responsive effects by sublethal doses of kepone. As described in Materials and Methods, ovariectomized wild-type mice were subjected to kepone (2.5 and 7.5 mg/kg), in combination with different stress responses. Previously, it was shown that ultrasonic sound stress or endoplasmic reticulum stress, known to induce stress-specific signals in tissues of mice (51, 52). In our initial analysis, we wanted to examine whether these stress inducers alter the levels of uterine Bip. As shown in Fig. 6Go, A and B, we indeed observed a dramatic induction of uterine Bip in mice, subjected to either stress. Next, we wanted to examine whether stress influences uterine cell proliferation in mice exposed to sublethal doses of kepone (2.5 and 7.5 mg/kg) or oil (as a vehicle control). As shown in Fig. 6CGo, both stress inducers were able to support increased uterine cell proliferation in the presence of either doses of kepone, compared with oil, although kepone-dependent alterations in the ER-stress group were lower in effect, suggesting that a negative influence appeared to be mediated by tunicamycin. Furthermore, our statistical analysis shows that dose-dependent increases of uterine cell proliferation by kepone in the ER-stress group is not significant, although individually they are significant against the oil (Fig. 6CGo). Overall, these results suggest that stress-inducing signals influence kepone-dependent growth regulation, presumably via alteration of uterine Bip.


Figure 6
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 6. Analysis of uterine Bip and actin expression by Western blotting in mice (three to four mice) after the ultrasonic sound stress (A) or ER-stress by injection of tunicamycin (1.0 mg/kg, sc) (B). C, Quantitative analysis of BrdU incorporation. Adult ovariectomized wild-type mice were subjected to stress either by ultrasonic sound or tunicamycin (1.0 mg/kg, sc) injection, and analyzed after 24 h. Statistical significance was performed using ANOVA (F = 87.4, df = 11 for sound-stress studies; F= 7.6, df = 11 for ER-stress studies), followed by the Dunnett’s t test. NS, Not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although kepone-induced actions in the uterus have been implicated estrogenically, the molecular mechanisms of this action remain poorly understood. In this regard, the highlight of the present investigation is that the degree of uterine estrogenicity by kepone can be potentially amplified by an endoplasmic reticulum protein Bip in the mouse uterus. The application of adenovirus-driven strategy using rAdBipS construct or stress-mediated signals appeared to cause sustained up-regulation of uterine Bip, which also resulted in a susceptible increase in uterine epithelial cell proliferation in mice given a suboptimal dose of kepone. This finding is highly significant with regard to the reproductive toxicity attributed to a large variety of polychlorinated hydrocarbons that still persists in the environment (1, 4, 5, 6). Based on existing literature (21, 22, 53, 54, 55, 56), it has been clearly shown that doses of the tested xenoestrogenic compounds used in the present study exhibit estrogen-like activities in rodent uterus viz. wet weights, epithelial thickness, and expressional regulation of estrogen-responsive genes. The basis for reproductive toxicity of these compounds was thought to be mediated by their interactions with the nuclear ERs of the target tissues. Consistent with this notion, our studies provide evidence that kepone essentially uses ER{alpha} in controlling the uterine growth response in mice. Furthermore, in conjunction with our previous studies (40), it is now evident to propose that E2 and kepone could elicit uterine Bip, which then mediates a cross talk with ER{alpha} via protein-protein interaction to control ER{alpha}-mediated nuclear gene transcription that is considered to be fundamental for manifestation of a full complement of estrogenic activity in the uterus.

Recently, Bip and Wnt-signaling genes have been identified as nonclassical targets because they do not involve nuclear ERs during the regulation by E2 or 4OH-E2 in the uteri of mice (39, 44). In the present study, we wanted to examine whether kepone, methoxychlor, and o,p’-DDT exhibit any nonclassical responses to these gene-signaling systems. Our analysis revealed that kepone selectively modulates uterine Bip mRNAs, without influencing Wnt-signaling genes, primarily through dose and time-dependent manners (Figs. 2Go and 3Go). In contrast, methoxychlor and o,p’-DDT were totally ineffective for these nonclassical gene targets (Fig. 2Go). Our observations with distinct regulation of uterine gene by estrogenic compounds are not surprising. Indeed, it has been reported that kepone and 4-OH-E2 were able to induce LF gene in the uteri of ER{alpha} null mice, whereas E2 was completely ineffective, suggesting that estrogenic compounds can have differential effects on uterine genes (20).

