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Potential role of HSP90 in mediating the interactions between estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) signaling pathways
Toxicology Letters 226 (2014) 6–13
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Potential role of HSP90 in mediating the interactions between estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) signaling pathways Ziwei Chang 1 , Ming Lu 1,2 , So-Sun Kim, Jang-Su Park ∗ Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Republic of Korea
h i g h l i g h t s • A reciprocal inhibiting crosstalk between the AhR and ER pathways was observed. • Cd up-regulated the E2-induced VTG and PCB77-induced CYP1A. • HSP90 participated in the reciprocal inhibiting interaction.
a r t i c l e
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Article history: Received 5 December 2013 Received in revised form 22 January 2014 Accepted 23 January 2014 Available online 31 January 2014 Keywords: Heat shock protein 90 Vitellogenin CYP1A Goldfish Primary culture
a b s t r a c t The estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) are ligand-activated transcription factors involved in estrogen or xenobiotic exposure, whereas the 90-kDa heat shock protein (HSP90), which is a ubiquitously expressed molecular chaperone, is involved in the signal transduction process. Although the interactions between these pathways have been under investigation, the mechanisms are unclear and the potential role of HSP90 in these interactions has not been reported. The results of goldfish primary hepatocytes showed that exposure to PCB77 and 17-estradiol (E2) alone induced significant protein expression of cytochrome P450 1A (CYP1A) and vitellogenin (VTG), respectively. On the other hand, the combined exposure to PCB77 and E2 led to the reduction of CYP1A and VTG compared to the single treatments. Although the AhRs and ERs were naturally induced during the co-treatment, the total amount of HSP90 binding to the receptors was not changed. Furthermore, while the HSP90 chaperon activity was blocked by the specific inhibitor (geldanamycin), reciprocal inhibition between AhR and ER pathways was not observed. These findings indicate a potential role of HSP90 where competition between AhR and ER for binding to HSP90 can occur and cause reciprocal inhibition. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Environmental pollution has increased substantially in recent decades. The most important pollutants include polychlorinated and polybrominated aromatics, heavy metals, phenols, organochlorine pesticides and estrogenic substances. Fish are valuable biomonitors of environmental pollution, and in recent studies, goldfish (Carassius auratus) have been found to be a useful species for examining the effects of toxic chemicals because they are widespread found widely in aquatic environments. Cytochrome P450 has also been proposed as a biochemical indicator of toxicant exposure in aquatic species. Cytochrome
∗ Corresponding author. Tel.: +82 51 510 2294; fax: +82 51 516 7421. E-mail address: [email protected] (J.-S. Park). 1 Both authors contributed equally to this work. 2 Present address: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China. 0378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2014.01.032
P4501A (CYP1A) is the most important gene family involved in the metabolism of xenobiotics and is used as a biomarker of exposure to the organic pollutants (Kim et al., 2007). The fish CYP1A gene is used widely to identify the effects of polycyclic aromatic hydrocarbons. The molecular mechanism of CYP1A activation has been studied extensively. Hahn (1998) reviewed the transcriptional regulation of CYP1A in fish through a transcription factor known as the aryl hydrocarbon receptor (AhR). Potent agonist binding to the receptor results in the disassociation of AhR from a chaperone complex consisting of heat shock protein 90 (HSP90), followed by the translocation of AhR to the nucleus and binding to a dioxin response element together with the aryl hydrocarbon receptor nucleus translocator (ARNT) (Gu et al., 2000; Hahn, 2001; Ma, 2007). In fish, another sensitive biomarker, vitellogenin (VTG) is used frequently to evaluate the effects of endocrine-disrupting chemicals (EDCs) (Lee et al., 2006; Radice et al., 2004). VTG is normally produced by female animals in response to normal estradiol cycles but is not normally synthesized in males (Vaccaro et al., 2005). The
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estrogenic control of VTG is mediated by binding the most potent estrogen, 17--estradiol (E2), to the ER. The ER–E2 complex binds with high affinity to specific DNA sequences known as the estrogenresponsive elements located in the regulatory regions of the VTG gene, leading to this activation and expression (Berg et al., 2004; Guevel et al., 2000). When exposed to heat shock or other insults, such as oxidative stress or toxic substances, organisms synthesize a common set of proteins known as heat shock proteins (HSPs). HSPs are ubiquitous and highly conserved proteins that can be grouped into several families according to their apparent molecular mass (Georgopoulos and Welch, 1993; Wei et al., 2013). Of these, HSP90 is a molecular chaperone that is involved in multiple signal transduction pathways (Soshilov and Denison, 2011). At least 100 proteins are regulated by HSP90, including the key mediators of signal transduction and cell cycle control, steroid hormone receptors, and tyrosine and serine/threonine kinases (Nollen and Morimoto, 2002; Sidera et al., 2008). HSP90 is a chaperone protein that is important for maintaining its client proteins including AhR and ER proteins, in a functional conformation for its biological function (To et al., 2011). The association with HSP90 is required for the AhR to assume a conformation that is optimal for ligand binding (Pongratz et al., 1992). Moreover, the 70 kDa heat shock proteins, HSP70s, has been shown to play a major role in the cellular response and promote the ability of organisms to survive various environmental stresses, and evaluation for HSP70 has been used as a biomarker of effect of pollutant.(Padmini and Usha Rani, 2008). Organisms in the environment are exposed to various mixtures of chemicals, and the response of the biomarkers can be affected by mixed exposure. The present study examined the biomarker responses of VTG, CYP1A and HSP70. Although the properties of the corresponding inducers and their ability to induce the proteins through the receptors are clear, details of the molecular and cellular processes underpinning these effects as well as the interactions between them are less evident. Therefore, the interactions between the biomarkers and the factors affecting these responses were investigated. Moreover, little is known regarding the role of HSP90 in modulating the interactions between AhR and ER. Geldanamycin (GA), a quite specific inhibitor of HSP90, was used to examine the role HSP90. GA can bind specifically to the ATP-binding site of HSP90, resulting in the inhibition of the HSP90 interaction with the target proteins (Powers and Workman, 2007). 2. Materials and methods 2.1. Animals Male goldfish (C. auratus) (200–300 g, n = 5) were purchased from a local market and maintained in dechlorinated freshwater tanks. The fish were kept in an aquarium at 20 ◦ C in an aquatic trunk with dechlorinated tap water for three days and fed once per day. All studies were carried out in accordance with the national and institutional guidelines for the protection of animal welfare. The project was approved by the Committee on Ethics of Pusan National University.
