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-Hydroxylase Gene
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Address all correspondence and requests for reprints to: You-Hua Xie or Yuan Wang, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Yueyang Road 320, Shanghai 200031, China. E-mail: yhxie@sibs.ac.cn or wangy{at}sibs.ac.cn.
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ABSTRACT |
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-hydroxylase (CYP7A1) catalyzes a rate-limiting step in bile acid synthesis in liver, and its gene transcription is under complex regulation by multiple nuclear receptors in response to bile acids, cholesterol derivatives, and hormones. The liver receptor homolog-1 (LRH-1), a member of the fushi tarazu factor 1 subfamily of nuclear receptors, has emerged as an essential regulator for the expression of cyp7a1. In this report, we demonstrate Prox1, a prospero-related homeobox transcription factor, identified through a yeast two-hybrid screening, can directly interact with human LRH-1 (hLRH-1) and suppresses hLRH-1-mediated transcriptional activation of human cyp7a1 gene. Biochemical analysis demonstrates that Prox1 interacts with both the ligand binding domain (LBD) and the DNA binding domain (DBD) of hLRH-1. An LRKLL motif in Prox1 is important for the interaction with the LBD but not the DBD of hLRH-1. In hLRH-1 LBD, helices 2 and 10 are essential for Prox1 recruitment. The suppression by Prox1 on the transcriptional activity of hLRH-1 can be mediated through its interaction with the LBD or the DBD of hLRH-1. Gel shift assays reveal that Prox1 impairs the binding of hLRH-1 to the promoter of human cyp7a1 gene. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, HNF3ß, and HNF4 (6, 7, 8). Likewise, LRH-1 can be a major player in a regulatory cascade that governs pancreatic development because it was recently identified as a direct downstream target of PDX-1, an essential transcriptional factor for pancreatic development, differentiation, and function (9).
In adults, an increasing number of LRH-1 target genes have been discovered, which are mainly involved in bile acid and cholesterol homeostasis. LRH-1 is an essential regulator for the expression of the cholesterol 7--hydroxylase (CYP7A1) gene that encodes a rate-limiting enzyme in the classical pathway of bile acid synthesis. An LRH-1 consensus binding site is located within the core promoter of both the murine and human cyp7a1 genes and has been demonstrated to mediate the regulation by LRH-1 in HepG2 cells (5, 10, 11). Other LRH-1 targets in bile acid and cholesterol homeostasis include the genes for sterol 12--hydroxylase (CYP8B1) (12, 13, 14) and several bile acid or cholesterol transporters such as multidrug resistance protein 3 (15), cholesterol ester transport protein (16), and scavenger receptor B1 (17). In addition, LRH-1 also appears to regulate the expression of the gene for aromatase in rodent ovary and human preadipocytes (18, 19).
Coregulators are critical for the biological functions of nuclear receptors by modulating their transcriptional activities. Several coregulators of the FTZ-F1 subfamily of nuclear receptors have been identified. Bonus, a Drosophila homolog of the mammalian transcription intermediary factors, can interact via an LXXLL motif to the activation function (AF)-2 domain in the ligand binding domain (LBD) of ßFTZ-F1 and inhibit ßFTZ-F1-dependent transcription (20). BmMBF-1 of Bombyx mori is a coactivator of BmFTZ-F1 and appears as a bridging factor between BmFTZ-F1 and the basic transcription machinery (21). Multiple coregulators of mammalian SF-1 have been described. The orphan nuclear receptor Dax-1 is found in the same tissues as SF-1 (22) and represses SF1-mediated transcriptional activation through its interaction with a repression domain at the carboxyl end of SF-1 LBD (23, 24, 25). Steroid receptor coactivator (SRC)-1 potentiates the activity of SF-1 through its interaction with the AF-2 domain and an upstream proximal interaction domain at residues 187–245 (26, 27). Other proteins, including Wilms’ tumor 1, early growth response-1, multiprotein bridging factor (MBF)-1, pituitary homeobox 1, glucocorticoid receptor-interacting protein 1/transcriptional intermediary factor 2, cAMP response element binding protein (CREB)-binding protein/p300, transcriptional regulating protein 132, dead box protein 103kD, and GATA binding protein 4 can also modulate the activity of SF-1 (25, 28, 29, 30, 31, 32, 33, 34, 35).
Compared with SF-1, much less is known about the coregulators of LRH-1. MBF-1 is a coactivator of LRH-1, and its interaction with LRH-1 requires the DNA binding domain (DBD) and the FTZ-F1 box but not the hinge region and the LBD (36). The orphan nuclear receptor SHP (short heterodimer partner) suppresses the transcriptional activity of LRH-1 through an interaction with the AF-2 domain in LRH-1 LBD (37). When bile acid levels are high, SHP can mediate a classic negative feedback loop through its interaction with LRH-1 and decreases the transcription of cyp7a1 (10, 11). However, loss of SHP in mice impairs but does not eliminate the negative feedback regulation of bile acid synthesis, suggesting the existence of other regulatory pathways (38, 39).
Many coregulators interact with the LBD of nuclear receptors. Structural analysis reveals that the LBD of hormone-bound nuclear receptors forms a tightly packed structure in which the ligand binding pocket and the AF-2 domain are important for coregulator recruitment (40, 41). However, specific ligands for LRH-1 or SF-1 have not been clearly demonstrated. Biochemical data indicate that AF-2 and helix 10 in the LBD are essential for the transcriptional activity of zebra fish LRH-1 homolog, zFF1a (42). Recently, the three-dimensional structure of mouse LRH-1 (mLRH-1) LBD was resolved. Examination of this structure reveals that an active ligand binding pocket is constitutively formed in the absence of a ligand (43). With this structure model, it becomes possible to investigate the special structural requirements for coregulator recruitment by LRH-1 LBD.
