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An Essential Role for Liver ERα in Coupling Hepatic Metabolism to the Reproductive Cycle.

Della Torre S, Mitro N, Fontana R, Gomaraschi M, Favari E, Recordati C, Lolli F, Quagliarini F, Meda C, Ohlsson C, Crestani M, Uhlenhaut NH, Calabresi L, Maggi A - Cell Rep (2016)

Bottom Line: We show that this receptor regulates the synthesis of cholesterol transport proteins, enzymes for lipoprotein remodeling, and receptors for cholesterol uptake.Additionally, ERα is indispensable during proestrus for the generation of high-density lipoproteins efficient in eliciting cholesterol efflux from macrophages.We propose that a specific interaction with liver X receptor α (LXRα) mediates the broad effects of ERα on the hepatic lipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy; Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133 Milan, Italy.

No MeSH data available.


Related in: MedlinePlus

ERα and LXRα Functional Interaction in Liver(A) Recruitment of ERα (upper) and LXRα (lower) by conventional ChIP followed by qPCR. ChIP was done using ERα antibody, LXRα antibody, or normal rabbit IgG as negative control. After reverse cross-link, the purified ChIP-enriched fragments were amplified using qPCR, with primers that target the selected regions (see Supplemental Experimental Procedures). For each gene, the recruitment of ERα or LXRα is expressed as ratio of the fold enrichment relative to occupancy in IgG-precipitated samples versus the FE of the negative control FoxL2: an exonic region not bound by any nuclear receptor in this cell type.(B) ERα-LXRα cross-coupling over the course of the mouse reproductive cycle. In P, the high concentration of circulating estrogens enhances ERα binding to DNA, thereby promoting LXRα binding and transcriptional activity. In M, circulating estrogens are low, ERα binding to DNA is loosened, and LXRα binding to DNA and transcriptional activity are reduced.The data indicate mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus SYN at P.
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fig6: ERα and LXRα Functional Interaction in Liver(A) Recruitment of ERα (upper) and LXRα (lower) by conventional ChIP followed by qPCR. ChIP was done using ERα antibody, LXRα antibody, or normal rabbit IgG as negative control. After reverse cross-link, the purified ChIP-enriched fragments were amplified using qPCR, with primers that target the selected regions (see Supplemental Experimental Procedures). For each gene, the recruitment of ERα or LXRα is expressed as ratio of the fold enrichment relative to occupancy in IgG-precipitated samples versus the FE of the negative control FoxL2: an exonic region not bound by any nuclear receptor in this cell type.(B) ERα-LXRα cross-coupling over the course of the mouse reproductive cycle. In P, the high concentration of circulating estrogens enhances ERα binding to DNA, thereby promoting LXRα binding and transcriptional activity. In M, circulating estrogens are low, ERα binding to DNA is loosened, and LXRα binding to DNA and transcriptional activity are reduced.The data indicate mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus SYN at P.