in 6-well plates in L-15 medium at density of 0.75 × 106 cells/well using a slight modifications of the methods of reported elsewhere (Takemura and Kim, 2001. The media was changed every 24 h. 2.4. Exposure of hepatocytes Approximately 48 h after initiating the primary culture, the hepatocytes were exposed to different concentrations (1, 10, 50, 100 and 200 nM) of CdCl2 , 17estradiol (E2) and PCB77, singly and in combination. Exposed to the solvent ethanol only was used as the control for each experiment. The media were replaced with fresh media containing the respective test chemicals and concentrations every 24 h. After exposure, the medium and cells were harvested for protein level analysis. The medium was collected to measure the VTG or CYP1A concentrations, and the cells were harvested to measure the levels of HSP70, AhR1, AhR2 and ER protein expression. 2.5. Protein level analysis A sandwich enzyme-linked immunosorbent assay system (ELISA) was used to measure the VTG levels using the methods described elsewhere (Li et al., 2005). The same method with slight modifications was performed for HSP70 and CYP1A analysis. Standard curves of HSP70, CYP1A and VTG were plotted using the measurements of 10 dilutions of the purified proteins (Fig. S1). The levels of HSP70, CYP1A and VTG were quantified by ELISA using the followed linear equations: Y = 0.079 + 0.0021X; Y = 0.0088 + 0.0031X and Y = 0.0592 + 0.0029X, respectively. Briefly, the cells were harvested in a cell suspension buffer, centrifuged, and resuspended in RIPA buffer. The cell suspension was incubated for 30 min at 4 ◦ C and then centrifuged at 16,000 g for 10 min at 4 ◦ C. The supernatant was coated on NUNC-immunoTM plates. After washing three times with PBST buffer (PBS with 0.05% Tween 20), the cells were blocked with PBSTG (PBST with 1% Gelatin) for one hour at 37 ◦ C and washed again. The polyclonal antibodies of anti-goldfish HSP70, CYP1A, VTG, AhR1/2 and ER, which were prepared in this laboratory (Chang et al., 2011; Lu et al., 2013), were used for specific binding. Goat anti-mouse IgG conjugated with alkaline phosphatase (sigma) was used as the secondary antibody. Finally, phosphatase substrate solutions (KPL) were added, and the absorbance at 490 nm was measured on an ELISA reader after a reaction time of 10 min. Statistical analysis was performed using the SPSS (version 11.0) program. The significant differences between the means were examined using a one-way analysis of variance (ANOVA). A p value <0.05 was considered significant. 2.6. Immunoprecipitation The partial cDNA, which consisted of 192 base pairs corresponding to aa112-175 of C. auratus HSP90 was amplified from the liver tissue, with the primers designed based on the published sequences (Accession number: DQ872650). Recombinant partial HSP90 were produced in Escherichia coli BL21 (DE3) and purified. The mice were injected with the protein to obtain the polyclonal antibodies for the production of anti-HSP90 antibodies. An immunoprecipitation experiment was performed, as described previously (Li et al., 2006). Briefly, after pre-clearing with the protein A-Sepharose beads, equal amounts of the protein lysate were immunoprecipitated with the anti-HSP90 antibodies for 2 h at 4 ◦ C. The immune complexes were collected for 2 h with 40 l of protein A-Sepharose and washed five times. The samples were processed by Western blotting. Equal amounts of the protein lysate and immune complexes were resolved by SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 5% BSA in a TBS-T buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) and probed with the respective HSP90, AhR2 or ER antibodies. GADPH was used as the loading control for the immunoprecipitation experiments. Anti-mouse IgG (sigma A3562) was used as the secondary antibody. The blots were developed by enhanced chemiluminescence (Western blue stabilized substrate for alkaline phosphatase, Promega). The analysis was performed using the software ImageQuant TL (GE Healthcare).
2.2. Chemicals and reagents
3. Results
Geldanamycin (GA), resorufin ethyl ether and nicotinamide adenine dinucleotide phosphate (NADPH), CdCl2 , E2 and PCB77 were purchased from Sigma–Aldrich (South Korea). All the other chemicals were of the highest commercially available grade.
3.1. Single treatments of HSP70, VTG and CYP1A protein expression
2.3. Isolation and primary culture of hepatocytes Five fish were euthanized with an overdose of MS222 (Sigma), and their liver tissue was isolated carefully and perfused with hepatocyte buffer (HB, 136.9 mM NaCl, 5.4 mM KCl, 0.81 mM MgSO4 , 0.44 mM KH2 PO4 , 0.33 mM Na2 HPO4 , 5.0 mM NaHCO3 , pH 7.6) for 10 min at room temperature. The liver tissue was then digested with HB buffer containing collagenase (0.3 mg/ml) (Sigma, St. Louis, MO). The softened tissue was minced, and sieved through a nylon mesh (50 m). The resulting cell suspension was washed three times and centrifuged three times at 50 g for 90 s at 10 ◦ C with HB containing 1.5 mM CaCl2 . The hepatocytes were cultured and plated
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The levels of HSP70, VTG and CYP1A in the cultured primary hepatocytes were quantified by ELISA using the polyclonal antibody. Ethanol in the medium had no observable effect on the protein expressions in hepatocyte culture. The level of HSP70 induced by 10 nM, 50 nM, 100 nM and 200 nM CdCl2 increased significantly after 48 h and 96 h of exposure compared to the control (Fig. 1A). In contrast, no obvious increase in the level of HSP70 was observed in the hepatocytes treated with E2 or PCB77 (Fig. 1B and C). The cells treated with either Cd or PCB77 showed a similar level
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Fig. 1. Expression of HSP70, CYPA1 and VTG in a primary culture of goldfish hepatocytes exposed to different treatments. The concentrations of 17-estradiol (E2), cadmium and PCB77 ranged from 0 to 200 nM. Protein expression was measured by ELISA at 48 h and 96 h after the treatments. The results are reported as the mean ± S.E.M (n = 4–6 fish). An asterisk indicates a significant different from controls if p < 0.05 determined by ANOVA.
of VTG synthesis and no increase was observed at different concentrations ranging from 1 nM to 200 nM (Fig. 1D and E). On the other hand, treatment with E2 led to a significant concentrationdependent increase in the induction of VTG (Fig. 1F). For the induction of CYP1A, after 48 h and 96 h exposure to different concentrations of PCB77, the cells showed an increase compared to the untreated cells (Fig. 1G). No obvious induction of CYP1A was observed in the hepatocytes treated with CdCl2 or E2 (Fig. 1H and I). 3.2. Effects of the co-treatments on HSP70, VTG and CYP1A protein expression To determine the mutual impacts between the three chemicals of E2, CdCl2 and PCB77 on protein expression, a 100 nM agonist was selected as the control to determine if the other two chemicals affect the corresponding protein expression. The additional treatments of E2 or PCB77 in the 100 nM CdCl2 -induced cells showed no significant increase in HSP70 compared to those treated with CdCl2 alone (Fig. 2A and B). By varying the concentration of E2 or PCB77, a less obvious increase in HSP70 was observed at a low concentration of E2 or PCB77, but no increase or decrease was observed in the concentration range, 100–200 nM (Fig. 2A and B).
The hepatocytes were then exposed to 100 nM E2 in combination with CdCl2 or PCB77 (from 1 to 200 nM), using 100 nM E2 alone as the control. Interestingly, co-treatment with 100 nM E2 and 100 nM CdCl2 resulted in a 50% increase in the VTG level compared to the control (Fig. 2C). In contrast, additional exposure to PCB77 resulted in a decrease in the VTG levels compared to 100 nM E2 alone; the addition of 100 nM PCB77 resulted in the lowest level of VTG (Fig. 2D). In the experiments examining the effects of CdCl2 and E2 on PCB77-induced CYP1A protein expression, CdCl2 induced a significant concentration-dependent increase in the E2-induced CYP1A levels (Fig. 2E). Co-treatment of 10 nM PCB77 with 100 nM and 200 nM CdCl2 induced a 50% increase compared to PCB77 alone. On the other hand, a slight decrease was observed with 1 nM CdCl2 (Fig. 2E). In addition, co-exposure to E2 (1–200 nM) and 100 nM PCB77 resulted in a decrease in CYP1A protein expression (Fig. 2F). 3.3. Effects of the co-treatments on AhR and ER receptors The expression of goldfish ER, AhR1 and AhR2 proteins in the primary hepatocytes were measured to determine the effects of the combined treatments on the receptors of the AhR and ER signaling pathways (Fig. 3). Exposure to E2 alone produced a
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Fig. 2. Protein expression of HSP70, CYPA1 and VTG exposed to 17-estradiol (E2), cadmium and PCB77 in combination for 48 h or 96 h. The data is reported as a percentage of the single treatment control ± standard error of the mean (n = 8 fish). Values significantly different from the corresponding 48 h (*) or 96 h (#) values at p < 0.05 as determined by ANOVA.