Because LRH-1 is an important regulator of bile acid and cholesterol homeostasis, and because of its potential role in the development of pancreas and liver, identification of more tissue-specific coregulators of LRH-1 will have profound significance for elucidation of the biological functions of LRH-1. In this study, we carried out a yeast two-hybrid screening in an attempt to search for liver-specific coregulators that modulate the transcriptional activity of human LRH-1 (hLRH-1). We found Prox1, a prospero-related homeobox transcription factor, as a corepressor of hLRH-1. Prox1 can suppress hLRH-1-mediated transcriptional activation of human cyp7a1 gene in HepG2 cells through a direct interaction with hLRH-1. Biochemical analysis reveals that Prox1 can interact with not only the LBD but also the DBD of hLRH-1. These two interactions are mutually independent in vitro and in cells. The molecular mechanism for the suppression by Prox1 on the transcriptional activity of hLRH-1 was investigated in detail.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) in the LBD is disrupted because this domain displays an autonomous transactivation that results in a high background in the screening (data not shown). Approximately 1.6 x 106 yeast transformants were screened, and nine strong positive clones were verified by retransformation into a yeast strain producing the bait protein (data not shown). The cDNA inserts in these clones were sequenced. Blast searches revealed that three clones encode an identical fragment corresponding to the aa55–553 fragment of human Prox1, a transcription factor related to Drosophila melanogaster prospero (Fig. 1B).
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, lanes 3 and 5). To examine whether full-length hLRH-1 and Prox1 interact in HepG2 cells, we performed a coimmunoprecipitation experiment. Flag-Prox1 was produced in HepG2 cells by transient transfection. To increase the cellular amount of hLRH-1, HepG2 cells were also transfected with an expression plasmid for hLRH-1. A rabbit anti-hLRH-1 polyclonal antibody against the aa244–495 fragment of hLRH-1 was used to immunoprecipitate hLRH-1. As shown in Fig. 2B, Flag-Prox1 was detected in the precipitate by a mouse anti-Flag monoclonal antibody (lane 2), suggesting that hLRH-1 and Prox1 associate in HepG2 cells. Such an interaction between hLRH-1 and Prox1 in HepG2 cells was supported by a mammalian two-hybrid assay. The hinge region and the LBD of hLRH-1 (aa141–495) were fused to the DBD of Gal4 (Gal4-DBD), whereas the full-length Prox1 or Prox155–553 was fused to the VP16-AD (activation domain of herpes simplex virus VP16). Expression plasmids for Gal4-hLRH-1141–495 and VP16-Prox1 were cotransfected into HepG2 cells along with the pG5Luc reporter. As shown in Fig. 2C, VP16-Prox1 or VP16-Prox155–553 could augment Gal4-hLRH-1141–495-mediated transactivation of the reporter gene, indicating that Prox1 interacts with hLRH-1 in HepG2 cells. Similar results were obtained with cotransfection and reporter gene assays in another hepatic cell line Huh7 (data not shown).
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, GFP proteins were evenly distributed in cells (panel A). Both GFP-Prox1 and the endogenous hLRH-1 were found present in the nuclei of HepG2 cells (panels E, F, G, and H). However, hLRH-1 could also be detected in the cytoplasm of HepG2 cells by the anti-hLRH-1 antibody (panels F, G, and H). Nuclear colocalizations of exogenously introduced GFP-Prox1 and hLRH-1 were observed in nonhepatic COS-7 cells (panels I, J, K, and L). Interestingly, hLRH-1 is exclusively nuclear in COS-7 cells.
Prox1 Interacts with Both the DBD and the LBD of hLRH-1
Which domain(s) of hLRH-1 does Prox1 interact with? To answer the question, we used a mammalian two-hybrid assay. Fragments of hLRH-1 representing the hinge region (141–256), the LBD (256–495) or the DBD (aa34–141) were fused to Gal4 DBD, respectively. Each construct and the expression plasmid for VP16-Prox155–553 were cotransfected into HepG2 cells along with pG5Luc. As shown in Fig. 3A, when Gal4-hLRH-1DBD or Gal4-hLRH-1LBD was used with VP16-Prox155–553, a 5.3-fold or a 6.9-fold elevated reporter activity was observed, compared with the respective control with VP16 (upper and middle panels). A much lower reporter activation (about 1.5-fold) was observed when Gal4-hLRH-1hinge was used (lower panel), which was barely higher than the additive activities of Gal4/VP16-Prox155–553 and Gal4-hLRH-1hinge/VP16. Thus, it is unlikely that the hinge region interacts with Prox155–553. The interaction between hLRH-1 DBD and Prox155–553 was unexpected because the DBD was not included in the bait for the yeast two-hybrid screening. Therefore, glutathione-S-transferase (GST) pull-down assays were performed to confirm the interaction. GST-hLRH-1186–495 containing the complete LBD and GST-hLRH-1DBD could be produced in soluble form and were used as bait proteins. As shown in Fig. 3B, GST-hLRH-1186–495 and GST-hLRH-1DBD interacted with the labeled, full-length Prox1, respectively (lanes 2 and 5), whereas GST did not (lanes 3 and 6). Taken together, these results indicate that Prox1 can interact with not only the LBD but also the DBD of hLRH-1.