Accumulating evidence suggests that natural and environmental estrogens modulate uterine genes without involving ERs (20, 21, 22, 38, 39, 44). These studies are primarily based on the observations that ER{alpha} null or wild-type mice, in which ER functions are silenced by ER antagonist ICI 182,780 (ICI), manifest expression of uterine genes in response to estrogenic compounds. Our findings of up-regulation of uterine Bip by kepone in wild-type and ER{alpha}(–/–) mice [Fig. 3CGo (i)] in the presence or absence of ICI consistently suggest that a similar ER-independent mechanism is operative for kepone-dependent regulation of uterine Bip in mice.

Our findings of uterine regulation of Bip expression and the increase in uterine wet weight and cell proliferation (Fig. 4Go, A–C) requiring concentrations (15–30 mg/kg) or multiple injections (3x) of kepone (7.5 mg/kg) in mice are consistent with the existing literature (13, 20, 21, 22). Moreover, consistent with our observations in the present study, multiple injections of kepone at 7.5 mg/kg have exhibited additive effects on uterine LF gene expression (20). However, to our knowledge there is no report to indicate that multiple injections of kepone at this dose can cause bioaccumulation of this xenoestrogen in the body. Moreover, one may raise concern about the toxicity of kepone in the body because this compound is known to affect liver and kidney detoxification systems, which may indirectly affect hormone metabolism. However, to our knowledge there is no evidence to suggest that estrogenic effects (viz. such as DNA and protein synthesis, cell proliferation, uterine weight increase, etc.) of this compound using the doses in the present study in mice are due to an induced cytotoxicity through other secondary organs. Furthermore, it should also be recognized that we have compared the effects of kepone in the wild-type and ER{alpha}(–/–) mice. Thus, the differences as observed by kepone in respect to uterine growth between these mice (Fig. 4Go) should not be considered as nonspecific effects because such effects should also be seen in both mice.

Bip, as a molecular chaperone, primarily functions to direct appropriate protein folding and assembly and intracellular trafficking (42). Studies have shown that Bip is an abundant protein during the growth regulatory conditions in both normal and tumor tissues (57, 58). In the present study, because Bip was not regulatory by o,p-DDT and methoxychlor in the mouse uterus (Fig. 2Go, B and C), and because uterine Bip is specifically altered by kepone, our further studies of Bip in conjunction with uterine growth precludes studies involving estrogenicity of o,p’-DDT and methoxychlor. Previously, we have shown that Bip plays an essential role via protein-protein interaction, to control nuclear ER{alpha} functions in respect to gene transcription and growth regulation by E2 (40). Consistent with these results, we observed that kepone regulates uterine Bip, in the absence of ER{alpha} [Fig. 3CGo (i)]. In addition, Bip molecularly interacts with ER{alpha} under the direction of kepone (Fig. 3DGo), suggesting that Bip may regulate kepone-dependent ER{alpha} function. This was strongly supported by our observation that kepone-dependent ER{alpha}-mediated gene expression is abrogated after suppression of uterine Bip via adenovirus approaches (Fig. 5EGo). Furthermore, it was observed that regulation of uterine Bip is closely followed by uterine growth in the presence of kepone in the wild-type mice (Figs. 3Go, A and B, vs. 4Go, A–C), and this is again strongly compromised after knockdown of uterine Bip expression (Fig. 5DGo), suggesting that uterine Bip is critically involved in this regulation. In contrast, studies also showed that up-regulation of uterine Bip did not correlate with the growth response in the presence of kepone in ER{alpha}(–/–) mice [Figs. 3CGo (i) vs. 4, A and D], suggesting that Bip must cooperate with ER{alpha} to have uterine growth under the direction of kepone. This result is further consistent with the observation that forced expression of uterine Bip in the wild-type mice did not lead to cell proliferation in the presence of oil. Overall, these results suggest that kepone-dependent uterine growth response used a molecular cross talk between Bip and ER{alpha}.