significant concentration-dependent increase in ER protein expression, whereas cadmium and PCB77 alone did not alter ER protein expression (Fig. 3A). Combining E2 (100 nM) with various concentrations of cadmium or PCB77 did not result in a change in ER protein expression (Fig. 3A). For the AhR receptor, exposure to PCB77 alone caused a small increase in AhR1 expression. On the other hand, a concentration-dependent significant increase in AhR2 protein expression was observed (Fig. 3B and C). By comparing with 100 nM PCB77 alone, the co-treatments of 100 nM PCB77 with various concentrations of E2 or cadmium caused slight but non-significant elevation of no AhR1/2 proteins (Fig. 3B). 3.4. HSP90 participates in modulating the interactions The published sequences of HSP90 showed high homology to other teleosts, which was classified as HSP90. HSP90, as the constitutive form, has been associated with cellular transformation and
signal transduction. Partial goldfish HSP90 was expressed in E. coli and purified. The selected sequences were specific to HSP90 and involved multiple epitopes (data not shown), that are suitable for antibody development. As expected, the anti-HSP90 antibody produced specifically recognized goldfish HSP90 in the hepatocytes (Fig. S2) and was used in subsequent experiments. The experiments were performed to determine if both AhR and ER receptors could bind to the same HSP90-containing complex. Anti-AhR2 antibodies were selected for the immunoprecipitation experiments because of the important role of AhR2 in the AhR signaling pathway. No ER proteins were detected in the complex of AhR2 and HSP90, (Fig. 4A). Therefore, a single HSP90-containing complex could not bind to both AhR and ER receptors. To examine the interaction between HSP90 and AhR and ER, HSP90 was immunoprecipitated with the HSP90 antibody, and its associated proteins were determined by immunoblot analysis. In the hepatocytes, a PCB77 treatment decreased the association of AhR with
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Fig. 3. Expression of ER, AhR1 and AhR2 protein expression in a primary culture of goldfish hepatocytes exposed to cadmium, E2 and PCB77 at 1, 10, 50, 100 and 200 nM, singly and in combination, for 6 h. The protein levels were quantified by ELISA with the corresponding antibodies. The data is reported as a percentage of the single treatment control ± standard error of the mean (n = 6 fish). An asterisk indicates a significant different from controls if p < 0.05 determined by ANOVA.
HSP90 significantly, which is believed to have led to the lower protein stability of AhR (Fig. 4B, lane 2). No change in the total amount of Hsp90 was observed during the co-treatment of 100 nM E2 with 100 nM PCB77 (Fig. 4B, lane 4). Compared to the single treatment of PCB77, the level of AhR2 receptor binding with HSP90 was almost unchanged. On the other hand, a slight decrease in AhR2 was observed after a co-treatment with 100 nM CdCl2 and 100 nM PCB77, suggesting the further dissociation AhR from HSP90 (Fig. 4B, lane 3). A similar pattern of E2 bound to HSP90 was observed in the E2-induced hepatocytes by the additional treatment of PCB77 or Cd (Fig. 4B, lane 4–6). On the other hand, a slight increase in the AhR2-HSP90 complex was also observed in the hepatocytes treated with E2 alone compared to the untreated cells (Fig. 4C, lane 1 and 6). Similarly, ER-HSP90 was increased during the treatment with PCB77 (Fig. 4D, lane 1 and 2). Moreover, an increase in HSP90 protein expression was observed in the hepatocytes exposed to CdCl2 , either under a single treatment or when co-treated with PCB77 and E2 (Fig. 4E, lane 3 and 5). 3.5. Effect of GA To determine if the inhibition of Hsp90 affects the competition for Hsp90, the experiments were repeated in the hepatocytes treated concurrently with GA. The addition of GA alone did not affect CYP1A or VTG protein expression significantly compared to the control group (Fig. 5A and B). On the other hand, the GA treatment depressed the PCB77-induced CYP1A protein expression and E2-induced VTG protein expression significantly compared to the PCB77 group. Our results suggested reciprocal inhibitory behavior between CYP1A and VTG during the co-treatment of PCB77, but in
the presence of GA, no change of CYP1A protein expression was observed between the hepatocytes treated by PCB77 alone or cotreated with PCB77 and E2 (Fig. 5A). Similarly, when GA was added, the protein expression of VTG was not changed in the cells coexposed to E2 and PCB77 compared to the E2-treated cells (Fig. 5B). In the immunoprecipitation experiments, the addition of GA largely decreased the amount of AhR-Hsp90 complex and ER-Hsp90 complex in the hepatocytes (Fig. 5C), which is significantly different from the results shown in Fig. 4. 4. Discussion In aquatic environments, a series of biomarkers for various contaminants have been established (Yan et al., 2012). The biomarker responses might be affected, either directly or indirectly, by the presence of other classes of chemicals. The present study examined the interactions between VTG, CYP1A and HSP70, as well as the potential factors affecting these biomarker responses. HSP90 is often found associated with the functional domains of client proteins, including ligand binding. Although HSP90 can maintain the conformation of the function domains of the AhR or ER receptors to their activation, the role of HSP90 in AhR-ER crosstalk is unknown. HSP90 is a potential mediator of AhR and ER crosstalk because it interacts with both AhR and ER, and represses the receptor. Based on this evidence, this study attempted to find a link between the different signaling pathways induced by a co-treatment of the pollutants. The determination of protein expression (VTG, CYP1A and HSP70) showed that the heavy metal (cadmium) up-regulated the E2-induced VTG and PCB77-induced CYP1A responsive proteins
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Fig. 4. Immunoprecipitation assay showing that HSP90 participated in the interaction between the AhR and ER pathway. (A) total cell lysates were prepared from the primary goldfish hepatocytes as described in Section 2. For immunoprecipitation, the protein was incubated with anti-AhR2. HSP90, ER and AhR2 in the immunoprecipitate were visualized using the respective antibodies. (B) For each immunoprecipitation experiment, a total of 1 mg of protein was incubated with goldfish anti-HSP90 pre-bound to Protein A. A representative blot from 3 independent experiments is shown. GADPH was used as the loading control for the immunoprecipitation experiments. The concentration of the chemicals was at 100 nM. (C–E) Visual expression densitometric analysis of the bands as the mean percentage of the control integrated density using ImageQuant TL software.
Fig. 5. Effect of GA (1 g/ml) on the cross-talk between AhR and ER signaling pathways. CYP1A (A) and VTG (B) protein expression in primary cultures was detected by ELISA after 48 h. PCB77 and E2 were treated with the concentration of 100 nM. The data is reported as a percentage of the single treatment control ± standard error of the mean (n = 8 fish). C. An immunoprecipitation assay was performed as described in the legend in Fig. 4B. Hsp90 was immunoprecipitated from the hepatocytes after treatment with GA, or in combination with 100 nM PCB and E2. A representative blot from 3 independent experiments is shown.
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Fig. 6. (A) CYP1A (or VTG) was activated transcriptionally when a ligand, PCB77 (or E2) binds the cytoplasmic AhR (or ER) complex consisting of AhR (or ER) and cochaperones containing HSP90. The free receptors without the assistance of HSP90 were unable to bind to the ligands. Ligand binding leads to the dissociation of the receptor-HSP90 complex, allowing the translocation of the receptor to the nucleus and binding to the dioxin-responsive elements, DRE (or the estrogen response element, ERE), in the CYP1A enhancer region. On the other hand, when treated with PCB77 and E2 together, there was competition for HSP90 between AhR and ER, resulting in reciprocal inhibition of the two pathways. (B) A heavy metal treatment induced oxidative stress in the cells, which caused the generation of misfolded proteins. Because of the important of HSP90 in stabilizing the non-native states of the proteins, the increased concentration of unfolded proteins under stress conditions causes the displacement of ER (or AhR) from HSP90. As a result, the protein expression of CYP1A induced by PCB77 or vitellogenin induced by E2 was up-regulated by cadmium.