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). These mutations were chosen based on the structure of mLRH-1 LBD, which suggests that they are unlikely to destabilize the packed structure but may influence the interaction with coregulators. Comparable amounts of the wild-type and the mutant GST-hLRH-1186–495 were used in pull down assays (Fig. 3D, lower panel). As shown in Fig. 3D, upper panel, mutations in H3 and H4 (lanes 5 and 6) decreased but did not eliminate the interaction with Prox1. In contrast, mutations in H2 and H10 (lanes 4 and 7) eliminated the interaction with Prox1, suggesting that H2 and H10 are essential for Prox1 recruitment.
The LRKLL Motif of Prox1 Is Important for the Interaction with hLRH-1 LBD But Not DBD
The domain organization of Prox1 has not been fully defined. Prox1 contains a unique homeo-prospero domain located at the C terminus that is supposed to bind DNA (46). Prox155–553 derived from the yeast two-hybrid screening falls within the N-terminal two thirds in which functional domains have not been determined. Nevertheless, several putative elements are inside Prox155–553. Two I/LXXLL motifs are located at aa70–74 (LRKLL) and aa93–97 (ISQLL), and a glutamine-rich element (Q-rich) within aa211–260 (Fig. 1B).
Pull-down assays were performed to examine whether these elements are involved in the interaction with hLRH-1 LBD. Several truncated Prox1 proteins were in vitro translated and labeled. Equal amounts of GST-hLRH-1186–495 and GST were used in pull-down assays. As shown in Fig. 4A, Prox155–553 interacted with GST-hLRH-1186–495 efficiently (lane 2). Prox155–333 containing all putative elements fully retained the ability to interact with GST-hLRH-1186–495 (lane 5). In contrast, Prox176–333 in which the LRKLL motif is excluded displayed a very weak interaction with GST-hLRH-1186–495 (lane 8) and Prox155–253, in which the Q-rich element is disrupted also showed a greatly diminished interaction with GST-hLRH-1186–495 (lane 11).
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ARKAL or ISQLL ASQAL) were made to one of or both (double mutant, DM) motifs in VP16-Prox155–553. The effect of the mutagenesis on the interaction between Prox1 and hLRH-1 LBD was examined with a mammalian two-hybrid assay. As shown in Fig. 4B, the ARKAL mutant or the double mutant Prox155–553 showed a markedly decreased ability to interact with Gal4-hLRH-1141–495 compared with the wild-type Prox155–553. On the contrary, the ASQAL mutant only displayed a slight destructive effect. These results demonstrate the importance of the LRKLL motif in the interaction between Prox1 and hLRH-1 LBD in intact cells. In comparison, the ISQLL motif plays little or no role in the interaction with hLRH-1 LBD.
Are the I/LXXLL motifs also important for the interaction between Prox1 and hLRH-1 DBD? Results of a mammalian two-hybrid assay showed that mutations in the I/LXXLL motifs have no apparent destructive influence on the interaction between Prox1 and Gal4-hLRH-1DBD (Fig. 4C), because with Gal4-hRH-1DBD, the wild-type, the ARKAL mutant, or the double mutant VP16-Prox155–553 activated the reporter similarly in HepG2 cells. Therefore, the I/LXXLL motifs are dispensable for the interaction with hLRH-1 DBD.
The weak interaction between GST-hLRH-1186–495 and Prox155–253 suggests that the Q-rich element may play an important role in the interaction with hLRH-1 LBD. It is also possible that the disruption of the Q-rich element might cause a conformational change that resulted in the observed diminished interaction (see Fig. 4A, lane 11). However, when the GST-fused Q-rich element (aa211–260) was used in a pull-down assay with the full-length hLRH-1, no interaction was observed (data not shown). Thus, whether the Q-rich element is directly involved in the interaction between Prox1 and hLRH-1 requires more detail analyses.
Prox1 Suppresses the Transcription of Human cyp7a1 Gene in HepG2 Cells
CYP7A1 is a liver-specific key enzyme in bile acid synthesis that is a major component of the complex cholesterol homeostasis in vivo. LRH-1 has been documented as an essential regulator for the expression of both the murine and human cyp7a1 genes (5, 10, 11). Whether Prox1 modulates the activity of human cyp7a1 promoter through hLRH-1 was tested with cotransfection and reporter gene assays. As shown in Fig. 5A, the cyp7a1 promoter was highly active in HepG2 cells (dark gray bars). The wild-type Flag-tagged Prox1 showed a dose-dependent suppression on the cyp7a1 promoter activity, reaching a 77% reduction with 250 ng of the expression plasmid (light gray bars). Compared with the wild-type Flag-Prox1, the double mutant Flag-Prox1 (Prox1-DM) is a less potent repressor, reaching about 45% reduction with 250 ng of the expression plasmid (black bars). The different suppressive activities of the wild-type and the double mutant Flag-Prox1 were not owing to the different protein synthesis because their levels in HepG2 cells were comparable as demonstrated with Western blot (Fig. 5B, lanes 2 and 3). Thus, these results suggest that Prox1 interacts with hLRH-1 and suppresses the activity of human cyp7a1 promoter.
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, upper panel, lanes 3 and 4), which was apparently higher than those in control cells (lanes 1 and 2). The amount of the cyp7a1 transcript in these cells was determined with real-time quantitative PCR. As shown in Fig. 5C, lower panel, compared with the levels in control cells, a 51.3% or a 31.1% reduction in the amount of the cyp7a1 transcript were detected in cells overexpressing Prox1 or Prox1-DM, respectively. These results demonstrate a physiological role of the interaction between Prox1 and hLRH-1 in the regulation of the cyp7a1 gene expression in HepG2 cells.
