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Endocrinology Vol. 143, No. 1 55-61
Copyright © 2002 by The Endocrine Society

RECEPTORS

Ligands Have Various Potential Effects on the Degradation of Pregnane X Receptor by Proteasome

Hisashi Masuyama, Hideshi Inoshita, Yuji Hiramatsu and Takafumi Kudo

Department of Obstetrics and Gynecology, Okayama University Medical School, Okayama 700-8558, Japan

Address all correspondence and requests for reprints to: Hisashi Masuyama, M.D., Ph.D., Department of Obstetrics and Gynecology, Okayama University Medical School, 2-5-1 Shikata, Okayama 700-8558, Japan. E-mail: masuyama{at}cc.okayama-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The degradations of several nuclear receptors are involved in the proteasome-mediated pathway. In our recent experiments, we found that mouse pregnane X receptor (PXR) interacted with suppressor for gal1 (SUG1), a component of the proteasome, in a progesterone-dependent manner, but that endocrine-disrupting chemicals (EDCs), phthalic acid and nonylphenol, which activated PXR-mediated transcription, did not enhance this interaction. PXR protein levels were markedly increased in the presence of proteasome inhibitors, suggesting that PXR may be degraded by proteasome. Furthermore, in the absence of ongoing protein synthesis, there is much slower degradation of PXR in the presence of phthalic acid compared with that in the presence of progesterone. The transient expression studies demonstrated that overexpression of wild-type SUG1 generated proteolytic PXR fragments, and these productions were blocked by a proteasome inhibitor. Functionally, expression of SUG1 inhibited PXR- and progesterone-mediated transcription. Moreover, in the presence of EDCs, SUG1 had no effect on the transcription. These findings indicate that the interaction between PXR and SUG1 may be involved in the proteasome-mediated degradation. Moreover, an EDC strongly blocks the degradation of PXR compared with progesterone, suggesting that EDCs may affect PXR-mediated transcription of target genes through up-regulation of the PXR protein level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AN ENDOCRINE-DISRUPTING chemical (EDC) has been defined as an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body, which are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior (1). These chemicals can alter endocrine functions through a variety of mechanisms, including steroid hormone receptor-mediated changes in protein synthesis, interfering with membrane receptor binding, steroidogenesis, or synthesis of other hormones (2). Although major chemicals, such as phthalates, alkylphenols, bisphenol A, and dichlorodiphenyltrichloroethane, have been shown to disrupt estrogenic actions mainly through their binding to ER or androgen receptors (2), it is not clear whether EDCs directly affect endocrine functions in vivo.

Pregnane X receptor (PXR), a new member of steroid receptor superfamily, has been shown to mediate the genomic effects of several steroid hormones, such as progesterone, pregnenolone, glucocorticoid, synthetic glucocorticoids and antiglucocorticoids, and xenobiotics in the mouse, rat, and human (3, 4, 5, 6, 7, 8, 9). Like nonsteroid hormone receptors, it binds as a heterodimer with RXR to specific DNA sequences, including the upstream region of the cytochrome P-450 3A (CYP3A) gene family (3, 4, 5, 8), which are monooxygenases responsible for the oxidative metabolism of certain endogenous substrates and xenobiotics (10, 11). Recently, we have demonstrated that some EDCs activated PXR-mediated transcription, perhaps through the interaction with coactivators (12). PXR is thought to regulate cytochrome P450 3A family genes, which play important roles in steroid metabolism (10, 11), suggesting that there may be a novel pathway of EDCs that affect endocrine functions.

We and others have demonstrated that several putative cofactor proteins, including steroid hormone receptor coactivator 1 (13), receptor interacting protein 140 (14), and suppressor for gal 1 (SUG1) (15), interacted with PXR in a natural steroid-dependent manner (3, 4, 12). Expression of steroid hormone receptor coactivator 1 and receptor interacting protein 140 augments ligand-activated transcription by a variety of nuclear receptors, indicating that these proteins have a transcriptional coactivator role (16, 17, 18). Although yeast SUG1 was originally identified as a transcription factor (19) and interacts with various nuclear receptors (VDR, RXR, and TR) in ligand-dependent manner (15, 18, 20), more recent evidence indicates that this protein is actually a component of the 26S proteasome complex (21), and overexpression of this protein enhanced the degradation of VDR (22), suggesting that SUG1 may be involved in the degradation of nuclear receptors by proteasome.