significantly. Previous studies have suggested that the induction of HSP70 by heavy metals is related to oxidative stress (Chang et al., 2011; Radice et al., 2004). When treated with heavy metals, the cells undergo oxidative stress, which generates misfolded proteins in the cell (Malhotra et al., 2008). Meanwhile, the correct folding of misfolded proteins depends on the presence of heat shock proteins (Kim et al., 2007). HSP90s can combine with misfolded proteins and help them fold correctly by acting as molecular chaperones (Vabulas et al., 2010). The increased unfolded proteins under stress conditions cause the displacement of ER (or AhR) from HSP90 due to the important role of HSP90 in stabilizing the non-native states of the proteins. Although there was a slight increase in the amount of HSP90 in the cells, these results revealed a decrease in the ER (or AhR)-HSP90 complexes after a co-treatment with cadmium compared to a single treatment of E2 or PCB77. Under stress environments, such as heat shock or heavy metals, the induced HSP90 participates mainly in folding the newly synthesized proteins and refolding the denatured proteins, even though there was competition with the ER (or AhR)-heterocomplexes (Fig. 6B). Based on the possible mechanisms explained above, cadmium can only promote E2-mediated ER or PCB-mediated AhR; it does not induce VTG or CYP1A expression. On the other hand, the slight increase in the HSP90-ER complex during the single treatment of PCB77 (Fig. 4B and D, lane 2) was attributed to an increase in free HSP90s in the cells treated with PCB77 alone. Another important finding in the present study was the reciprocal inhibitory interaction between the ER and AhR signaling pathways. Several studies reported the anti-estrogenic
properties of the AhR ligand. Previous studies also suggested specific interactions between AhR and ER (Arukwe et al., 2001; Klinge et al., 2000; Mortensen et al., 2006; Wormke et al., 2003). Mortensen and Arukwe (2007) reported a reciprocal inhibitory interaction between the estrogen- and Ah-Receptor signaling pathways in salmon primary hepatocytes exposed to nonylphenol and 3,3,4,4 -tetrachlorobiphenyl. These simultaneous interactions were confirmed in a later study by exposing the cells to E2 and benzo[a]pyrene (Yan et al., 2012). In general, the present data is consistent with previous studies of other teleost species. Several reports suggest that the AhR agonist induces estrogenic activity directly through AhR–ER interactions (Liu et al., 2006; Shipley and Waxman, 2006), but there is still some controversy. This study focused on the chaperone proteins that exist in both the AhR and ER pathways. The results revealed reciprocal inhibition between the model ER agonist, E2 and AhR agonist, PCB77. The data revealed the full induction of AhR (or ER) during a single treatment of 100 nM PCB77 (or E2), but there was no change in AhR2 (or ER) protein expression after co-treatment. This is consistent with previous results (Lu et al., 2013), in that the AhR2 in goldfish mainly up-regulates the expression of the CYP1A protein. In mammals, ligand binding leads to a rapid decrease in AhR by proteasome (Pollenz, 2002; Wentworth et al., 2004). On the other hand, the study in trout hepatocytes reported that a higher AhR mRNA and protein content was induced by the agonist (Aluru et al., 2005), which is consistent with the present results. According to the immunoprecipitation experiments, the amount of AhR2 (or ER) binding with HSP90 was equal to the single treatment of PCB77 (or E2). A possible explanation for the inhibition is the reduced activation of the receptors. In the experiments by the addition of the HSP90 inhibitor (GA), reciprocal inhibitory behavior between AhR and ER was barely observed, highlighting the important role of HSP90 in the inhibition. As shown in Fig. 6A, without the assistance of the HSP90-containing complex, the receptors alone were unable to bind the ligands. Ligand binding leads to the dissociation of the receptor-HSP90 complex (Ma, 2007). On the other hand, during the co-treatment of PCB77 and E2, competition between AhR and ER exists, resulting in the repression of VTG and CYP1A. Therefore, the reciprocal inhibiting interaction between the ER-AhR pathways is caused by competition with the free HSP90-containg complex. In conclusion, cadmium promotes PCB77 (AhR agonist)mediated CYP1A protein expression and 17-estradiol (E2, ER agonist)-mediated VTG protein expression in goldfish primary hepatocytes. On the other hand, reciprocal inhibiting crosstalk between the AhR and ER pathways was observed. AhR-ER crosstalk also has a complex interaction with the heat shock response. The 90-kDa heat shock protein (HSP90) participating in modulating crosstalk between the AhR and ER pathways was examined using immune-precipitation experiments. HSP90 mediates the ligand–receptor interactions because the free receptors were unable to bind their ligands without the assistance of a HSP90containing complex. These findings of a mechanism among AhR, ER and heat shock pathways provide novel insights into the signaling pathways. Transparency document The Transparency document associated with this article can be found in the online version. Conflict of interest The authors declare that there are no conflicts of interest.
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Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-007643). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. 2014.01.032. References Aluru, N., Vuori, K., Vijayan, M.M., 2005. Modulation of Ah receptor and CYP1A1 expression by alpha-naphthoflavone in rainbow trout hepatocytes. Comparative Biochemistry and Physiology Toxicology & Pharmacology 141, 40–49. Arukwe, A., Yadetie, F., Male, R., Goksoyr, A., 2001. In vivo modulation of nonylphenol-induced zonagenesis and vitellogenesis by the antiestrogen, 3,3 4,4 -tetrachlorobiphenyl (PCB-77) in juvenile fish. Environmental Toxicology and Pharmacology 10, 5–15. Berg, H., Modig, C., Olsson, P.E., 2004. 17beta-estradiol induced vitellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char (Salvelinus alpinus). Reproductive Biology and Endocrinology 2, 62. Chang, Z., Lu, M., Lee, K.W., Oh, B.S., Bae, M.J., Park, J.S., 2011. Influence of divalent metal ions on E2-induced ER pathway in goldfish (Carassius auratus) hepatocytes. Ecotoxicology and Environmental Safety 74, 2233–2239. Georgopoulos, C., Welch, W.J., 1993. Role of the major heat shock proteins as molecular chaperones. Annual Review of Cell Biology 9, 601–634. Gu, Y.Z., Hogenesch, J.B., Bradfield, C.A., 2000. The PAS superfamily: sensors of environmental and developmental signals. Annual Review of Pharmacology and Toxicology 40, 519–561. Guevel, R.L., Petit, F.G., Goff, P.L., Metivier, R., Valotaire, Y., Pakdel, F., 2000. Inhibition of rainbow trout (Oncorhynchus mykiss) estrogen receptor activity by cadmium. Biology of Reproduction 63, 259–266. Hahn, M.E., 1998. The aryl hydrocarbon receptor: a comparative perspective. Comparative Biochemistry and Physiology Part C, Pharmacology, Toxicology & Endocrinology 121, 23–53. Hahn, M.E., 2001. Dioxin toxicology and the aryl hydrocarbon receptor: insights from fish and other non-traditional models. Marine Biotechnology 3, S224–S238. Kim, H.J., Hwang, N.R., Lee, K.J., 2007. Heat shock responses for understanding diseases of protein denaturation. Molecules and Cells 23, 123–131. Klinge, C.M., Kaur, K., Swanson, H.I., 2000. The aryl hydrocarbon receptor interacts with estrogen receptor alpha and orphan receptors COUP-TFI and ERRalpha1. Archives of Biochemistry and Biophysics 373, 163–174. Lee, Y.M., Seo, J.S., Kim, I.C., Yoon, Y.D., Lee, J.S., 2006. Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol, 4-tert-octylphenol) modulate expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus. Biochemical and Biophysical Research Communications 345, 894–903. Li, C.R., Kim, K.T., Kang, Y.J., Kim, A., Kang, S.W., Park, J.S., 2005. A novel enzyme-linked immunosorbent assay system for the quantitative analysis of Carassius auratus vitellogenin. Chemosphere 59, 997–1003. Li, L., Li, Z., Howley, P.M., Sacks, D.B., 2006. E6AP and calmodulin reciprocally regulate estrogen receptor stability. The Journal of Biological Chemistry 281, 1978–1985. Liu, S., Abdelrahim, M., Khan, S., Ariazi, E., Jordan, V.C., Safe, S., 2006. Aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha in MCF-7 breast cancer cells. Biological Chemistry 387, 1209–1213. Lu, M., Chang, Z., Bae, M.J., Oh, S.M., Chung, K.H., Park, J.S., 2013. Molecular characterization of the aryl hydrocarbon receptor (AhR) pathway in goldfish (Carassius auratus) exposure to TCDD: the mRNA and protein levels. Fish & Shellfish Immunology 35, 469–475. Ma, Q., 2007. Aryl hydrocarbon receptor degradation-promoting factor (ADPF) and the control of the xenobiotic response. Molecular Interventions 7, 133–137.