Suppression by Prox1 Can Be Mediated by the Interaction with the DBD or the LBD of hLRH-1
Because Prox1 can interact with both the DBD and the LBD of hLRH-1, we next ask whether each interaction contributes to the suppression by Prox1 on the transcriptional activity of hLRH-1. To separately investigate the interaction between Prox1 and hLRH-1 LBD or the DBD, two reporter assay systems (pG5Luc and pCYP7A1Luc) were used (Fig. 6, A and B).
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, Gal4-hLRH-1141–495 activated the reporter strongly. The wild-type Prox1 markedly suppressed Gal4-hLRH-1141–495-mediated activation of the reporter, whereas the double mutant Prox1 showed no suppression. The results suggest that Prox1 can exert its suppressive activity through its interaction with hLRH-1 LBD. Moreover, the lack of suppression by the double mutant Prox1 on the activity of Gal4-hLRH-1141–495 contrasts with its moderate suppression on the cyp7a1 promoter described earlier, implying that the interaction between hLRH-1 DBD and Prox1 might also contribute to the suppression on the cyp7a1 promoter in HepG2 cells.
To examine the functional interaction between hLRH-1 DBD and Prox1, a fusion protein containing hLRH-1 DBD and VP16-AD was constructed. Expression plasmid for VP16-hLRH-1DBD was transfected along with the cyp7a1 promoter reporter into HepG2 cells. As shown in Fig. 6B, VP16-hLRH-1DBD could activate the cyp7a1 promoter about 2-fold. The wild-type as well as the double mutant Prox1 markedly suppressed VP16-hLRH-1DBD-mediated activation of the cyp7a1 promoter. The suppression by the double mutant Prox1 is consistent with the results described earlier, in which the double mutant Prox1 interacted efficiently with hLRH-1 DBD (see Fig. 4C). Thus, Prox1 can also suppress the transcriptional activity of hLRH-1 through its interaction with hLRH-1 DBD.
Prox1 Impairs the DNA Binding of hLRH-1
How does the interaction between Prox1 and hLRH-1 suppress the cyp7a1 promoter activity? One of the possibilities is that the interaction between Prox1 and hLRH-1 affects the DNA binding of hLRH-1. This hypothesis was tested by EMSA using in vitro-translated Prox1 and hLRH-1 proteins. A short fragment of human cyp7a1 promoter (–122 to –140) that contains the hLRH-1 consensus binding site was end-labeled and used as a probe. As shown in Fig. 7, hLRH-1 binds specifically to this probe (lanes 2, 9, and 10), whereas neither the wild-type nor the double mutant Prox1 was able to bind to this probe (lanes 7 and 8). The specific binding of hLRH-1 (lane 2) was impaired by an increasing amount of the wild-type Prox1 (lanes 3 and 4). A comparable amount of the double mutant Prox1 was less effective than wild-type Prox1 but still had the ability to impair hLRH-1 binding to the probe (lanes 5 and 6). The results clearly indicate that Prox1 can prevent hLRH-1 from binding to its target site on the cyp7a1 promoter. Moreover, the results also suggest that the interaction between Prox1 and hLRH-1 DBD plays a major role in impairing the DNA binding of hLRH-1 because the double mutant Prox1 also greatly decreases the specific binding of hLRH-1.
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DISCUSSION |
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The overall domain organization of Prox1 has not been deciphered except the C-terminal DBD. We demonstrate that the aa55–333 fragment of Prox1 is required and sufficient for the interaction with hLRH-1 LBD. Primary features in the region include two I/LXXLL motifs and a Q-rich element (Fig. 1B). The LXXLL motif present in many coregulators often interacts with AF-2 in the LBD of nuclear receptors (40, 41, 49). Therefore, the fact that AF-2 of hLRH-1 LBD is not essential for the interaction with Prox1 differs from mechanisms commonly found for many other nuclear receptors. Moreover, mutagenesis of H3 and H4 in hLRH-1 LBD indicates that they are not essential or play minor roles in the interaction (Fig. 3D). Because H3, H4, and H12 take part in the formation of the ligand binding pocket of mLRH-1 LBD (43), these results indicate that the ligand binding pocket is not directly required for the recruitment of Prox1. On the contrary, mutagenesis of H2 and H10 establishes their importance in Prox1 recruitment (Fig. 3D). H10 is conserved in the FTZ-F1 subfamily of nuclear receptors, and L454L455 in H10 have been reported to be crucial for the activity of zebra fish LRH-1 homolog, zFF1a (42). Moreover, H10 is also localized on the outer surface of mLRH-1 LBD (43), suggesting that it can be involved in the interaction with FTZ-F1-specific coregulators. The requirement of H2 reflects the unique tertiary structure of LRH-1 LBD. The relatively long H2 in mLRH-1 LBD forms an additional fourth and outmost layer and is important for coregulator recruitment (43). Thus, Prox1 is likely recruited to the outer surface of hLRH-1 LBD primarily via H2 and may also require H10.
The Q-rich element is also present in other coregulators such as SRC-1. The Q-rich region of SRC-1 can mediate a ligand-independent activity of the androgen receptor through its interaction with the N-terminal AF-1 domain of the androgen receptor (50). The diminished interaction between hLRH-1 and Prox1 containing a truncated Q-rich element (Fig. 4A) suggests that the Q-rich element may participate in the interaction with hLRH-1, although improper protein folding due to the truncation should not be excluded. However, GST-pull down assays failed to demonstrate an interaction between hLRH-1 and the Q-rich element alone (data not shown). Thus, the requirement of the Q-rich element for the interaction with hLRH-1 remains uncertain. It is possible that in the presence of the preceding LRKLL motif, the Q-rich element may enhance the interaction of Prox1 with hLRH-1 LBD.