Mentions: Next, we investigated the hypothesis that ERα-dependent modulation of LXRα transcriptional activity was due to a competition between ERα and LXRα for common co-regulators. The mutual interference of ERα and LXRα in the recruitment of common co-activators was studied using fluorescence resonance energy transfer (FRET; Figures 5E and 5F). In this assay, we observed that the addition of increasing amounts of ERα protein progressively augmented the competition with LXRα for steroid receptor coactivator 1 (SRC-1), nuclear receptor interacting protein (RIP140), transcriptional intermediary factor 2 (TIF2), and thyroid hormone receptor-associated protein (TRAP220). No effect was seen in the recruitment of PPARγ coactivator 1-alpha (PGC-1α) and transcriptional regulator CBP (CBP) by LXRα. No differences in co-activator recruitment were observed in the presence of vehicle (DMSO) or E2 (5 nM), which indicates that the competition was ERα dependent but ligand independent. This further confirmed the antagonist activity of unliganded ERα reported in Figures 5A and 5C. To better evaluate the physiological significance of the two assays, we measured the relative concentrations of ERα and LXRα in the liver by qPCR; we found the results to be 1:13 at P and 1:15 at E, as the concentrations of the two mRNAs change across the estrous cycle. In the transfection and FRET assays, ERα interference was observed with a stoichiometry of at least 1:5, indicating that in the in vitro assays, the proportion between ERα and LXRα was significantly different than in liver. This led us to verify the potential for interaction of the two receptors on the promoter of the genes responsive to the presence/absence of ERα. Prior studies in liver on chromatin immunoprecipitation (ChIP) with ERα (Gao et al., 2008) and LXRα (Boergesen et al., 2012) supported the idea of a cross-coupling between the two hepatic receptors, showing that they recognized overlapping sites (see Figure S7) in the promoter/enhancer of several genes of lipid metabolism. Therefore, we carried out a series of ChIP-qPCR studies on female livers harvested at each phase of the estrous cycle. Figure 6 shows that ERα and LXRα recognize and bind the same regions of chromatin and that the reproductive cycle has a significant influence on the extent to which both receptors interact with the promoter/enhancer of several of the genes relevant for CH metabolism. Most important is the finding that, in general, ERα maximal binding to the promoter/enhancer of the genes studied occurs at P, when estrogen production is highest; the observation that the target DNA co-precipitated with ERα at E shows that the receptor association with these promoters persists in time. For most of the promoters/enhancers studied, LXRα binding was generally higher at P, thus indicating the potential for a positive co-operation between the two hormonally regulated transcription factors. These observations led us to propose that, at the end of the follicular phase, during P, the high levels of estrogens promote the binding of ERα to the DNA in regions proximal to LXR-binding sites facilitating the transcription of LXRα target genes. The decreased concentration of estrogens loosens ERα interactions with both the DNA and LXRα: the consequence is the decreased synthesis of mRNA observed at M (Figure 6D). Consistent with the expression studies, the association of ERα with the Srebp1-1c promoter did not change significantly with the cycle; quite unexpected, however, was the finding that the highest fluctuation of LXRα binding across the cycle was observed on the Srebp-1c promoter, in spite of the fact that the amount of Srebp-1c mRNA is not influenced by the cycle.


An Essential Role for Liver ERα in Coupling Hepatic Metabolism to the Reproductive Cycle.

Della Torre S, Mitro N, Fontana R, Gomaraschi M, Favari E, Recordati C, Lolli F, Quagliarini F, Meda C, Ohlsson C, Crestani M, Uhlenhaut NH, Calabresi L, Maggi A - Cell Rep (2016)

ERα and LXRα Functional Interaction in Liver(A) Recruitment of ERα (upper) and LXRα (lower) by conventional ChIP followed by qPCR. ChIP was done using ERα antibody, LXRα antibody, or normal rabbit IgG as negative control. After reverse cross-link, the purified ChIP-enriched fragments were amplified using qPCR, with primers that target the selected regions (see Supplemental Experimental Procedures). For each gene, the recruitment of ERα or LXRα is expressed as ratio of the fold enrichment relative to occupancy in IgG-precipitated samples versus the FE of the negative control FoxL2: an exonic region not bound by any nuclear receptor in this cell type.(B) ERα-LXRα cross-coupling over the course of the mouse reproductive cycle. In P, the high concentration of circulating estrogens enhances ERα binding to DNA, thereby promoting LXRα binding and transcriptional activity. In M, circulating estrogens are low, ERα binding to DNA is loosened, and LXRα binding to DNA and transcriptional activity are reduced.The data indicate mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus SYN at P.
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fig6: ERα and LXRα Functional Interaction in Liver(A) Recruitment of ERα (upper) and LXRα (lower) by conventional ChIP followed by qPCR. ChIP was done using ERα antibody, LXRα antibody, or normal rabbit IgG as negative control. After reverse cross-link, the purified ChIP-enriched fragments were amplified using qPCR, with primers that target the selected regions (see Supplemental Experimental Procedures). For each gene, the recruitment of ERα or LXRα is expressed as ratio of the fold enrichment relative to occupancy in IgG-precipitated samples versus the FE of the negative control FoxL2: an exonic region not bound by any nuclear receptor in this cell type.(B) ERα-LXRα cross-coupling over the course of the mouse reproductive cycle. In P, the high concentration of circulating estrogens enhances ERα binding to DNA, thereby promoting LXRα binding and transcriptional activity. In M, circulating estrogens are low, ERα binding to DNA is loosened, and LXRα binding to DNA and transcriptional activity are reduced.The data indicate mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus SYN at P.
Mentions: Next, we investigated the hypothesis that ERα-dependent modulation of LXRα transcriptional activity was due to a competition between ERα and LXRα for common co-regulators. The mutual interference of ERα and LXRα in the recruitment of common co-activators was studied using fluorescence resonance energy transfer (FRET; Figures 5E and 5F). In this assay, we observed that the addition of increasing amounts of ERα protein progressively augmented the competition with LXRα for steroid receptor coactivator 1 (SRC-1), nuclear receptor interacting protein (RIP140), transcriptional intermediary factor 2 (TIF2), and thyroid hormone receptor-associated protein (TRAP220). No effect was seen in the recruitment of PPARγ coactivator 1-alpha (PGC-1α) and transcriptional regulator CBP (CBP) by LXRα. No differences in co-activator recruitment were observed in the presence of vehicle (DMSO) or E2 (5 nM), which indicates that the competition was ERα dependent but ligand independent. This further confirmed the antagonist activity of unliganded ERα reported in Figures 5A and 5C. To better evaluate the physiological significance of the two assays, we measured the relative concentrations of ERα and LXRα in the liver by qPCR; we found the results to be 1:13 at P and 1:15 at E, as the concentrations of the two mRNAs change across the estrous cycle. In the transfection and FRET assays, ERα interference was observed with a stoichiometry of at least 1:5, indicating that in the in vitro assays, the proportion between ERα and LXRα was significantly different than in liver. This led us to verify the potential for interaction of the two receptors on the promoter of the genes responsive to the presence/absence of ERα. Prior studies in liver on chromatin immunoprecipitation (ChIP) with ERα (Gao et al., 2008) and LXRα (Boergesen et al., 2012) supported the idea of a cross-coupling between the two hepatic receptors, showing that they recognized overlapping sites (see Figure S7) in the promoter/enhancer of several genes of lipid metabolism. Therefore, we carried out a series of ChIP-qPCR studies on female livers harvested at each phase of the estrous cycle. Figure 6 shows that ERα and LXRα recognize and bind the same regions of chromatin and that the reproductive cycle has a significant influence on the extent to which both receptors interact with the promoter/enhancer of several of the genes relevant for CH metabolism. Most important is the finding that, in general, ERα maximal binding to the promoter/enhancer of the genes studied occurs at P, when estrogen production is highest; the observation that the target DNA co-precipitated with ERα at E shows that the receptor association with these promoters persists in time. For most of the promoters/enhancers studied, LXRα binding was generally higher at P, thus indicating the potential for a positive co-operation between the two hormonally regulated transcription factors. These observations led us to propose that, at the end of the follicular phase, during P, the high levels of estrogens promote the binding of ERα to the DNA in regions proximal to LXR-binding sites facilitating the transcription of LXRα target genes. The decreased concentration of estrogens loosens ERα interactions with both the DNA and LXRα: the consequence is the decreased synthesis of mRNA observed at M (Figure 6D). Consistent with the expression studies, the association of ERα with the Srebp1-1c promoter did not change significantly with the cycle; quite unexpected, however, was the finding that the highest fluctuation of LXRα binding across the cycle was observed on the Srebp-1c promoter, in spite of the fact that the amount of Srebp-1c mRNA is not influenced by the cycle.

Bottom Line: We show that this receptor regulates the synthesis of cholesterol transport proteins, enzymes for lipoprotein remodeling, and receptors for cholesterol uptake.Additionally, ERα is indispensable during proestrus for the generation of high-density lipoproteins efficient in eliciting cholesterol efflux from macrophages.We propose that a specific interaction with liver X receptor α (LXRα) mediates the broad effects of ERα on the hepatic lipid metabolism.

View Article: PubMed Central - PubMed

Affiliation: Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy; Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133 Milan, Italy.

No MeSH data available.


Related in: MedlinePlus