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Malhotra, J.D., Miao, H., Zhang, K., Wolfson, A., Pennathur, S., Pipe, S.W., Kaufman, R.J., 2008. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proceedings of the National Academy of Sciences of the United States of America 105, 18525–18530. Mortensen, A.S., Arukwe, A., 2007. Interactions between estrogen- and Ah-receptor signalling pathways in primary culture of salmon hepatocytes exposed to nonylphenol and 3,3 ,4,4 -tetrachlorobiphenyl (congener 77). Comparative Hepatology 6, 2. Mortensen, A.S., Tolfsen, C.C., Arukwe, A., 2006. Gene expression patterns in estrogen (nonylphenol) and aryl hydrocarbon receptor agonists (PCB-77) interaction using rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture. Journal of Toxicology and Environmental Health Part A 69, 1–19. Nollen, E.A., Morimoto, R.I., 2002. Chaperoning signaling pathways: molecular chaperones as stress-sensing ‘heat shock’ proteins. Journal of Cell Science 115, 2809–2816. Padmini, E., Usha Rani, M., 2008. Impact of seasonal variation on HSP70 expression quantitated in stressed fish hepatocytes. Comparative Biochemistry and Physiology Part B: Biochemistry & Molecular Biology 151, 278–285. Pollenz, R.S., 2002. The mechanism of AH receptor protein down-regulation (degradation) and its impact on AH receptor-mediated gene regulation. Chemico-Biological Interactions 141, 41–61. Pongratz, I., Mason, G.G., Poellinger, L., 1992. Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. The Journal of Biological Chemistry 267, 13728–13734. Powers, M.V., Workman, P., 2007. Inhibitors of the heat shock response: biology and pharmacology. FEBS Letters 581, 3758–3769. Radice, S., Fumagalli, R., Chiesara, E., Ferraris, M., Frigerio, S., Marabini, L., 2004. Estrogenic activity of procymidone in rainbow trout (Oncorhynchus mykiss) hepatocytes: a possible mechanism of action. Chemico-Biological Interactions 147, 185–193. Shipley, J.M., Waxman, D.J., 2006. Aryl hydrocarbon receptor-independent activation of estrogen receptor-dependent transcription by 3-methylcholanthrene. Toxicology and Applied Pharmacology 213, 87–97. Sidera, K., Gaitanou, M., Stellas, D., Matsas, R., Patsavoudi, E., 2008. A critical role for HSP90 in cancer cell invasion involves interaction with the extracellular domain of HER-2. The Journal of Biological Chemistry 283, 2031–2041. Soshilov, A., Denison, M.S., 2011. Ligand displaces heat shock protein 90 from overlapping binding sites within the aryl hydrocarbon receptor ligand-binding domain. The Journal of Biological Chemistry 286, 35275–35282. Takemura, A., Kim, B.H., 2001. Effects of estradiol-17beta treatment on in vitro and in vivo synthesis of two distinct vitellogenins in tilapia. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 129, 641–651. To, K.K., Robey, R., Zhan, Z., Bangiolo, L., Bates, S.E., 2011. Upregulation of ABCG2 by romidepsin via the aryl hydrocarbon receptor pathway. Molecular Cancer Research 9, 516–527. Vabulas, R.M., Raychaudhuri, S., Hayer-Hartl, M., Hartl, F.U., 2010. Protein folding in the cytoplasm and the heat shock response. Cold Spring Harbor Perspectives in Biology 2, a004390. Vaccaro, E., Meucci, V., Intorre, L., Soldani, G., Di Bello, D., Longo, V., Gervasi, P.G., Pretti, C., 2005. Effects of 17beta-estradiol, 4-nonylphenol and PCB 126 on the estrogenic activity and phase 1 and 2 biotransformation enzymes in male sea bass (Dicentrarchus labrax). Aquatic Toxicology 75, 293–305. Wei, T., Gao, Y., Wang, R., Xu, T., 2013. A heat shock protein 90 beta isoform involved in immune response to bacteria challenge and heat shock from Miichthys miiuy. Fish & Shellfish Immunology 35, 429–437. Wentworth, J.N., Buzzeo, R., Pollenz, R.S., 2004. Functional characterization of aryl hydrocarbon receptor (zfAHR2) localization and degradation in zebrafish (Danio rerio). Biochemical Pharmacology 67, 1363–1372. Wormke, M., Stoner, M., Saville, B., Walker, K., Abdelrahim, M., Burghardt, R., Safe, S., 2003. The aryl hydrocarbon receptor mediates degradation of estrogen receptor alpha through activation of proteasomes. Molecular and Cellular Biology 23, 1843–1855. Yan, Z., Lu, G., He, J., 2012. Reciprocal inhibiting interactive mechanism between the estrogen receptor and aryl hydrocarbon receptor signaling pathways in goldfish (Carassius auratus) exposed to 17beta-estradiol and benzo[a]pyrene. Comparative Biochemistry and Physiology Toxicology & Pharmacology 156, 17–23.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Potential role of HSP90 in mediating the interactions between estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) signaling pathways Ziwei Chang 1 , Ming Lu 1,2 , So-Sun Kim, Jang-Su Park ∗ Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Republic of Korea
h i g h l i g h t s • A reciprocal inhibiting crosstalk between the AhR and ER pathways was observed. • Cd up-regulated the E2-induced VTG and PCB77-induced CYP1A. • HSP90 participated in the reciprocal inhibiting interaction.
a r t i c l e
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Article history: Received 5 December 2013 Received in revised form 22 January 2014 Accepted 23 January 2014 Available online 31 January 2014 Keywords: Heat shock protein 90 Vitellogenin CYP1A Goldfish Primary culture
a b s t r a c t The estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) are ligand-activated transcription factors involved in estrogen or xenobiotic exposure, whereas the 90-kDa heat shock protein (HSP90), which is a ubiquitously expressed molecular chaperone, is involved in the signal transduction process. Although the interactions between these pathways have been under investigation, the mechanisms are unclear and the potential role of HSP90 in these interactions has not been reported. The results of goldfish primary hepatocytes showed that exposure to PCB77 and 17-estradiol (E2) alone induced significant protein expression of cytochrome P450 1A (CYP1A) and vitellogenin (VTG), respectively. On the other hand, the combined exposure to PCB77 and E2 led to the reduction of CYP1A and VTG compared to the single treatments. Although the AhRs and ERs were naturally induced during the co-treatment, the total amount of HSP90 binding to the receptors was not changed. Furthermore, while the HSP90 chaperon activity was blocked by the specific inhibitor (geldanamycin), reciprocal inhibition between AhR and ER pathways was not observed. These findings indicate a potential role of HSP90 where competition between AhR and ER for binding to HSP90 can occur and cause reciprocal inhibition. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Environmental pollution has increased substantially in recent decades. The most important pollutants include polychlorinated and polybrominated aromatics, heavy metals, phenols, organochlorine pesticides and estrogenic substances. Fish are valuable biomonitors of environmental pollution, and in recent studies, goldfish (Carassius auratus) have been found to be a useful species for examining the effects of toxic chemicals because they are widespread found widely in aquatic environments. Cytochrome P450 has also been proposed as a biochemical indicator of toxicant exposure in aquatic species. Cytochrome
∗ Corresponding author. Tel.: +82 51 510 2294; fax: +82 51 516 7421. E-mail address: [email protected] (J.-S. Park). 1 Both authors contributed equally to this work. 2 Present address: Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China. 0378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2014.01.032
P4501A (CYP1A) is the most important gene family involved in the metabolism of xenobiotics and is used as a biomarker of exposure to the organic pollutants (Kim et al., 2007). The fish CYP1A gene is used widely to identify the effects of polycyclic aromatic hydrocarbons. The molecular mechanism of CYP1A activation has been studied extensively. Hahn (1998) reviewed the transcriptional regulation of CYP1A in fish through a transcription factor known as the aryl hydrocarbon receptor (AhR). Potent agonist binding to the receptor results in the disassociation of AhR from a chaperone complex consisting of heat shock protein 90 (HSP90), followed by the translocation of AhR to the nucleus and binding to a dioxin response element together with the aryl hydrocarbon receptor nucleus translocator (ARNT) (Gu et al., 2000; Hahn, 2001; Ma, 2007). In fish, another sensitive biomarker, vitellogenin (VTG) is used frequently to evaluate the effects of endocrine-disrupting chemicals (EDCs) (Lee et al., 2006; Radice et al., 2004). VTG is normally produced by female animals in response to normal estradiol cycles but is not normally synthesized in males (Vaccaro et al., 2005). The