Besides the LBD, Prox1 also interacts with hLRH-1 DBD. Other coregulators interacting with the DBD of SF-1 or LRH-1 have been reported or suggested (25, 36). Interestingly, it appears that the aa55–553 fragment of Prox1 can interact with both the LBD and the DBD of hLRH-1. Unlike the interaction with hLRH-1 LBD, the LRKLL motif is not required for the interaction with hLRH-1 DBD (Fig. 4C), suggesting that the domain of Prox1 involved in the interaction with hLRH-1 DBD is not identical with that with hLRH-1 LBD. The precise domain of Prox1 for the interaction with hLRH-1 DBD remains to be identified.
The independent interaction with hLRH-1 LBD and DBD makes Prox1 unique among known coregulators of the FTZ-F1 subfamily of nuclear receptors. Other coregulators interact with one domain/motif (MBF-1, DP103, SHP, Bonus) or require two cooperative domains/motifs (Dax-1, SRC-1, transcriptional regulating protein 132) (20, 23, 26, 29, 33, 34, 36, 37). Our findings indicate that dual molecular mechanisms govern the suppression by Prox1 on the activity of human cyp7a1 promoter through its interaction with hLRH-1 (Fig. 6). On one hand, Prox1 can impair the DNA binding of hLRH-1 (Fig. 7). In this case, the DNA binding by Prox1 is not required for the impairment to take effect, which is similar to Drosophila prospero. Prospero is reported to interact with several homeodomain containing proteins and differentially modulate their DNA binding properties in vitro without binding to DNA by itself (51). On the other hand, Prox1 can be recruited to hLRH-1 LBD, which may result in the hindrance of the binding by coactivators. It is noteworthy that, although these two mechanisms seem to work independently as demonstrated in our experiments, they probably synergize in vivo to achieve an efficient suppression on the transcriptional activity of hLRH-1.
So far, Prox1 as a coregulator of factors other than the FTZ-F1 subfamily of nuclear receptors has not been experimentally demonstrated. On the other hand, Prox1 as a corepressor of hLRH-1 may reflect only one aspect of the biological function of the interaction between Prox1 and hLRH-1. It is possible that hLRH-1 is a coregulator of Prox1 as well and modulates the expression of the genes regulated by Prox1. Prospero is reported to bind to a CAYNNCY consensus sequence (51). However, using in vitro-translated Prox1, we were unable to detect the binding of Prox1 to this sequence (data not shown). Known target genes of Prox1 are limited. Prox1 has been reported to regulate the activities of the lens-specific six3 and 
-crystallin gene promoters (52, 53). However, whether they are direct targets of Prox1 remains uncertain. Determination of the consensus binding sequence of Prox1 will facilitate the study of Prox1 target genes and the potential coregulation by hLRH-1.
One possible implication from the suppression by Prox1 on the transcription of the cyp7a1 gene is that there might be two connected pathways in liver that negatively modulate bile acid synthesis through the hLRH-1 response element in the cyp7a1 promoter. The orphan nuclear receptor SHP is known to act as a tissue-specific corepressor of LRH-1 and mediates the classic negative feedback loop of bile acids on the cyp7a1 promoter (10, 11). Other pathways for negative feedback regulation on bile acid synthesis have been suggested based on the studies with the shp knockout mice (38, 39). Inactivation of SHP disturbs but does not eliminate the negative feedback regulation on bile acid synthesis. Because the promoter of the shp gene is activated by LRH-1 in hepatocytes (54), Prox1 may modulate the shp promoter activity through LRH-1. It will be important to find out the signaling pathway that regulates the suppression by Prox1 on the transcription of the cyp7a1 gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The complete hLRH-1 cDNA (4) was cloned in the pcDNA3 vector (Invitrogen Life Technologies, Carlsbad, CA). pDBLeu-LRH-1141–481 was made by subcloning the aa141–481 fragment of hLRH-1 amplified by PCR in the SalI/SpeI sites of pDBLeu (Invitrogen Life Technologies). Fragments of hLRH-1 representing aa34–141, aa141–257, aa256–495, and aa141–495 were inserted in the BamHI/XbaI or EcoRI/XbaI sites of the pM vector (CLONTECH) (55), in frame to Gal4-DBD. The aa34–141 fragment of hLRH-1 was also inserted in the BamHI/XbaI sites of the pVP16 vector (CLONTECH) (55), in frame to the activation domain of VP16 (VP16-AD).
Full-length human Prox1 cDNA was obtained by PCR from a Marathon pre-made human liver cDNA mixture (CLONTECH) and cloned in the BamHI/EcoRI sites of pcDNA3. The full-length Prox1 cDNA was also inserted into the BglII/EcoRI sites of pEGFP-N2 (CLONTECH), in frame to the coding sequence of GFP. Flag-Prox1 was generated by PCR using primers containing the coding sequence for the Flag tag, and inserted in the EcoRI/XbaI sites of pcDNA3. Partial cDNAs of Prox1 were inserted in the BamHI/EcoRI sites of pcDNA3. The resulting constructs represent fragments aa55–553, aa55–333, aa55–253, and aa76–333, respectively. The aa55–553 fragment of Prox1 was also inserted in the BamHI/XbaI sites of pVP16, in frame to VP16-AD. Mutagenesis of one of or both I/LXXLL motifs in VP16-Prox155–553 or Flag-Prox1 was achieved by overlapping PCR with primers containing point mutations that change LRKLL (aa70–74) to ARKAL and ISQLL (aa93–97) to ASQAL, respectively. After PCR, Flag-Prox1-DM was inserted in the EcoRI/XbaI sites of pcDNA3 and each mutant Prox155–553 was subcloned in the BamHI/XbaI sites of pVP16. Flag-luciferase was generated by PCR using primers containing the coding sequence for the Flag tag and inserted in the EcoRI/XhoI sites of pcDNA3.