The major thrust of this work was to demonstrate that sustained levels of uterine Bip can be detrimental to kepone’s action in mice. This idea was primarily evolved from our previous report that Bip is regulatory for estrogen-dependent ER{alpha}-mediated uterine growth (40) and from our present findings that kepone at a sublethal dose (7.5 mg/kg) is unable to sustain up-regulation of uterine Bip expression (Figs. 2Go and 3Go) and cell proliferation (Fig. 4Go) in mice. This was further supported by our observations of ER{alpha}-dependent enhanced uterine cell proliferation and gene expression in mice after exposure to a sublethal dose of kepone, in combination with forced expression of uterine Bip via an adenovirus-driven strategy (Fig. 5Go, B, C, and E). Overall, our studies, using both sense or antisense adenoviruses for Bip, suggest that heightened expression of Bip is strongly correlated with the ability of kepone to induce ER{alpha}-dependent uterine cell proliferation and gene expression (Fig. 5Go, B–E). In this regard, it should be noted that a similar sort of enhancement of ligand-dependent sensitivity for nuclear receptor functional activities has been reported for some xenobiotic compounds (59).

It is known that the regulation of cellular Bip occurs under a variety of conditions, including stress, chemical toxicity, treatment with Ca2+ ionophores, and inhibitors of glycosylation, that all influence endoplasmic reticulum (42). Furthermore, it has been shown that the effects of chronic stress cause enhanced uterine growth response by E2 in rats (60), therefore, we speculated that stress-induced uterine Bip may support uterine estrogenicity for a suboptimal dose of kepone. Consistent with this speculation, indeed our observations showed that the increase of uterine cell proliferation occurs in correlation with heightened levels of uterine Bip in mice that were subjected to stress in combination with kepone at suboptimal doses (2.5 or 7.5 mg/kg) (Fig. 6Go). Although, in general, less responsive effects for cell proliferation in the ER-stress group appear to suggest that tunicamycin may impose an inhibition of cell proliferation, as implicated in the literature (61, 62). Overall, these results revealed that endogenous Bip via stress-related signals contributes to the establishment of uterine estrogenicity for kepone.

In summary, studies provide novel evidence that Bip can be considered a major regulator to amplify estrogenic potency for a weak xenoestrogen. Furthermore, because Bip is regulatory by a variety of signals in the body, including stress and cancer, these may thus act as plausible risk factors to produce enhanced estrogenicity for xenoestrogen, which should be a major health concern.


   Footnotes
 
This work was supported in part by the National Institutes of Health Grants HD37830 and ES07814.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 19, 2007

Abbreviations: BrdU, Bromodeoxyuridine; CMV, cytomegalovirus; E2, estradiol-17ß; ER, estrogen receptor; GFP, green fluorescence protein; ICI, ICI-182,780 or [7(9-4,4,5,5,5-pentafluoropenylsulfinyl) nonyl-estra-1,3,4(10-triene-3,17-diol)]; LF, lactoferrin; o,p’-DDT, 1-(o-chlorophenyl)-1-(p-chlorophenyl) 2,2,2-trichloroethane; rAdBipAs, recombinant adenoviral plasmids for the antisense Bip; rAdBipS, recombinant Bip-sense cDNA construct; rAdGFP, recombinant adenoviral plasmids for the green fluorescence protein control; rpL7, ribosomal protein L7; SFRP-2, secreted frizzled related protein 2.

Received April 25, 2007.