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estrogenic control of VTG is mediated by binding the most potent estrogen, 17--estradiol (E2), to the ER. The ER–E2 complex binds with high affinity to specific DNA sequences known as the estrogenresponsive elements located in the regulatory regions of the VTG gene, leading to this activation and expression (Berg et al., 2004; Guevel et al., 2000). When exposed to heat shock or other insults, such as oxidative stress or toxic substances, organisms synthesize a common set of proteins known as heat shock proteins (HSPs). HSPs are ubiquitous and highly conserved proteins that can be grouped into several families according to their apparent molecular mass (Georgopoulos and Welch, 1993; Wei et al., 2013). Of these, HSP90 is a molecular chaperone that is involved in multiple signal transduction pathways (Soshilov and Denison, 2011). At least 100 proteins are regulated by HSP90, including the key mediators of signal transduction and cell cycle control, steroid hormone receptors, and tyrosine and serine/threonine kinases (Nollen and Morimoto, 2002; Sidera et al., 2008). HSP90 is a chaperone protein that is important for maintaining its client proteins including AhR and ER proteins, in a functional conformation for its biological function (To et al., 2011). The association with HSP90 is required for the AhR to assume a conformation that is optimal for ligand binding (Pongratz et al., 1992). Moreover, the 70 kDa heat shock proteins, HSP70s, has been shown to play a major role in the cellular response and promote the ability of organisms to survive various environmental stresses, and evaluation for HSP70 has been used as a biomarker of effect of pollutant.(Padmini and Usha Rani, 2008). Organisms in the environment are exposed to various mixtures of chemicals, and the response of the biomarkers can be affected by mixed exposure. The present study examined the biomarker responses of VTG, CYP1A and HSP70. Although the properties of the corresponding inducers and their ability to induce the proteins through the receptors are clear, details of the molecular and cellular processes underpinning these effects as well as the interactions between them are less evident. Therefore, the interactions between the biomarkers and the factors affecting these responses were investigated. Moreover, little is known regarding the role of HSP90 in modulating the interactions between AhR and ER. Geldanamycin (GA), a quite specific inhibitor of HSP90, was used to examine the role HSP90. GA can bind specifically to the ATP-binding site of HSP90, resulting in the inhibition of the HSP90 interaction with the target proteins (Powers and Workman, 2007). 2. Materials and methods 2.1. Animals Male goldfish (C. auratus) (200–300 g, n = 5) were purchased from a local market and maintained in dechlorinated freshwater tanks. The fish were kept in an aquarium at 20 ◦ C in an aquatic trunk with dechlorinated tap water for three days and fed once per day. All studies were carried out in accordance with the national and institutional guidelines for the protection of animal welfare. The project was approved by the Committee on Ethics of Pusan National University.
in 6-well plates in L-15 medium at density of 0.75 × 106 cells/well using a slight modifications of the methods of reported elsewhere (Takemura and Kim, 2001. The media was changed every 24 h. 2.4. Exposure of hepatocytes Approximately 48 h after initiating the primary culture, the hepatocytes were exposed to different concentrations (1, 10, 50, 100 and 200 nM) of CdCl2 , 17estradiol (E2) and PCB77, singly and in combination. Exposed to the solvent ethanol only was used as the control for each experiment. The media were replaced with fresh media containing the respective test chemicals and concentrations every 24 h. After exposure, the medium and cells were harvested for protein level analysis. The medium was collected to measure the VTG or CYP1A concentrations, and the cells were harvested to measure the levels of HSP70, AhR1, AhR2 and ER protein expression. 2.5. Protein level analysis A sandwich enzyme-linked immunosorbent assay system (ELISA) was used to measure the VTG levels using the methods described elsewhere (Li et al., 2005). The same method with slight modifications was performed for HSP70 and CYP1A analysis. Standard curves of HSP70, CYP1A and VTG were plotted using the measurements of 10 dilutions of the purified proteins (Fig. S1). The levels of HSP70, CYP1A and VTG were quantified by ELISA using the followed linear equations: Y = 0.079 + 0.0021X; Y = 0.0088 + 0.0031X and Y = 0.0592 + 0.0029X, respectively. Briefly, the cells were harvested in a cell suspension buffer, centrifuged, and resuspended in RIPA buffer. The cell suspension was incubated for 30 min at 4 ◦ C and then centrifuged at 16,000 g for 10 min at 4 ◦ C. The supernatant was coated on NUNC-immunoTM plates. After washing three times with PBST buffer (PBS with 0.05% Tween 20), the cells were blocked with PBSTG (PBST with 1% Gelatin) for one hour at 37 ◦ C and washed again. The polyclonal antibodies of anti-goldfish HSP70, CYP1A, VTG, AhR1/2 and ER, which were prepared in this laboratory (Chang et al., 2011; Lu et al., 2013), were used for specific binding. Goat anti-mouse IgG conjugated with alkaline phosphatase (sigma) was used as the secondary antibody. Finally, phosphatase substrate solutions (KPL) were added, and the absorbance at 490 nm was measured on an ELISA reader after a reaction time of 10 min. Statistical analysis was performed using the SPSS (version 11.0) program. The significant differences between the means were examined using a one-way analysis of variance (ANOVA). A p value <0.05 was considered significant. 2.6. Immunoprecipitation The partial cDNA, which consisted of 192 base pairs corresponding to aa112-175 of C. auratus HSP90 was amplified from the liver tissue, with the primers designed based on the published sequences (Accession number: DQ872650). Recombinant partial HSP90 were produced in Escherichia coli BL21 (DE3) and purified. The mice were injected with the protein to obtain the polyclonal antibodies for the production of anti-HSP90 antibodies. An immunoprecipitation experiment was performed, as described previously (Li et al., 2006). Briefly, after pre-clearing with the protein A-Sepharose beads, equal amounts of the protein lysate were immunoprecipitated with the anti-HSP90 antibodies for 2 h at 4 ◦ C. The immune complexes were collected for 2 h with 40 l of protein A-Sepharose and washed five times. The samples were processed by Western blotting. Equal amounts of the protein lysate and immune complexes were resolved by SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 5% BSA in a TBS-T buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) and probed with the respective HSP90, AhR2 or ER antibodies. GADPH was used as the loading control for the immunoprecipitation experiments. Anti-mouse IgG (sigma A3562) was used as the secondary antibody. The blots were developed by enhanced chemiluminescence (Western blue stabilized substrate for alkaline phosphatase, Promega). The analysis was performed using the software ImageQuant TL (GE Healthcare).
2.2. Chemicals and reagents
3. Results
Geldanamycin (GA), resorufin ethyl ether and nicotinamide adenine dinucleotide phosphate (NADPH), CdCl2 , E2 and PCB77 were purchased from Sigma–Aldrich (South Korea). All the other chemicals were of the highest commercially available grade.
3.1. Single treatments of HSP70, VTG and CYP1A protein expression
2.3. Isolation and primary culture of hepatocytes Five fish were euthanized with an overdose of MS222 (Sigma), and their liver tissue was isolated carefully and perfused with hepatocyte buffer (HB, 136.9 mM NaCl, 5.4 mM KCl, 0.81 mM MgSO4 , 0.44 mM KH2 PO4 , 0.33 mM Na2 HPO4 , 5.0 mM NaHCO3 , pH 7.6) for 10 min at room temperature. The liver tissue was then digested with HB buffer containing collagenase (0.3 mg/ml) (Sigma, St. Louis, MO). The softened tissue was minced, and sieved through a nylon mesh (50 m). The resulting cell suspension was washed three times and centrifuged three times at 50 g for 90 s at 10 ◦ C with HB containing 1.5 mM CaCl2 . The hepatocytes were cultured and plated
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The levels of HSP70, VTG and CYP1A in the cultured primary hepatocytes were quantified by ELISA using the polyclonal antibody. Ethanol in the medium had no observable effect on the protein expressions in hepatocyte culture. The level of HSP70 induced by 10 nM, 50 nM, 100 nM and 200 nM CdCl2 increased significantly after 48 h and 96 h of exposure compared to the control (Fig. 1A). In contrast, no obvious increase in the level of HSP70 was observed in the hepatocytes treated with E2 or PCB77 (Fig. 1B and C). The cells treated with either Cd or PCB77 showed a similar level
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Fig. 1. Expression of HSP70, CYPA1 and VTG in a primary culture of goldfish hepatocytes exposed to different treatments. The concentrations of 17-estradiol (E2), cadmium and PCB77 ranged from 0 to 200 nM. Protein expression was measured by ELISA at 48 h and 96 h after the treatments. The results are reported as the mean ± S.E.M (n = 4–6 fish). An asterisk indicates a significant different from controls if p < 0.05 determined by ANOVA.