For GST fusion proteins, the aa186–495 and the aa34–141 fragments of hLRH-1 were amplified and inserted in the EcoRI site of the pGEX-3X vector, respectively (Amersham Pharmacia Biotech, Uppsala, Sweden). Mutagenesis of the helices in hLRH-1 LBD was achieved by overlapping PCR with primers containing point mutations (Fig. 3C). After PCR, each mutant hLRH-1186–495 was inserted in the EcoRI site of pGEX-3X.
PCR amplifications were performed with the high fidelity Pyrobest polymerase (TaKaRa, Dalian, China). Plasmids constructed using amplified fragments were verified by sequencing.
Yeast Two-Hybrid Screening
PROQUEST two-hybrid system (Invitrogen Life Technologies) was used to screen a PROQUEST human adult liver cDNA library according to the supplier’s protocol using pDBLeu-hLRH-1141–481 as a bait plasmid.
Cell Culture, Transfection, and Reporter Gene Analysis
COS-7 and human hepatoma cell lines HepG2 and Huh7 (ATCC, Manassas, VA) were grown in DMEM (Invitrogen Life Technologies) supplemented with 10% (vol/vol) fetal calf serum (Invitrogen Life Technologies) at 37 C and 5% CO2. The day before transfection, cells were plated onto 35-mm dishes at a density of 60–70% confluency. Transfection was carried out with the calcium-phosphate precipitation method (56). In general, 0.5 µg of a luciferase reporter and 0.5 µg of an expression plasmid were cotransfected along with 0.3 µg of a ß-galactosidase expression plasmid pCMV-lacZ (Promega) that served as an internal control to monitor cell viability and normalize transfection efficiencies among different transfections. When necessary, an appropriate amount of the empty vector (pM, pVP16, or pcDNA3) was added to ensure an equal quantity of DNA among different transfections. Forty-eight hours post transfection, cells were harvested and lysed in 1x reporter lysis buffer (Promega). Luciferase activities were determined with luciferase assay system (Promega). ß-Galactosidase activities were measured according to a standard colorimetric method (56). Luciferase activities of different transfections were normalized by ß-galactosidase activities. Each transfection was performed in duplicate dishes and repeated at least three times.
Western Blot
HepG2 cells were transfected with 2.5 µg of an expression plasmid for Flag-Prox1 or Flag-Prox1-DM along with 0.3 µg of pCMV-lacZ. Forty-eight hours post transfection, a small proportion of the cells were removed for the measurement of the ß-galactosidase activity (56), which was used to normalize the transfection efficiency. The remaining cells were lysed. To ensure equal loading, the amount of the cell lysate from each transfection that would be subjected to 8% SDS-PAGE was adjusted based on the ß-galactosidase activity. After electrophoresis, proteins were transferred onto nitrocellulose membrane (PROTRAN, Schleicher & Schuell GmbH, Dassel, Germany). Immunoblotting was carried out with a mouse anti-Flag monoclonal antibody M2 (1:1500 dilution, Sigma, St. Louis, MO), followed by a rabbit antimouse Ig/horseradish peroxidase (1:1000; Dako, Carpinteria, CA) as the secondary antibody. SDS-PAGE, protein transfer, and immunoblotting were performed according to standard methods (56). Peroxidase activities were detected by the ECL reaction with the Western blot luminol reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) according to the manufacturer’s instruction.
Protein Expression
GST fusion proteins were produced in Escherichia coli BL21(DE3) (Novagen, LaJolla, CA) cultures by induction with 0.1 mM isopropyl-ß-D-thiogalactoside for 4 h at 37 C. After centrifugation, bacterial pellets were resuspended in PBS supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and lysed by sonication. Cell debris was removed by centrifugation. Supernatant was incubated with 60 µl of the glutathione-Sepharose 4B bead (Amersham Pharmacia Biotech) for 1 h at 4 C with gentle agitation. After washing, the beads three times with PBS, GST fusion proteins were eluted at room temperature on a rotating wheel for 30 min with elution buffer [25 mM reduced glutathione, 50 mM Tris-HCl (pH 8.0), 0.1% Triton X-100]. Protein purity was checked on coomassie brilliant blue-stained 10% SDS-PAGE (56). Purified proteins were quantified with the standard Bradford method (Bio-Rad, Hercules, CA). The 6xHis-tagged hLRH-1244–495 protein was a generous gift from Prof. H. M. Wu (Shanghai, China).
Prox1 and hLRH-1 proteins were synthesized in vitro with the TNT Quick Coupled Transcription/translation System (Promega) in the presence of [35S] methionine (Amersham Pharmacia) according to the manufacturer’s protocol. In case different labeled proteins were used and compared in pull-down or binding assays, the relative quantity of each protein was estimated by running 1 µl of the reaction mix on 10% SDS-PAGE. After electrophoresis, the gel was dried and exposed to x-ray film (56). The relative quantity of each protein was estimated by density calculation of the corresponding band on scanned gel image using Totalab software.
GST Pull-Down Assay
Pull-down assays were performed with a purified GST fusion protein and an appropriate [35S]-labeled protein. Briefly, 2 µg of the GST fusion protein bound to glutathione-Sepharose beads were incubated with 10 µl of the labeled protein in 150 µl of binding buffer [200 mM KCl, 20 mM HEPES (pH 7.9), 0.1% Nonidet P-40 (NP-40), 5 mM MgCl2, 0.2% BSA, 10% glycerol, 1 mM PMSF] at 4 C for 1.5 h. Unbound and nonspecifically bound proteins were removed by washing five times with wash buffer [200 mM KCl, 20 mM HEPES (pH 7.9), 0.1% NP-40, 5 mM MgCl2, 1 mM PMSF]. Beads were resuspended in an equal volume of 2x sodium dodecyl sulfate (SDS) loading buffer (TaKaRa). Bound proteins were detached off the beads by boiling, and resolved on 10% SDS-PAGE. One microliter of the labeled protein (10% input) was run alongside on the gel. Gel was dried and exposed to x-ray film for 12–48 h.
EMSA
EMSA was performed with in vitro-translated full-length Prox1 and hLRH-1. Complementary oligonucleotides (5'-CTTAGTTCAAGGCCAGTTA-3', corresponding to –122 to –144 of human cyp7a1 promoter; only the sense strand is presented) containing a consensus hLRH-1 binding site (underlined) were annealed and [32P] end-labeled as probe. The reaction volume of EMSA is 20 µl. Before the addition of the probe, proteins were incubated on ice for 20 min in a binding buffer containing 10 mM HEPES (pH 7.6), 1 µg of poly(dI·dC) (Roche Molecular Biochemicals, Indianapolis, IN), 120 mM KCl, 7% glycerol, 1 mM dithiothreitol, and 5 mM MgCl2. After the addition of the probe (2 x 104 cpm), the mixture was further incubated at 16 C for 15 min. For competition assays, unlabeled wild-type or mutant probe (5'-CTTAGTTtccataCAGTTA-3', mutations in lowercase letters; only the sense strand is presented) was incubated with hLRH-1 on ice for 20 min before the addition of the labeled probe. Finally, the mixture was separated by electrophoresis on a 5% polyacrylamide gel at 8 V/cm gel length, with 0.5x TBE as the electrophoresis buffer (56). After electrophoresis, gel was dried and exposed to x-ray film. EMSA was repeated at least three times.
Preparation and Purification of Anti-LRH-1 Antibody
6xHis-tagged hLRH-1244–495 protein was used to immunize rabbits according to a standard protocol (57). To purify anti-LRH-1 antibody, GST-LRH-1186–495 was cross-linked onto glutathione-Sepharose 4B beads. In brief, GST-LRH-1186–495 bound beads were sequentially washed with 100 mM borate sodium (pH 8.0) at room temperature for 5 min and 200 mM triethanolamine (pH 8.2) at room temperature for 5 min. Then the beads were gently shaken in 200 mM triethanolamine (pH 8.2)/40 mM dimethylpimelimidate at room temperature for 1 h. After the cross-linking reaction, the beads were washed twice with 40 mM ethanolamine (pH 8.2) at room temperature for 30 min, 100 mM borate sodium (pH 8.0) at room temperature for 5 min, and 20 mM reduced glutathione/50 mM Tris-HCl (pH 8.0) at 4 C for 30 min. The antisera were loaded onto a column packed with GST-LRH-1186–495 cross-linked beads. The column was sequentially washed twice with PBS/2 M NaCl (pH 7.3) and PBS. The anti-LRH-1 antibody was eluted with 50 mM glycine (pH 2.5). The pH of the eluate was adjusted with 0.05 volume of 2 M Tris-HCl (pH 8.0) immediately after elution.
Coimmunoprecipitation
HepG2 cells were transiently transfected with the expression plasmids for Flag-Prox1 and hLRH-1 using the Fugene reagent (Roche) according to the manufacturer’s protocol (5 µg of plasmid per 100-mm dish). Forty-eight hours post transfection, cells were washed once with ice-cold PBS. Cells were lysed in 0.5 ml of a modified RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 0.03% SDS, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, and Roche cocktail inhibitors] at 4 C for 15 min. After centrifugation at 10,000 rpm for 10 min, the supernatant was diluted in 2 vol dilution buffer [24% glycerol, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, and Roche cocktail inhibitors]. The extracts were first cleared with the preimmune rabbit serum (sc-2027, Santa Cruz) and protein A-Sepharose for 1 h at 4 C. Then, 10 mg of protein were incubated overnight with 10 µg of the rabbit anti-hLRH-1 antibody or the preimmune rabbit serum and 25 µl of protein A-Sepharose at 4 C. After washing four times with 0.5 ml of wash buffer [8% glycerol, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, and Roche cocktail inhibitors), the pellets were resuspended in an equal volume of 2x SDS loading buffer (TaKaRa), boiled for 5 min, and separated on a 8% SDS-polyacrylamide gel. Proteins were transferred onto nitrocellulose membrane (PROTRAN) and immunoblotted with the mouse anti-Flag monoclonal antibody M2 (1:1000 dilution, Sigma). Immunoblotting of hLRH-1 or Flag-Prox1 in 500 µg of the starting protein (1/20 input) was performed with the rabbit anti-hLRH-1 antibody (1:400 dilution) or the mouse anti-Flag monoclonal antibody M2 (1:1000 dilution, Sigma), followed by the rabbit Ig (1:1000, Dako) or the rabbit antimouse Ig/horseradish peroxidase (1:1000, Dako) as the secondary antibody (56).