Accepted for publication July 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Simonich SL, Hites RA 1995 Global distribution of persistent organochlorine compounds. Science 269:1851–1854[Abstract/Free Full Text]
  2. Schapira A 2006 DDT: a polluted debate in malaria control. Lancet 368:2111–2113[CrossRef][Medline]
  3. Gunasekaran K, Sahu SS, Jambulingam P, Das PK 2005 DDT indoor residual spray, still an effective tool to control Anopheles fluviatilis-transmitted Plasmodium falciparum malaria in India. Trop Med Int Health 10:160–168[CrossRef][Medline]
  4. Manz M, Wenzel KD, Dietze U, Schuurmann G 2001 Persistent organic pollutants in agricultural soils of central Germany. Sci Total Environ 277:187–198[CrossRef][Medline]
  5. Backe C, Cousins IT, Larsson P 2004 PCB in soils and estimated soil-air exchange fluxes of selected PCB congeners in the south of Sweden. Environ Pollut 128:59–72[CrossRef][Medline]
  6. Chen Y, Wang C, Wang Z 2005 Residues and source identification of persistent organic pollutants in farmland soils irrigated by effluents from biological treatment plants. Environ Int 31:778–783[CrossRef][Medline]
  7. Zhang H, Lu Y, Dawson RW, Shi Y, Wang T 2005 Classification and ordination of DDT and HCH in soil samples from the Guanting Reservoir, China. Chemosphere 60:762–769[Medline]
  8. Ferrante MC, Cirillo T, Naso B, Clausi MT, Lucisano A, Cocchieri RA 2007 Polychlorinated biphenyls and organochlorine pesticides in seafood from the Gulf of Naples (Italy). J Food Prot 70:706–715[Medline]
  9. Safe SH, Pallaroni L, Yoon K, Gaido K, Ross S, Saville B, McDonnell D 2001 Toxicology of environmental estrogens. Reprod Fertil Dev 13:307–315[CrossRef][Medline]
  10. Singleton DW, Khan SA 2003 Xenoestrogen exposure and mechanisms of endocrine disruption. Front Biosci 8:s110–s118
  11. Safe S 1990 Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Crit Rev Toxicol 21:51–88[Medline]
  12. Wolff MS, Toniolo P, Lee E, Rivera M, Dubin N 1993 Blood levels of organochlorine residues and the risk of breast cancer. J Natl Cancer Inst 85:648–652[Abstract/Free Full Text]
  13. Das SK, Paria BC, Johnson DC, Dey SK 1997 Embryo-uterine interactions during implantation: potential sites of interference by environmental toxins. In: Sipes G, McQueen CA, Gandolfi AJ, eds. Comprehensive toxicology. Reproductive and endocrine toxicology. Vol. 10. New York: Elsevier Science Ltd.; 317–328
  14. Davis DL, Bradlow HL, Wolff M, Woodruff T, Hoel DG, Anton-Culver H 1993 Medical hypothesis: xenoestrogens as preventable causes of breast cancer. Environ Health Perspect 101:372–377[Medline]
  15. Safe S 1995 Environmental and dietary estrogens and human health: is there a problem? Environ Health Perspect 103:346–351[Medline]
  16. Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, Tong W, Shi L, Perkins R, Sheehan DM 2000 The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol Sci 54:138–153[Abstract/Free Full Text]
  17. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  18. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson JA 1998 Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 139:4252–4263[Abstract/Free Full Text]
  19. Safe S, Connor K, Ramamoorthy K, Gaido K, Maness S 1997 Human exposure to endocrine-active chemicals: hazard assessment problems. Regul Toxicol Pharmacol 26(1 Pt 1):52–58
  20. Das SK, Taylor JA, Korach KS, Paria BC, Dey SK Lubahn DB 1997 Estrogenic responses in estrogen receptor-alpha deficient mice reveal a distinct estrogen signaling pathway. Proc Natl Acad Sci USA 94:12786–12791[Abstract/Free Full Text]
  21. Das SK, Tan J, Johnson DC, Dey SK 1998 Differential spatiotemporal regulation of lactoferrin and progesterone receptor genes in the mouse uterus by primary estrogen, catechol estrogen, and xenoestrogen. Endocrinology 139:2905–2915[Abstract/Free Full Text]