of VTG synthesis and no increase was observed at different concentrations ranging from 1 nM to 200 nM (Fig. 1D and E). On the other hand, treatment with E2 led to a significant concentrationdependent increase in the induction of VTG (Fig. 1F). For the induction of CYP1A, after 48 h and 96 h exposure to different concentrations of PCB77, the cells showed an increase compared to the untreated cells (Fig. 1G). No obvious induction of CYP1A was observed in the hepatocytes treated with CdCl2 or E2 (Fig. 1H and I). 3.2. Effects of the co-treatments on HSP70, VTG and CYP1A protein expression To determine the mutual impacts between the three chemicals of E2, CdCl2 and PCB77 on protein expression, a 100 nM agonist was selected as the control to determine if the other two chemicals affect the corresponding protein expression. The additional treatments of E2 or PCB77 in the 100 nM CdCl2 -induced cells showed no significant increase in HSP70 compared to those treated with CdCl2 alone (Fig. 2A and B). By varying the concentration of E2 or PCB77, a less obvious increase in HSP70 was observed at a low concentration of E2 or PCB77, but no increase or decrease was observed in the concentration range, 100–200 nM (Fig. 2A and B).
The hepatocytes were then exposed to 100 nM E2 in combination with CdCl2 or PCB77 (from 1 to 200 nM), using 100 nM E2 alone as the control. Interestingly, co-treatment with 100 nM E2 and 100 nM CdCl2 resulted in a 50% increase in the VTG level compared to the control (Fig. 2C). In contrast, additional exposure to PCB77 resulted in a decrease in the VTG levels compared to 100 nM E2 alone; the addition of 100 nM PCB77 resulted in the lowest level of VTG (Fig. 2D). In the experiments examining the effects of CdCl2 and E2 on PCB77-induced CYP1A protein expression, CdCl2 induced a significant concentration-dependent increase in the E2-induced CYP1A levels (Fig. 2E). Co-treatment of 10 nM PCB77 with 100 nM and 200 nM CdCl2 induced a 50% increase compared to PCB77 alone. On the other hand, a slight decrease was observed with 1 nM CdCl2 (Fig. 2E). In addition, co-exposure to E2 (1–200 nM) and 100 nM PCB77 resulted in a decrease in CYP1A protein expression (Fig. 2F). 3.3. Effects of the co-treatments on AhR and ER receptors The expression of goldfish ER, AhR1 and AhR2 proteins in the primary hepatocytes were measured to determine the effects of the combined treatments on the receptors of the AhR and ER signaling pathways (Fig. 3). Exposure to E2 alone produced a
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Fig. 2. Protein expression of HSP70, CYPA1 and VTG exposed to 17-estradiol (E2), cadmium and PCB77 in combination for 48 h or 96 h. The data is reported as a percentage of the single treatment control ± standard error of the mean (n = 8 fish). Values significantly different from the corresponding 48 h (*) or 96 h (#) values at p < 0.05 as determined by ANOVA.
significant concentration-dependent increase in ER protein expression, whereas cadmium and PCB77 alone did not alter ER protein expression (Fig. 3A). Combining E2 (100 nM) with various concentrations of cadmium or PCB77 did not result in a change in ER protein expression (Fig. 3A). For the AhR receptor, exposure to PCB77 alone caused a small increase in AhR1 expression. On the other hand, a concentration-dependent significant increase in AhR2 protein expression was observed (Fig. 3B and C). By comparing with 100 nM PCB77 alone, the co-treatments of 100 nM PCB77 with various concentrations of E2 or cadmium caused slight but non-significant elevation of no AhR1/2 proteins (Fig. 3B). 3.4. HSP90 participates in modulating the interactions The published sequences of HSP90 showed high homology to other teleosts, which was classified as HSP90. HSP90, as the constitutive form, has been associated with cellular transformation and
signal transduction. Partial goldfish HSP90 was expressed in E. coli and purified. The selected sequences were specific to HSP90 and involved multiple epitopes (data not shown), that are suitable for antibody development. As expected, the anti-HSP90 antibody produced specifically recognized goldfish HSP90 in the hepatocytes (Fig. S2) and was used in subsequent experiments. The experiments were performed to determine if both AhR and ER receptors could bind to the same HSP90-containing complex. Anti-AhR2 antibodies were selected for the immunoprecipitation experiments because of the important role of AhR2 in the AhR signaling pathway. No ER proteins were detected in the complex of AhR2 and HSP90, (Fig. 4A). Therefore, a single HSP90-containing complex could not bind to both AhR and ER receptors. To examine the interaction between HSP90 and AhR and ER, HSP90 was immunoprecipitated with the HSP90 antibody, and its associated proteins were determined by immunoblot analysis. In the hepatocytes, a PCB77 treatment decreased the association of AhR with
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Fig. 3. Expression of ER, AhR1 and AhR2 protein expression in a primary culture of goldfish hepatocytes exposed to cadmium, E2 and PCB77 at 1, 10, 50, 100 and 200 nM, singly and in combination, for 6 h. The protein levels were quantified by ELISA with the corresponding antibodies. The data is reported as a percentage of the single treatment control ± standard error of the mean (n = 6 fish). An asterisk indicates a significant different from controls if p < 0.05 determined by ANOVA.
HSP90 significantly, which is believed to have led to the lower protein stability of AhR (Fig. 4B, lane 2). No change in the total amount of Hsp90 was observed during the co-treatment of 100 nM E2 with 100 nM PCB77 (Fig. 4B, lane 4). Compared to the single treatment of PCB77, the level of AhR2 receptor binding with HSP90 was almost unchanged. On the other hand, a slight decrease in AhR2 was observed after a co-treatment with 100 nM CdCl2 and 100 nM PCB77, suggesting the further dissociation AhR from HSP90 (Fig. 4B, lane 3). A similar pattern of E2 bound to HSP90 was observed in the E2-induced hepatocytes by the additional treatment of PCB77 or Cd (Fig. 4B, lane 4–6). On the other hand, a slight increase in the AhR2-HSP90 complex was also observed in the hepatocytes treated with E2 alone compared to the untreated cells (Fig. 4C, lane 1 and 6). Similarly, ER-HSP90 was increased during the treatment with PCB77 (Fig. 4D, lane 1 and 2). Moreover, an increase in HSP90 protein expression was observed in the hepatocytes exposed to CdCl2 , either under a single treatment or when co-treated with PCB77 and E2 (Fig. 4E, lane 3 and 5). 3.5. Effect of GA To determine if the inhibition of Hsp90 affects the competition for Hsp90, the experiments were repeated in the hepatocytes treated concurrently with GA. The addition of GA alone did not affect CYP1A or VTG protein expression significantly compared to the control group (Fig. 5A and B). On the other hand, the GA treatment depressed the PCB77-induced CYP1A protein expression and E2-induced VTG protein expression significantly compared to the PCB77 group. Our results suggested reciprocal inhibitory behavior between CYP1A and VTG during the co-treatment of PCB77, but in
the presence of GA, no change of CYP1A protein expression was observed between the hepatocytes treated by PCB77 alone or cotreated with PCB77 and E2 (Fig. 5A). Similarly, when GA was added, the protein expression of VTG was not changed in the cells coexposed to E2 and PCB77 compared to the E2-treated cells (Fig. 5B). In the immunoprecipitation experiments, the addition of GA largely decreased the amount of AhR-Hsp90 complex and ER-Hsp90 complex in the hepatocytes (Fig. 5C), which is significantly different from the results shown in Fig. 4. 4. Discussion In aquatic environments, a series of biomarkers for various contaminants have been established (Yan et al., 2012). The biomarker responses might be affected, either directly or indirectly, by the presence of other classes of chemicals. The present study examined the interactions between VTG, CYP1A and HSP70, as well as the potential factors affecting these biomarker responses. HSP90 is often found associated with the functional domains of client proteins, including ligand binding. Although HSP90 can maintain the conformation of the function domains of the AhR or ER receptors to their activation, the role of HSP90 in AhR-ER crosstalk is unknown. HSP90 is a potential mediator of AhR and ER crosstalk because it interacts with both AhR and ER, and represses the receptor. Based on this evidence, this study attempted to find a link between the different signaling pathways induced by a co-treatment of the pollutants. The determination of protein expression (VTG, CYP1A and HSP70) showed that the heavy metal (cadmium) up-regulated the E2-induced VTG and PCB77-induced CYP1A responsive proteins
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Fig. 4. Immunoprecipitation assay showing that HSP90 participated in the interaction between the AhR and ER pathway. (A) total cell lysates were prepared from the primary goldfish hepatocytes as described in Section 2. For immunoprecipitation, the protein was incubated with anti-AhR2. HSP90, ER and AhR2 in the immunoprecipitate were visualized using the respective antibodies. (B) For each immunoprecipitation experiment, a total of 1 mg of protein was incubated with goldfish anti-HSP90 pre-bound to Protein A. A representative blot from 3 independent experiments is shown. GADPH was used as the loading control for the immunoprecipitation experiments. The concentration of the chemicals was at 100 nM. (C–E) Visual expression densitometric analysis of the bands as the mean percentage of the control integrated density using ImageQuant TL software.
Fig. 5. Effect of GA (1 g/ml) on the cross-talk between AhR and ER signaling pathways. CYP1A (A) and VTG (B) protein expression in primary cultures was detected by ELISA after 48 h. PCB77 and E2 were treated with the concentration of 100 nM. The data is reported as a percentage of the single treatment control ± standard error of the mean (n = 8 fish). C. An immunoprecipitation assay was performed as described in the legend in Fig. 4B. Hsp90 was immunoprecipitated from the hepatocytes after treatment with GA, or in combination with 100 nM PCB and E2. A representative blot from 3 independent experiments is shown.
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Fig. 6. (A) CYP1A (or VTG) was activated transcriptionally when a ligand, PCB77 (or E2) binds the cytoplasmic AhR (or ER) complex consisting of AhR (or ER) and cochaperones containing HSP90. The free receptors without the assistance of HSP90 were unable to bind to the ligands. Ligand binding leads to the dissociation of the receptor-HSP90 complex, allowing the translocation of the receptor to the nucleus and binding to the dioxin-responsive elements, DRE (or the estrogen response element, ERE), in the CYP1A enhancer region. On the other hand, when treated with PCB77 and E2 together, there was competition for HSP90 between AhR and ER, resulting in reciprocal inhibition of the two pathways. (B) A heavy metal treatment induced oxidative stress in the cells, which caused the generation of misfolded proteins. Because of the important of HSP90 in stabilizing the non-native states of the proteins, the increased concentration of unfolded proteins under stress conditions causes the displacement of ER (or AhR) from HSP90. As a result, the protein expression of CYP1A induced by PCB77 or vitellogenin induced by E2 was up-regulated by cadmium.
significantly. Previous studies have suggested that the induction of HSP70 by heavy metals is related to oxidative stress (Chang et al., 2011; Radice et al., 2004). When treated with heavy metals, the cells undergo oxidative stress, which generates misfolded proteins in the cell (Malhotra et al., 2008). Meanwhile, the correct folding of misfolded proteins depends on the presence of heat shock proteins (Kim et al., 2007). HSP90s can combine with misfolded proteins and help them fold correctly by acting as molecular chaperones (Vabulas et al., 2010). The increased unfolded proteins under stress conditions cause the displacement of ER (or AhR) from HSP90 due to the important role of HSP90 in stabilizing the non-native states of the proteins. Although there was a slight increase in the amount of HSP90 in the cells, these results revealed a decrease in the ER (or AhR)-HSP90 complexes after a co-treatment with cadmium compared to a single treatment of E2 or PCB77. Under stress environments, such as heat shock or heavy metals, the induced HSP90 participates mainly in folding the newly synthesized proteins and refolding the denatured proteins, even though there was competition with the ER (or AhR)-heterocomplexes (Fig. 6B). Based on the possible mechanisms explained above, cadmium can only promote E2-mediated ER or PCB-mediated AhR; it does not induce VTG or CYP1A expression. On the other hand, the slight increase in the HSP90-ER complex during the single treatment of PCB77 (Fig. 4B and D, lane 2) was attributed to an increase in free HSP90s in the cells treated with PCB77 alone. Another important finding in the present study was the reciprocal inhibitory interaction between the ER and AhR signaling pathways. Several studies reported the anti-estrogenic
properties of the AhR ligand. Previous studies also suggested specific interactions between AhR and ER (Arukwe et al., 2001; Klinge et al., 2000; Mortensen et al., 2006; Wormke et al., 2003). Mortensen and Arukwe (2007) reported a reciprocal inhibitory interaction between the estrogen- and Ah-Receptor signaling pathways in salmon primary hepatocytes exposed to nonylphenol and 3,3,4,4 -tetrachlorobiphenyl. These simultaneous interactions were confirmed in a later study by exposing the cells to E2 and benzo[a]pyrene (Yan et al., 2012). In general, the present data is consistent with previous studies of other teleost species. Several reports suggest that the AhR agonist induces estrogenic activity directly through AhR–ER interactions (Liu et al., 2006; Shipley and Waxman, 2006), but there is still some controversy. This study focused on the chaperone proteins that exist in both the AhR and ER pathways. The results revealed reciprocal inhibition between the model ER agonist, E2 and AhR agonist, PCB77. The data revealed the full induction of AhR (or ER) during a single treatment of 100 nM PCB77 (or E2), but there was no change in AhR2 (or ER) protein expression after co-treatment. This is consistent with previous results (Lu et al., 2013), in that the AhR2 in goldfish mainly up-regulates the expression of the CYP1A protein. In mammals, ligand binding leads to a rapid decrease in AhR by proteasome (Pollenz, 2002; Wentworth et al., 2004). On the other hand, the study in trout hepatocytes reported that a higher AhR mRNA and protein content was induced by the agonist (Aluru et al., 2005), which is consistent with the present results. According to the immunoprecipitation experiments, the amount of AhR2 (or ER) binding with HSP90 was equal to the single treatment of PCB77 (or E2). A possible explanation for the inhibition is the reduced activation of the receptors. In the experiments by the addition of the HSP90 inhibitor (GA), reciprocal inhibitory behavior between AhR and ER was barely observed, highlighting the important role of HSP90 in the inhibition. As shown in Fig. 6A, without the assistance of the HSP90-containing complex, the receptors alone were unable to bind the ligands. Ligand binding leads to the dissociation of the receptor-HSP90 complex (Ma, 2007). On the other hand, during the co-treatment of PCB77 and E2, competition between AhR and ER exists, resulting in the repression of VTG and CYP1A. Therefore, the reciprocal inhibiting interaction between the ER-AhR pathways is caused by competition with the free HSP90-containg complex. In conclusion, cadmium promotes PCB77 (AhR agonist)mediated CYP1A protein expression and 17-estradiol (E2, ER agonist)-mediated VTG protein expression in goldfish primary hepatocytes. On the other hand, reciprocal inhibiting crosstalk between the AhR and ER pathways was observed. AhR-ER crosstalk also has a complex interaction with the heat shock response. The 90-kDa heat shock protein (HSP90) participating in modulating crosstalk between the AhR and ER pathways was examined using immune-precipitation experiments. HSP90 mediates the ligand–receptor interactions because the free receptors were unable to bind their ligands without the assistance of a HSP90containing complex. These findings of a mechanism among AhR, ER and heat shock pathways provide novel insights into the signaling pathways. Transparency document The Transparency document associated with this article can be found in the online version. Conflict of interest The authors declare that there are no conflicts of interest.
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