We have examined how EDCs affect endocrine function. We checked whether EDCs affect the turnover of PXR, because PXR played some roles in steroid metabolism with CYP3A1, and EDCs have no effect on the interaction between PXR and SUG1, a component of proteasome, although PXR interacted with SUG1 in the presence of progesterone. First, we examined whether the degradation of PXR was mediated through the proteasome pathway. We also examined whether the EDCs tested in this study affected this proteasome-mediated degradation. Finally, we used a transient transfection assay to determine whether the interaction of PXR with SUG1 is involved in this degradation system. The results suggest that PXR is degraded by proteasome and that EDCs block this proteasome-mediated degradation of PXR, which may result in up-regulation of the PXR level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of two-hybrid expression vectors and ß-galactosidase assays
All two-hybrid plasmids constructs used the pAS1 (23) and pAD-GAL4 yeast expression vectors (Stratagene, La Jolla, CA). The AS1-PXR construct has been previously described (12). A point mutation, K196H, was introduced into the mouse SUG1 cDNA with oligonucleotide-directed mutagenesis (22). The K196H mutant of SUG1 was confirmed by DNA sequencing and subcloned into the pAD-GAL4 vector for examination in the two-hybrid assay. The wild-type and mutant pAD-mSUG1 were cotransformed with wild-type and mutant pAS1-PXR into the yeast strain Hf7c. Transformants were plated on medium lacking leucine and tryptophan (SC-Leu-Trp) and were grown for 4 d at 30 C to select for yeast that had acquired both plasmids. Triplicate independent colonies from each plate were grown overnight in 2 ml SC-Leu-Trp with or without the indicated concentrations of progesterone, phthalic acid, nonylphenol or bisphenol A. Cells were harvested and assayed for ß-galactosidase activity as described previously (24).

Inhibitors and ligands
Z-Leu-Leu-Leu-H (MG132) was purchased from Peptides International, Inc. (Louisville, KY). E64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane), adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S) and dimethylsulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). Clasto-lactacystin ß-lactone (ß-lactone) was obtained from Calbiochem-Novabiochem Co. (San Diego, CA). All inhibitors were maintained in DMSO at a final concentration of 50 mM and stored at -20 C. Isopropylidenediphenol (bisphenol A), phthalic acid bis-(2-ethylhexel ester) (phthalic acid), and progesterone were purchased from Sigma (St. Louis, MO). 4-Nonylphenol (nonylphenol), which is a mixture of compounds with branched side chains, phthalic acid benzyl n-butyl ester (benzyl butyl phthalate), phthalic acid diisodecyl ester (diisodecyl phthalate), and phthalic acid di-n-octyl ester (di-n-octyl phthalate) were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). The purities of all chemicals except 4-nonylphenol were more than 98%. All inhibitors and (2) ligands were added simultaneously to the culture media at the indicated concentrations.

Nuclear extracts and Western analysis
Nuclear extracts were obtained from BALB-MC cells essentially by the method of Shapiro et al. (25) and stored at -80 C. Equivalent amounts of nuclear protein from each extract were solubilized in SDS buffer and analyzed by Western blot analysis as previously described (22) using goat polyclonal antibody for PXR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Transient transfection studies
COS-7 cells and BALB-MC cells were cultured in DMEM without phenol red supplemented with 10% charcoal-striped calf serum. The (CYP3A1)2-thymidine kinase (tk)-chloramphenicol acetyltransferase (CAT), containing two copies of the CYP3A1 motif, which is a direct repeat of the nonsteroid nuclear receptor half-site sequence AGTTCA separated by a three-nucleotide spacer (3), and the pSG5-PXR expression plasmid containing full-length mouse PXR cDNA were obtained from Dr. S. A. Kliewer (3). Wild-type and mutant (K196H) mSUG1 constructs have been described previously (18, 22). COS-7 cells were cotransfected with 1 µg of a reporter gene construct ((CYP3A1) 2-tk-CAT) and 0.5 µg of a receptor expression vector (pSG5-PXR) or empty vector (pSG5). BALB-MC cells were also cotransfected with 10 µg pSG5-PXR or pSG5. In all transfections, liposome-mediated transfections were accomplished using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Transfected cells were treated either with vehicle alone or with the indicated concentrations of steroid hormones or EDCs for 36 h. COS-7 cell extracts were prepared and assayed for CAT activity. The amount of CAT was determined using a CAT ELISA kit (5 Prime->3 Prime, Inc., Boulder, CO) according to the manufacturer’s protocol. The nuclear extracts of BALB-MC cells were prepared and analyzed by Western blotting as mentioned above. The ß-galactosidase staining kit (Invitrogen, Carlsbad, CA) was also used to check the percentage of transiently transfected cells in BALB-MC cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Natural steroid-dependent interaction between PXR and SUG1
As illustrated in Fig. 1AGo, SUG1 interacted with PXR in a concentration-dependent manner with increasing amounts of progesterone (2) in the two-hybrid system. Half-maximal ß-galactosidase activity occurred at approximately 10-7 M. In contrast, all EDCs, including phthalic acid and nonylphenol, which activated PXR-mediated transcription (12), had no effect on the interaction between PXR and SUG1. Mutant SUG1 (K196H), which does not interact with other nuclear receptors (15, 22), did not affect the interaction in the presence of any ligands (Fig. 1BGo). In addition, pregnenolone and dexamethasone enhanced the interaction between PXR and SUG1 (26), suggesting that the interaction between PXR and SUG1 is dependent on natural steroids.



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  		Figure 1. Natural steroid-dependent interaction between PXR and SUG1 in a two-hybrid system. A, Yeast expressing the pAS1-PXR and pAD-SUG1 two-hybrid plasmids were grown for 24 h at 30 C in the absence and presence of increasing concentrations of progesterone or EDCs. The PXR-SUG1 interaction was assessed in a ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures. B, The pAD-SUG1 (wild-type or mutant) plasmid was cotransformed with pAS1-PXR into the yeast strain Hf7c. Relative growth of yeast on histidine-deficient plates was assessed after 4 d at 30 C. Wild-type or mutant SUG1 interaction with PXR was quantitated in a ß-galactosidase assay after overnight growth in a selection medium (SC-Leu-Trp) in the presence of 10-6 M progesterone or EDCs. Results are presented as the mean ± SD of triplicate independent cultures.

 
Effect of proteasome inhibitors on PXR protein levels in BALB-MC cells
Because SUG1 is completely identical to p45, a component of the regulatory subunit of the proteasome (21), and because we have shown that the degradation of VDR was involved in the SUG1-proteasome system (22), we examined whether PXR is also a target for proteasome-mediated degradation. Three cell lines, human colon cancer (COLO320 DM) cells, mouse mammary cancer (BALB-MC) cells, and uterine cervical cancer (HeLa) cells, were examined to determine which cell lines expressed PXR. As shown in Fig. 2AGo, PXR was abundant in COLO320 DM cells and BALB-MC cells, but it was not detected in HeLa cells. Thus, PXR protein levels were qualitatively examined in BALB-MC cells that had been exposed to a variety of proteasome inhibitors. Interestingly, PXR protein levels were markedly increased in the presence of 0.05 mM MG132 or ß-lactone, which strongly inhibit proteasome activities (27, 28), with or without progesterone (Fig. 2BGo). In contrast, PXR levels did not change in response to the lysosomal protease inhibitor (E64) (28). The proteasome inhibitors MG132 and ß-lactone had no effect on PXR-mediated transcription using transient transfection assay (data not shown), suggesting that these inhibitors are not ligands for mouse PXR. PXR degradation was also examined in the absence of ongoing protein synthesis (Fig. 2CGo). BALB-MC cells were treated with cycloheximide (10 mg/ml), and the effect of progesterone or EDCs, phthalic acid, nonylphenol, and bisphenol A on PXR turnover was determined. In the absence of progesterone, the PXR protein was rapidly degraded (t1/2, <4 h), and the degradation of progesterone-occupied PXR proceeded at a somewhat slower rate (t1/2, >8 h), similar to previous findings showing ligand induced stability of the VDR (22, 29, 30). Pregnenolone and dexamethasone, which are other well known mouse PXR ligands of steroid hormones (3, 12), had almost the same effects on PXR turnover compared with that in the presence of progesterone (data not shown). Moreover, phthalic acid and nonylphenol, which activated PXR-mediated transcription, but did not enhance the interaction with SUG1, strongly blocked the degradation of PXR. Bisphenol A, which had no effect on either transcription or interaction with SUG1, did not affect the degradation of PXR, and the turnover of bisphenol A-occupied PXR was compatible with that of the unliganded receptor. We also examined the synergism effects of EDCs and progesterone on PXR degradation (Fig. 2DGo). In the presence of progesterone and EDC, phthalic acid, or nonylphenol, PXR degradation was slower than that in the presence of progesterone alone. However, there was no change in PXR turnover in the presence of progesterone and bisphenol A compared with progesterone alone. In addition, the presence of both phthalic acid and nonylphenol completely blocked the PXR degradation. As phthalic acid has been demonstrated to show no estrogenic activity in in vitro assay (31), we checked the effects of other phthalates on the PXR degradation (Fig. 2EGo). Butyl benzyl phthalate, which has an estrogenic effect (31), completely blocked PXR degradation; however, other phthalates, diisodecyl phthalate and di-n-octyl phthalate, which have no estrogenic activity (31), did not affect this degradation.



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  		Figure 2. The effect of proteasome inhibitors on PXR protein levels in BALB-MC cells. A, Nuclear extracts of subconfluent BALB-MC, COLO320 DM, and HeLa cells were prepared as described in Materials and Methods. Equivalent amounts of each extract were resolved by 10% SDS-PAGE, and PXR protein levels were determined by Western blotting using anti-PXR antibody. B, Subconfluent BALB-MC cells were treated with DMSO or various protease inhibitors for 6 h, and nuclear extracts were prepared as described in Materials and Methods. Equivalent amounts of each extract were resolved by 10% SDS-PAGE, and PXR protein levels was determined by Western blotting using anti-PXR antibody. C, Subconfluent BALB-MC cells were treated with cycloheximide (10 mg/ml medium) for 10 min before inhibitor and ligand addition. Then, cells were treated in the presence of 10-6 M progesterone, EDCs, or vehicle (2 ). Nuclear extracts were prepared, and PXR protein levels were examined as described above. This level of cycloheximide inhibited more than 95% of 35S-labeled methionine incorporation into trichloroacetic acid-precipitated protein (data not shown). D, Subconfluent BALB-MC cells were treated with cycloheximide (10 mg/ml medium) for 10 min before inhibitor and ligand addition. Then, cells were treated in the presence of 10-6 M progesterone, EDCs, or vehicle for 16 h. Nuclear extracts were prepared, and PXR protein levels were examined as described above. E, Subconfluent BALB-MC cells were treated with cycloheximide (10 mg/ml medium) for 10 min before inhibitor and ligand addition. Then, cells were treated in the presence of 10-6 M progesterone, several phthalates, or vehicle for 16 h. Nuclear extracts were prepared, and PXR protein levels were examined as described above.

 
SUG1 overexpression enhances PXR proteolysis in BALB-MC cells
To test whether the SUG1-PXR interaction is involved in PXR degradation in this system, wild-type SUG1 and SUG1 (K196H), a mutant that does not interact with PXR (Fig. 1BGo), were transiently overexpressed in BALB-MC cells, and their effects on PXR protein levels were examined in Western immunoblots (Fig. 3AGo). In the absence of progesterone, overexpression of wild-type and mutant SUG1 did not significantly affect PXR protein levels (lane 1–3). However, in the presence of progesterone (2), novel proteolytic fragments of PXR (Fig. 3AGo, lane 5) were observed when BALB-MC cells were transfected with the wild-type SUG1 expression vector. PXR fragments were not observed in cells transfected with the SUG1 (K196H) mutant or in cells treated with phthalic acid. These findings suggest that a natural steroid-dependent interaction between PXR and SUG1 resulted in the generation of these proteolytic fragments of PXR. Similar studies using a proteasome inhibitor (ß-lactone) indicated that the PXR proteolytic fragments were generated by proteasome-mediated degradation. As shown in lane 4 of Fig. 3BGo, 0.05 mM ß-lactone completely blocked the formation of this progesterone and SUG1-dependent fragments. The production of this proteolytic fragment could also be blocked by treating the cells with the nonhydrolyzable ATP analog, ATP{gamma}S, indicating that both proteasome activity and ATP hydrolysis were required for this fragment of PXR in this system. Moreover, progesterone was also required for this phenomenon, because proteolytic fragments were not observed in cells transfected with wild-type SUG1 in the presence of phthalic acid. To check the percentage of the cells transiently transfected under this condition, a plasmid expressing lacZ (pcDNA3.1/His/lacZ) was also transfected into BALB-MC cells, and the cells were stained for ß-galactosidase expression according to the manufacturer’s protocol. The average transfection efficiency was about 15% (n = 3). As 15% of cells were transfected under this condition, this might be the reason why we did not observe a net decrease in the protein level of PXR by overexpression of wild-type SUG1.



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  		Figure 3. The enhancement of PXR proteolysis by SUG1 overexpression. A, BALB-MC cells on 150-mm plates were transfected with 10 µg pcDNA3 parent expression plasmid or pcDNA3 derivatives that express wild-type or mutant SUG1. The cells were treated with ethanol vehicle or 10-6 M progesterone for 6 h, and nuclear extracts were prepared. PXR protein levels were examined as described in Fig. 2Go. B, BALB-MC cells were transfected with 10 µg pcDNA3 parent expression plasmid or pcDNA3 derivatives that express wild-type mUG1. The cells were treated with 10-6 M progesterone or EDCs and exposed to 0.05 mM ß-lactone or 5 mM ATP{gamma}S for 6 h, and nuclear extracts were prepared. PXR protein levels were examined as described in Fig. 2Go.

 
Suppression of PXR-mediated transcription by overexpressed wild-type SUG1
To examine the effect of SUG1 on PXR-mediated trans-activation, SUG1 was expressed in a transient gene expression system. A PXR expression plasmid (SG5-PXR) and a reporter gene construct [(CYP3A1)2-tk-CAT] were introduced into COS-7 cells in the absence or presence of an expression vector that generates SUG1 (pcDNA3-SUG1) (Fig. 4A). Expression of SUG1 suppressed progesterone-mediated trans-activation of PXR, but had no effect on basal transcription. This effect depended on the amount of SUG1, and the maximal suppression was about 50% compared with similar transfections with the parent expression vector (pcDNA3). The suppressive effect on progesterone-dependent transcription was due to an interaction between PXR and SUG1, because the expression of mutant SUG1 (K196H), which does not interact with PXR, had no significant effect on PXR-mediated trans-activation in this system (Fig. 4BGo). Wild-type and mutant SUG1 did not affect PXR-mediated transcription in the presence of phthalic acid and nonylphenol.



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  		Figure 4. Suppression of progesterone/PXR-mediated trans-activation by wild-type SUG1. A, COS-7 cells were transfected with 1 µg of the (CYP3A1)2-tk-CAT reporter gene construct and 0.5 µg of the PXR expression plasmid together with an increasing amount of pcDNA-SUG1 (wild-type). The cells were treated with ethanol vehicle or 10-6 M progesterone for 36 h. The amount of CAT was determined with a CAT ELISA kit (5 Prime->3 Prime, Inc.) according to the manufacturer’s protocol. The results represent the mean ± SD of triplicate determinations. B, COS-7 cells were cotransfected with 1 µg of the (CYP3A1)2-tk-CAT reporter gene constructs, 1 µg of the SUG1 expression plasmids (wild-type or K196H) or empty vector, and 0.5 µg pSG5-PXR. The cells were treated with ethanol vehicle or 10-6 M progesterone or various EDCs for 36 h. The amount of CAT was determined with a CAT ELISA kit (5 Prime->3 Prime, Inc.) according to the manufacturer’s protocol. The results represent the mean ± SD of triplicate determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proteasome is a major cytosolic and nuclear protease complex that is responsible for an ATP-dependent, extralysosomal proteolytic pathway. This complex is responsible for the degradation of most cellular proteins, and proteasome activity is necessary for cell viability (32, 33). Proteasome is highly conserved throughout eukaryotic evolution, and it exists as two major complexes: 20S proteasome, which contains multiple peptidase activities, and 26S proteasome, which contains the 20S subunit as well as a 19S regulatory complex composed of multiple adenosine triphosphatases and components necessary for binding protein substrates (32, 33). To date, a wide variety of substrates for proteasome have been identified, including rate-limiting enzymes such as ornithine decarboxylase; transcriptional regulators such as c-Jun, p53, and nuclear factor-{kappa}B; and critical regulatory proteins such as cyclins and tk receptors (32, 33). In addition, we and others have demonstrated that the proteasome system might be involved in the degradation of nuclear receptors, including VDR, PR, and ER (22, 34, 35). Several recent studies have focused on the involvement of the proteasome pathway in ligand-mediated degradation of ER{alpha}, and ligand-mediated down-regulation of the PR and RXR has also been reported (34, 35, 36). On the other hand, other receptors, such as the VDR and the AR, have been shown to be up-regulated in the presence of their cognate ligand in most cell contexts (22, 37).

In the present study some evidence that PXR might be degraded by the proteasome system was obtained. First, a variety of relatively selective inhibitors of the proteasome pathway, including MG-132 and ß-lactone, dramatically increased the steady state levels of native PXR protein in nuclear extracts obtained from BALB-MC cells. Secondly, overexpression of wild-type SUG1 in BALB-MC cells resulted in the appearance of proteolytic PXR derivatives. PXR-SUG1 interaction was also required for the formation of these proteolytic fragments, because overexpression of the SUG (K196H) mutant did not produce a similar effect. Moreover, the interaction between PXR and SUG1 requires natural steroids, and proteolytic PXR fragments were not observed in SUG1-transfected BALB-MC cells in the absence of a ligand or in the presence of an EDC, phthalic acid. Finally, the ß-lactone proteasome inhibitor abolished formation of the degraded PXR, indicating that this and SUG1-dependent products were the result of proteasome-mediated proteolysis. Taken together, these results suggest that SUG1 interacts with PXR and targets, either directly or indirectly, the PXR for degradation by the proteasome machinery. Generally, the polyubiquitin chain is important for specific targeting to the proteasome (32, 33). Several nuclear receptors have been reported to be polyubiquitinated and degraded by proteasome (34, 35). We are investigating whether the degradation of this receptor is also involved in the ubiquitin-mediated targeting system to proteasome.

A variety of putative pathways by which EDCs have effects on the endocrine system have been reported (1, 2). We have already shown one potential pathway by which EDCs may affect endocrine function through PXR-mediated changes in steroidogenesis (12). In our experiments we demonstrated that the interaction between PXR and SUG1 required natural steroids and that this interaction might be involved in proteasome-mediated degradation of PXR. Moreover, phthalic acid, an EDC, blocked the degradation of PXR by proteasome as well as activated PXR-mediated transcription, which results in the relative up-regulation of PXR protein levels that have positive impacts on PXR-mediated gene regulation. If so, it may be a potential mechanism of EDCs that affect endocrine function. However, we demonstrated that both phthalic acid and butyl benzyl phthalate affected PXR degradation, although butyl benzyl phthalate showed estrogenic activity, but phthalic acid did not in an in vitro assay (31). Moreover, although bisphenol A and nonylphenol are well known to have similar estrogenic activities (38, 39), we observed that only nonylphenol had some effect on PXR-mediated transcription (12), and PXR turnover and that bisphenol A had no effect. Thus, we have some questions about which phthalates are active molecules for PXR and why there are discrepancies between ER ligands and PXR ligands in EDCs. Further analysis is required to answer these questions.

In summary, we have presented some evidence that PXR is degraded by proteasome and that overexpression of SUG1 selectively altered PXR proteolysis in the presence of progesterone. These findings suggest the existence of a general mechanism for receptor down-regulation that may involve proteasome-mediated proteolysis with the interaction between PXR and SUG1. Moreover, although some EDCs enhanced PXR-mediated transcription, these chemicals had no impact on the interaction between PXR and SUG1 and strongly blocked PXR degradation by proteasome, suggesting that some EDCs affect the degradation of PXR, resulting, somehow, in transcriptional activity through up-regulation of PXR.


   Footnotes
 
This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan.

Abbreviations: ATP{gamma}S, Adenosine 5'-O-(3-thiotriphosphate); CAT, chloramphenicol acetyltransferase; CYP3A, cytochrome P-450 3A; DMSO, dimethylsulfoxide; EDC, endocrine-disrupting chemical; PXR, pregnane X receptor; SUG1, suppressor for gal1; tk, thymidine kinase.

Received May 14, 2001.

Accepted for publication September 11, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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