  22. Ghosh D, Taylor JA, Green JA, Lubahn DB 1999 Methoxychlor stimulates estrogen-responsive messenger ribonucleic acids in mouse uterus through a non-estrogen receptor (non-ER) {alpha} and non-ER ß mechanism. Endocrinology 140:3526–3533[Abstract/Free Full Text]
  23. Simons Jr SS, Oshima H, Szapary D 1992 Higher levels of control: modulation of steroid hormone-regulated gene transcription. Mol Endocrinol 6:995–1002[Free Full Text]
  24. Kupfer D, Bulger WH 1990 Inactivation of the uterine estrogen receptor binding of estradiol during P-450 catalyzed metabolism of chlorotrianisene (TACE). Speculation that TACE antiestrogenic activity involves covalent binding to the estrogen receptor. FEBS Lett 261:59–62[CrossRef][Medline]
  25. Meyers CY, Kolb VM, Gass GH, Rao BR, Roos CR, Dandliker WB 1988 Doisynolic-type acids: uterotrophically potent estrogens which compete poorly with estradiol for cytosolic estradiol receptors. J Steroid Biochem 31:393–404[CrossRef][Medline]
  26. Meyers CY, Lutfi HG, Adler S 1997 Transcriptional regulation of estrogen-responsive genes by non-steroidal estrogens: doisynolic and allenolic acids. J Steroid Biochem Mol Biol 62:477–489[CrossRef][Medline]
  27. Bulger WH, Muccitelli RM, Kupfer D 1978 Studies on the in vivo and in vitro estrogenic activities of methoxychlor and its metabolites. Role of hepatic mono-oxygenase in methoxychlor activation. Biochem Pharmacol 27:2417–2423[CrossRef][Medline]
  28. Cummings AM 1997 Methoxychlor as a model for environmental estrogens. Crit Rev Toxicol 27:367–379[Medline]
  29. Nagel SC, vom Saal FS, Thayer KA, Dhar MG, Boechler M, Welshons WV 1997 Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ Health Perspect 105:70–76[Medline]
  30. Sheehan DM, Willingham E, Gaylor D, Bergeron JM, Crews D 1999 No threshold dose for oestradiol-induced sex reversal of turtle embryos: how little is too much? Environ Health Perspect 107:155–159[Medline]
  31. Routledge EJ, White R, Parker MG, Sumpter JP 2000 Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) {alpha} and ERß. J Biol Chem 275:35986–35993[Abstract/Free Full Text]
  32. Mueller SO, Simon S, Chae K, Metzler M, Korach KS 2004 Phytoestrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor {alpha} (ER{alpha}) and ERß in human cells. Toxicol Sci 80:14–25[Abstract/Free Full Text]
  33. Nishikawa J, Saito K, Goto J, Dakeyama F, Matsuo M, Nishihara T 1999 New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator. Toxicol Appl Pharmacol 154:76–83[CrossRef][Medline]
  34. Smith CC, Taylor HS 2007 Xenoestrogen exposure imprints expression of genes (Hoxa10) required for normal uterine development. FASEB J 21:239–246[Abstract/Free Full Text]
  35. Tan J, Paria BC, Dey SK, Das SK 1999 Differential uterine expression of estrogen and progesterone receptors correlates with uterine preparation for implantation and decidualization in the mouse. Endocrinology 140:5310–5321[Abstract/Free Full Text]
  36. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677–15682[Abstract/Free Full Text]
  37. Koehler KF, Helguero LA, Haldosen LA, Warner M, Gustafsson JA 2005 Reflections on the discovery and significance of estrogen receptor ß. Endocr Rev 26:465–478[Abstract/Free Full Text]
  38. Henley DV, Korach KS 2006 Endocrine-disrupting chemicals use distinct mechanisms of action to modulate endocrine system function. Endocrinology 147(Suppl 6):S25–S32
  39. Das SK, Tan J, Raja S, Halder J, Paria BC, Dey SK 2000 Estrogen targets genes involved in protein processing, calcium homeostasis and Wnt signaling in the mouse uterus independent of estrogen receptor-{alpha} and -ß. J Biol Chem 275:28834–28842[Abstract/Free Full Text]
  40. Ray S, Hou X, Zhou HE, Wang H, Das SK 2006 Bip is a molecular link between the phase-I and phase-II estrogenic responses in uterus. Mol Endocrinol 20:1825–1837[Abstract/Free Full Text]
  41. Hartl FU 1996 Molecular chaperones in cellular protein folding. Nature 381:571–579[CrossRef][Medline]
  42. Kaufman RJ 1999 Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 13:1211–1233[Free Full Text]