Immunofluorescence
HepG2 and COS-7 cells were plated on glass coverslips at 20–30% confluency the day before transfection. Cells were transfected using the calcium-phosphate precipitation method (55). HepG2 cells were transfected with the expression plasmid for GFP or GFP-Prox1, and COS-7 cells with the expression plasmid for GFP-Prox1 and hLRH-1. Forty-eight hours post transfection, cells were fixed with 3.7% paraformaldehyde in PBS for 20 min and permeabilized by treatment with 0.2% Triton X-100 in PBS for 15 min. After blocking with blocking buffer (3% BSA, 1% goat serum, 0.1% Triton X-100 in PBS) for 1 h, cells were incubated with the rabbit anti-hLRH-1 antibody (1:200) in blocking buffer for 5 h at room temperature, followed by staining with the Alexa Fluor 546 goat antirabbit IgG(H+L) (Molecular Probes, Eugene, OR). Nuclei were stained with the DNA-specific dye 4',6'-diamidino-2-phenylindole (DAPI) (2 µg/ml) (Sigma) for 5 min at room temperature. The stained samples were examined by confocal microscopy (Leica Microsystems GmbH, Mannheim, Germany).
RT-PCR
HepG2 cells seeded at 60–70% confluency on 35-mm dishes were transfected with 2 µg of an expression plasmid for Flag-Prox1, Flag-Prox1-DM, or Flag-luciferase using the Fugene reagent (Roche) according to the manufacturer’s instruction. Each transfection was repeated three times. Forty-eight hours post transfection, total RNA was isolated with TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s protocol. Reverse transcription (RT) was carried out using random priming method. Briefly, 2 µg of total RNA were mixed with 4 µl of 5x RT buffer (Promega), 1.25 µl of deoxynucleotide triphosphates (10 mM), 3 µl of random hexamers (50 µM) (Promega), 100 U of Moloney murine leukemia virus reverse transcriptase (Promega) and diethylpyrocarbonate water up to 20 µl. The reaction mixture without RT buffer and RT was first heated for 5 min at 65 C, followed by 1 min on ice. After adding the enzyme and the buffer, the mixture was sequentially incubated for 10 min at 25 C and 70 min at 42 C. After the reaction, the reverse transcriptase was inactivated at 70 C for 15 min. RT-PCR was performed using the following primers. Prox1: forward, 5'-CTACATTCAGATGGAGAAGTACG-3'; reverse, 5'-CTTCACTATCCA GCTTGCAG-3'. hLRH-1: forward, 5'-GGAGATAAAGTGTCTGGGTACCAT-3'; reverse, 5'-AACTATCCATATATGAATAGC-3'. ß-Actin: forward, 5-CAACTCCATCATGAAGTGT GACG-3'; reverse, 5'-ACTCGTCATACTCCTGCTTGC-3'. PCRs were carried out using rTaq DNA polymerase (TaKaRa) with the following condition: 94 C for 45 sec, 54 C for 45 sec, and 72 C for 50 sec for 30 cycles for hLRH-1, 26 cycles for Prox1, and 18 cycles for ß-actin. The amplified products were separated on 1% agarose gel in 1x Tris-acetate-EDTA buffer and visualized by ethidium-bromide staining (56).
Real-time PCR
Fluorescence real-time PCR was performed using SYBR Green PCR Core Reagent (Applied Biosystems, Foster City, CA) on ABI PRISM 7900 system (PerkinElmer Life and Analytical Sciences, Inc., Boston, MA). The reaction (10 µl in volume) contained 1 µl of 10x SYBR Green PCR buffer, 0.8 µl of deoxynucleotide triphosphates (10 mM), 0.1 µl of AmpErase uracil-N-glycosylase (1 U/µl), 0.05 µl of AmpliTaq Gold DNA Polymerase (5 U/µl), 1.2 µl of MgCl2 (25 mM), 0.1 µl of forward and reverse primers (20 µM), 1 µl of the first-strand cDNA and 5.65 µl of double distilled H2O. PCR was initiated at 50 C for 2 min (uracil-N-glycosylase incubation) and 95 C for 10 min (hot start), followed by 40 cycles with denaturing at 95 C for 30 sec, annealing at 59 C for 30 sec and extension at 72 C for 30 sec. After PCR, a dissociation curve (melting curve) was constructed in the range of 65–95 C. All amplifications and detections were carried out in a MicroAmp optical 384-well reaction plate with optical adhesive covers (Applied Biosystems). PCR was done in triplicate, and SD representing experimental errors were calculated. All data were analyzed by the ABI PRISM SDS 2.0 software optimized for the instrument (PerkinElmer). Relative mRNA expression levels were calculated by 
CT analysis, using ß-actin mRNA for internal normalization. The following primers were used. CYP7A1: forward, 5'-ACACCATTCCAGC GACTTTCTG-3'; reverse, 5'-AGGCACTGGAAAGCCTCAGC-3'. ß-Actin: forward, 5'-CA TCCTCACCCTGAAGTACCC-3'; reverse, 5'-AGCCSTGGAT AGCAACGTACATG-3'.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: aa, Amino acids; AD, activation domain; AF-2, activation function 2; CYP7A1, cholesterol 7--hydroxylase; DAPI, 4',6'-diamidino-2-phenylindole; DBD, DNA binding domain; DM, double mutant; FTZ-F1, fushi tarazu factor 1; GFP, green fluorescence protein; GST, glutathione-S-transferase; hLRH, human LRH; LBD, ligand binding domain; LRH-1, liver receptor homolog 1; Luc, luciferase; MBF, multiprotein bridging factor; mLRH, mouse LRH; NP-40, Nonidet P-40; PMSF, phenylmethylsulfonyl fluoride; Prox1, prospero-related homeobox; RT, reverse transcription; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor-1; SHP, short heterodimer partner; SRC, steroid receptor coactivator; VP16, herpes simplex virus VP16.
Received for publication January 9, 2004. Accepted for publication June 10, 2004.
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REFERENCES |
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