  43. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90:11162–11186[Abstract/Free Full Text]
  44. Hou X, Tan Y, Li M, Dey SK, Das SK 2004 Canonical Wnt signaling is critical to estrogen mediated uterine growth. Mol Endocrinol 18:3005–3044
  45. Das SK, Chakraborty I, Paria BC, Wang-X-N, Plowman GD, Dey SK 1995 Amphiregulin is a implantation specific and progesterone-regulated gene in the mouse uterus. Mol Endocrinol 9:691–701[Abstract/Free Full Text]
  46. Tan J, Raja S, Davis MK, Tawfik O, Dey SK, Das SK 2002 Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation. Mech Dev 111:99–113[CrossRef][Medline]
  47. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B 1998 A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514[Abstract/Free Full Text]
  48. Rahman MA, Li M, Li P, Wang H, Dey SK, Das SK 2006 Hoxa-10 deficiency alters region-specific gene expression and perturbs differentiation of natural killer cells during decidualization. Dev Biol 290:105–117[CrossRef][Medline]
  49. Daikoku T, Tranguch S, Friedman DB, Das SK, Smith DF, Dey SK 2005 Proteomic analysis identifies immunophilin FKBP52 as a downstream target of Hoxa10 in the periimplantation mouse uterus. Mol Endocrinol 19:683–697[Abstract/Free Full Text]
  50. Hewitt SC, Deroo BJ, Hansen K, Collins J, Grissom S, Afshari CA, Korach KS 2003 Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen. Mol Endocrinol 17:2070–2083[Abstract/Free Full Text]
  51. Tometten M, Blois S, Kuhlmei A, Stretz A, Klapp BF, Arck PC 2006 Nerve growth factor translates stress response and subsequent murine abortion via adhesion molecule-dependent pathways. Biol Reprod 74:674–683[Abstract/Free Full Text]
  52. Kondoh M, Tsukada M, Kuronaga M, Higashimoto M, Takiguchi M, Himeno S, Watanabe Y, Sato M 2004 Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice. Toxicol Lett 148:133–139[CrossRef][Medline]
  53. Welch RM, Levin W, Conney AH 1969 Estrogenic action of DDT and its analogs. Toxicol Appl Pharmacol 14:358–367[CrossRef][Medline]
  54. Eroschenko VP, Rourke AW, Sims WF 1996 Estradiol or methoxychlor stimulates estrogen receptor (ER) expression in uteri. Reprod Toxicol 10:265–271[CrossRef][Medline]
  55. Bulger WH, Kupfer D 1985 Estrogenic activity of pesticides and other xenoblotics on the uterus and male reproductive tract. In: Thomas JA, Korach KS, McLachlan JA, eds. Endocrine toxicology. New York: Raven Press; 1–33
  56. Hammond B, Katzenellenbogen BS, Krauthammer N, McConnell J 1979 Estrogenic activity of the insecticide chlordecone (Kepone) and interaction with uterine estrogen receptors. Proc Natl Acad Sci USA 76:6641–6645[Abstract/Free Full Text]
  57. Simmons DG, Kennedy TG 2000 Induction of glucose-regulated protein 78 in rat uterine glandular epithelium during uterine sensitization for the decidual cell reaction. Biol Reprod 62:1168–1176[Abstract/Free Full Text]
  58. Lee AS 2007 GRP78 induction in cancer: therapeutic and prognostic implications. Cancer Res 67:3496–3499[Abstract/Free Full Text]
  59. Jansen MS, Nagel SC, Miranda PJ, Lobenhofer EK, Afshari CA, McDonnell DP 2004 Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition. Proc Natl Acad Sci USA 101:7199–7204[Abstract/Free Full Text]
  60. Gunin AG 1996 Effect of chronic stress on estradiol action in the uterus of ovariectomized rats. Eur J Obstet Gynecol Reprod Biol 66:169–174[CrossRef][Medline]
  61. Schraen-Maschke S, Zanetta JP 2003 Role of oligomannosidic N-glycans in the proliferation, adhesion and signalling of C6 glioblastoma cells. Biochimie 85:219–229[Medline]
  62. Harrison ML, Villemez CL 1981 Protein glycosylation is required for the proliferation of mitogen-activated lymphocytes. J Immunol 127:2616–2617[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ray, S.
Right arrow Articles by Das, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ray, S.
Right arrow Articles by Das, S. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals