Document Detail

Intrahepatic cholestasis of pregnancy levels of sulfated progesterone metabolites inhibit farnesoid X receptor resulting in a cholestatic phenotype.
Jump to Full Text
MedLine Citation:
PMID:  22961653     Owner:  NLM     Status:  MEDLINE    
CONCLUSION: Our results reveal a novel molecular interaction between ICP-associated levels of the 3β-sulfated progesterone metabolite epiallopregnanolone sulfate and FXR that couples the endocrine component of pregnancy in ICP to abnormal bile acid homeostasis.
Shadi Abu-Hayyeh; Georgia Papacleovoulou; Anita Lövgren-Sandblom; Mehreen Tahir; Olayiwola Oduwole; Nurul Akmal Jamaludin; Sabiha Ravat; Vanya Nikolova; Jenny Chambers; Clare Selden; Myrddin Rees; Hanns-Ulrich Marschall; Malcolm G Parker; Catherine Williamson
Related Documents :
12069923 - Neutral lipids and phospholipids in scots pine (pinus sylvestris) sapwood and heartwood.
2817343 - Quantitative determination of phospholipids using the dyes victoria blue r and b.
14610653 - Isovaleric acid accumulation in odontocete melon during development.
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2013-01-08
Journal Detail:
Title:  Hepatology (Baltimore, Md.)     Volume:  57     ISSN:  1527-3350     ISO Abbreviation:  Hepatology     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-06     Completed Date:  2013-04-04     Revised Date:  2013-07-11    
Medline Journal Info:
Nlm Unique ID:  8302946     Medline TA:  Hepatology     Country:  United States    
Other Details:
Languages:  eng     Pagination:  716-26     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 American Association for the Study of Liver Diseases.
Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Bile Acids and Salts / metabolism*
Cholestasis / chemically induced
Cholestasis, Intrahepatic / metabolism*
Cholic Acid
Mice, Inbred C57BL
Pregnancy Complications / metabolism*
Pregnanolone / analogs & derivatives*,  blood
Progesterone / metabolism*
Receptors, Cytoplasmic and Nuclear / agonists,  antagonists & inhibitors*
Sulfuric Acid Esters / blood*
Grant Support
P30874//Wellcome Trust
Reg. No./Substance:
0/Bile Acids and Salts; 0/Receptors, Cytoplasmic and Nuclear; 0/Sulfuric Acid Esters; 0/epiallopregnanolone sulfate; 0/farnesoid X-activated receptor; 128-20-1/Pregnanolone; 57-83-0/Progesterone; 81-25-4/Cholic Acid

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Hepatology
Journal ID (iso-abbrev): Hepatology
Journal ID (publisher-id): hep
ISSN: 0270-9139
ISSN: 1527-3350
Publisher: Wiley Subscription Services, Inc., A Wiley Company, Hoboken
Article Information
Copyright © 2012 American Association for the Study of Liver Diseases
Received Day: 18 Month: 4 Year: 2012
Accepted Day: 27 Month: 8 Year: 2012
Print publication date: Month: 2 Year: 2013
Electronic publication date: Day: 08 Month: 1 Year: 2013
Volume: 57 Issue: 2
First Page: 716 Last Page: 726
PubMed Id: 22961653
ID: 3592994
DOI: 10.1002/hep.26055

Intrahepatic Cholestasis of Pregnancy Levels of Sulfated Progesterone Metabolites Inhibit Farnesoid X Receptor Resulting in a Cholestatic Phenotype
Shadi Abu-Hayyeh1
Georgia Papacleovoulou1
Anita Lövgren-Sandblom2
Mehreen Tahir1
Olayiwola Oduwole1
Nurul Akmal Jamaludin1
Sabiha Ravat1
Vanya Nikolova1
Jenny Chambers1
Clare Selden3
Myrddin Rees4
Hanns-Ulrich Marschall5
Malcolm G Parker1
Catherine Williamson1
1Institute of Reproductive and Developmental Biology, Dept. of Surgery and Cancer, Faculty of Medicine, Imperial College LondonLondon, UK
2Department of Clinical Chemistry, Karolinska University Hospital HuddingeStockholm, Sweden
3UCL Institute of Liver and Digestive Health, Royal Free Hospital Campus University College Medical SchoolLondon, UK
4North Hampshire Hospital National Health Service TrustBasingstoke, Hampshire, UK
5Institute of Medicine, Department of Internal Medicine, Sahlgrenska Academy, University of GothenburgGothenburg, Sweden
Correspondence: Address reprint requests to: Catherine Williamson, Maternal and Fetal Disease Group, Institute of Reproductive and Developmental Biology, Dept. of Surgery and Cancer, Faculty of Medicine, Imperial College London, Du Cane Rd., London W12 0NN, UK. E-mail:; fax: 44-20-7594-2154..
Potential conflict of interest: Nothing to report.
Supported by Action Medical Research Grant SP4005, Genesis Research Trust, Lauren Page Trust, Biomedical Research Centre at Imperial College Healthcare NHS Trust, and The Wellcome Trust Grant P30874.

Intrahepatic cholestasis of pregnancy (ICP) is the commonest pregnancy-specific liver disease. It typically affects 0.5%-1.5% of pregnant women in Europe and is 2-3 times more prevalent in women of Chilean and Asian origin.1 ICP presents in the second or third trimester of pregnancy with maternal pruritus, raised liver transaminases, and serum bile acids.1 It may be complicated by fetal hypoxia, spontaneous preterm labor, and intrauterine death.2,3 Genetic variation has been reported in bile acid homeostasis-related genes, including the hepatic bile acid receptor, farnesoid X receptor (FXR; NR1H4)4 and its target genes, the bile salt export pump (BSEP; ABCB11),5,6 MDR3 (ABCB4),7-10 and MRP2 (ABCC2).11 However, women with ICP are usually asymptomatic when they are not pregnant and the disease phenotype is unmasked by pregnancy in the majority of cases. The symptoms and biochemical features of ICP usually resolve after delivery of the fetus, although ∼ 15% of affected women also develop cholestasis when given exogenous estrogens12 or the combined oral contraceptive pill.13 Thus, it is likely that reproductive hormones play a role in the etiology of ICP.

3α-Sulfated progesterone metabolites are raised in the serum and urine of women with ICP compared to those with uncomplicated pregnancy.14-17 Maximal serum concentrations for 3α-monosulfated and -disulfated progesterone metabolites that are elevated in ICP have been shown to reach 4.5 μM and 12 μM, respectively.15 Furthermore, these metabolites have been shown to be raised before the onset of the disease.18 It has been demonstrated that sulfated progesterone metabolites impair hepatic bile acid transport. We have recently shown that two monosulfated progesterone metabolites, allopregnanolone sulfate (PM4S) and epiallopregnanolone sulfate (PM5S), reduce Na+-dependent and Na+-independent influx of the primary bile acid taurocholate into primary human hepatocytes.19 They were shown to competitively inhibit taurocholate uptake by the principal sinusoidal bile acid transporter, Na+-taurocholate cotransporting polypeptide (NTCP), in NTCP-transfected Xenopus laevis oocytes19 and to reduce the efflux of bile acids from X. laevis oocytes expressing BSEP.20 Although it has been demonstrated that sulfated progesterone metabolites directly impair biliary transport of bile acids, it has not been established whether they influence hepatic pathways of bile acid homeostasis.

The nuclear receptor FXR plays a central role in hepatic bile acid homeostasis. In the presence of raised hepatocyte bile acid levels, FXR heterodimerizes with the retinoid X receptor (RXR) and regulates bile flow by inducing the expression of the canalicular transporters that mediate efflux of bile acids (BSEP), organic anions (MRP2), and phosphatidylcholine (MDR3) into bile.21,22 Another important FXR target gene is the short heterodimer partner (SHP), which represses NTCP23 and enzymes in the bile acid synthetic pathway (CYP7A1, CYP7B1).24 Thus, FXR prevents the accumulation of bile acids in hepatocytes by regulating their uptake, synthesis, and export.

We have shown that FXR function is impaired in murine pregnancy,25 and therefore we hypothesize that the levels of sulfated progesterone metabolites found in ICP abrogate FXR function despite the presence of raised serum and hepatocyte bile acids. Here we demonstrate that levels of the 3β-sulfated progesterone metabolite, epiallopregnanolone sulfate (PM5S), are supraphysiologically raised in ICP. Moreover, we show that PM5S can exacerbate cholic acid-induced hypercholanemia in the mouse and inhibit the hepatic induction of FXR target genes, thus reducing the expression and function of BSEP, and providing an explanation for the development of maternal cholestasis in ICP.

Materials and Methods
Human Serum Samples

This study conformed to the 1975 Declaration of Helsinki guidelines and permission was obtained from the Ethics Committee of the Hammersmith Hospitals NHS Trust, London (97/5197 and 08/H0707/21). Blood was collected from 15 women with ICP and 12 controls with uncomplicated pregnancy (between 33-41 weeks) (see Supporting Table 1 for patient details). Diagnostic criteria and sample preparation were as described.19,26

Animals and Treatments

Studies were conducted in accordance with the UK Animals (Scientific Procedures) Act of 1986. The 7 to 8-week-old C57BL6 female mice were administered by oral gavage either 200 μL of 20% cyclodextrin (vehicle), 50 mg/kg of cholic acid, or 50 mg/kg of cholic acid and 500 mg/kg of PM5S twice daily for 2 days: day 1 at 5:30 PM and 10:30 PM, day 2 at 8:30 AM and 1:30 PM. On the second day, tissues were harvested at 4 PM after an 8-hour fast.

Methods for further biochemical, molecular, and in vitro cell-culture experiments are described in the Supporting Information.

Levels of the 3β-Sulfated Progesterone Metabolite Epiallopregnanolone Sulfate Are Raised in ICP

We have previously shown that the levels of the sulfated progesterone metabolite epiallopregnanolone sulfate (PM5S) are increased in normal pregnancy relative to nonpregnant women.19 To investigate whether the levels of PM5S are further raised in ICP patients, UPLC/MSMS was used to assay PM5S serum concentrations in pregnant women with ICP or uncomplicated pregnancy (33-41 weeks). Serum from control and ICP cases had mean concentrations of PM5S of 6.3 μM and 21 μM, respectively (Fig. 1), corresponding to a significant 330% increase in PM5S levels in ICP. This result demonstrates for the first time that a 3β-sulfated progesterone metabolite is supraphysiologically raised in ICP at concentrations greater than those reported for the 3α-sulfated progesterone metabolites.15,18

Raised Serum Bile Acids in PM5S-Challenged Mice

To investigate whether PM5S can interfere with bile acid homeostasis, we studied the impact of PM5S on the ability of a mouse to metabolize cholic acid by comparing bile acid and gene expression levels in mice orally gavaged with either vehicle, cholic acid (CA), or CA and PM5S (CA+PM5S). Mice coadministered CA+PM5S had significantly raised serum PM5S (Table 1) and CA levels, and a trend for conjugated bile acids to be raised when compared to the vehicle or CA groups (Fig. 2A). Hepatic gene expression levels of Bsep, Mdr2, Ost-β, and Sult2a1 were significantly induced in the CA group (Fig. 2B). In contrast, induction of Bsep and Mdr2 expression was significantly abrogated and there was a trend for Ost-β and Sult2a1 expression to be reduced in the CA+PM5S group when compared to the CA group. There were no differences in Shp and Ntcp expression levels (Supporting Fig. 1). These data establish that PM5S can interfere with bile acid metabolism resulting in hypercholanemia and impaired induction of key hepatic bile acid-responsive genes consistent with cholestasis.

PM5S Inhibits FXR-mediated BSEP Expression and Function

We addressed the hypothesis that the increase in the levels of serum PM5S in ICP may abrogate FXR function by testing its effects on FXR-mediated bile acid transport. Huh7 cells were pretreated with the FXR agonist GW4064 ±PM5S for 24 hours, following which3H-taurocholic acid (3HTC) uptake and efflux were assessed. Intracellular3HTC levels did not vary significantly between all the treatment groups studied prior to the efflux assay (data not shown). However,3HTC efflux was significantly increased in GW4064-treated cells relative to the vehicle treated group, an increase that was abolished when cotreated with 25 or 50 μM PM5S (Fig. 3A). These data demonstrate that supraphysiological levels of PM5S, at concentrations similar to those observed in ICP, are capable of perturbing FXR-mediated bile acid efflux.

Bile acid efflux has been shown to be primarily mediated by BSEP.27 BSEP expression was investigated in Huh7 cells that were treated with GW4064 ±increasing doses of PM5S. GW4064 treatment resulted in a 1.5-fold increase in BSEP protein levels relative to vehicle-treated control cells. The addition of 10 μM PM5S inhibited the GW4064-mediated BSEP protein expression. Furthermore, GW4064 ±25-100 μM PM5S treatment completely abolished BSEP protein expression (Fig. 3B). Similarly GW4064-mediated BSEP gene expression was completely abolished by the addition of increasing PM5S doses, whereas PM5S significantly reduced CDCA-mediated BSEP expression at all concentrations tested, but without completely blunting the CDCA-induced component of expression (Fig. 3C). Importantly, the expression of BSEP was markedly reduced at both 10 and 25 μM PM5S, the levels observed in the majority of women with ICP, and therefore can account for, at least in part, the inhibition of GW4064-mediated bile acid efflux.

We investigated the expression of FGF19 in the Huh7 cells, as this has been shown to be an FXR target in human hepatocytes.28 GW4064 treatment resulted in a significant 2.8-fold increase in secreted FGF19. Cotreatment with 10 μM and 25 μM PM5S resulted in a trend towards a reduction in the levels of secreted FGF19, reaching significance with the addition of 50 μM and 100 μM PM5S (Fig. 3D). Similarly, the addition of 25-100 μM PM5S significantly reduced GW4064-mediated FGF19 gene expression in a dose-dependent manner (Fig. 3E). The expression of the FXR target genes, SHP and MDR3 was regulated in the same manner by PM5S, such that the GW4064-mediated SHP and MDR3 gene expression was significantly abrogated by PM5S in a dose-dependent manner (Fig. 3F). These data were confirmed in primary human hepatocytes with the exception of SHP (Supporting Fig. 2). A comparison of the cycle threshold values of quantitative polymerase chain reaction (qPCR) with those obtained from Huh7 cells reveal similar expression levels (Supporting Table 2).

We investigated whether PM5S exhibits any partial agonist activity by analyzing BSEP and SHP expression in primary human hepatocytes treated with PM5S or CDCA. BSEP expression was induced 7.5-fold by the FXR ligand CDCA, but it was also modestly increased by PM5S (Supporting Fig. 3A). PM5S was capable of inducing SHP expression, albeit to a lesser extent than CDCA (Supporting Fig. 3B). Thus, we conclude that PM5S is an antagonist of FXR with partial agonist activity.

Sulfated Progesterone Metabolites Are Competitive Inhibitors of Ligand-Dependent FXR Activation

To investigate the molecular mechanism by which PM5S inhibits FXR target gene expression, we used reporter assays to examine FXR activity in Huh7 cells in the presence of the FXR agonist CDCA ±PM5S. CDCA mediated a 3.5-fold increase in FXR transactivity (data not shown), which was abrogated by PM5S concentrations of ≥5 μM in a dose-dependent manner (Fig. 4A). This was confirmed using a BSEP-promoter-luciferase construct (Supporting Fig. 4). This result proved that FXR activity is sensitive to the actions of PM5S at concentrations found in ICP.

Given that ligand binding to the ligand-binding domain (LBD) of many nuclear receptors leads to the recruitment of transcriptional cofactors by means of LxxLL motifs,29 we established a cell-free homogeneous time-resolved fluorescence (HTRF) cofactor recruitment assay for the FXR-LBD. In this assay the fluorescence resonance energy transfer resulting from the binding of a fluorescently labeled LxxLL containing peptide derived from SRC-1 to a GST tagged-FXR-LBD was monitored in the presence of CDCA. The SRC-1 peptide was recruited to the FXR-LBD in a dose-dependent manner with an EC50 value of 9 μM and maximal normalized HTRF signal at 100 μM (data not shown), as previously reported.30 We next investigated the effect of PM5S on the recruitment of SRC-1 peptide achieved in the presence of CDCA. Based on the EC50 and maximal HTRF signal values in the presence of CDCA, we incubated GST-FXR-LBD with SRC-1 peptide in the presence of 9 μM or 100 μM of CDCA together with increasing concentrations of PM5S. We found that PM5S was able to inhibit the CDCA-mediated recruitment of the SRC-1 peptide to the FXR-LBD in a dose-dependent manner (Fig. 4B). Together, these data demonstrate that PM5S can directly antagonize FXR activity by competing with CDCA for the FXR-LBD and, as a consequence, inhibit the recruitment of cofactors possessing the LxxLL motif.

PM5S was able to transactivate FXR in a reporter assay in a dose-dependent manner, but was less efficacious than CDCA (Fig. 4C), generating an EC50 of >83 μM, compared to 4.6 μM for CDCA. Surprisingly, in contrast to CDCA, PM5S was unable to potentiate the recruitment of the SRC-1 peptide to the FXR-LBD in an HTRF cofactor peptide recruitment assay (Fig. 4D). These data establish that the progesterone metabolite PM5S functions as a partial agonist of FXR by competitively inhibiting ligand mediated activation. Thus, we conclude that the ability of PM5S to inhibit the recruitment of LxxLL peptides provides a mechanism for the antagonistic activity of PM5S but the molecular basis for the mild partial agonist activity has yet to be established.

3-Carbon β-Sulfated Progesterone Metabolites Are Modulators of FXR

To gain an insight into the structure-function relationship of sulfated progesterone metabolites and FXR, PM5S and a panel of progesterone-based compounds (Supporting Fig. 5) were assayed for their ability to activate FXR in a reporter assay. PM5S, epipregnanolone sulfate (EPS), and epiallo-pregnanediol 3-sodium sulfate (EPAS) were able to significantly transactivate FXR by 182%, 125%, and 183%, respectively, relative to vehicle-treated control (Fig. 5A). These data indicate that progesterone metabolites that have a sulfate group at the 3-carbon of the steroid ring in the β-position are able to function as FXR modulators. EPAS and EPS were able to transactivate FXR in a dose-dependent manner, generating EC50 values of 22.6 μM and 15.1 μM, respectively (Fig. 5A), which are lower than the value observed for PM5S. In an HTRF cofactor peptide recruitment assay, EPAS and EPS were unable to induce the recruitment of the SRC-1 peptide to the FXR-LBD (Fig. 5B), consistent with the results obtained for PM5S.

The antagonistic activity of EPAS and EPS was also examined in the FXR reporter assay and HTRF cofactor recruitment assay. In the presence of CDCA, both EPAS and EPS inhibited the activity of FXR (Fig. 5C) and the recruitment of the LxxLL SRC-1 peptide (Fig. 5D) in a dose-dependent manner, demonstrating antagonistic properties. These data demonstrate that FXR-LBD-mediated activity is sensitive to progesterone metabolites sulfated at the 3-carbon in the β-position and that these progesterone-based compounds can function as antagonists with weak agonist activity.

Modulation of Endogenous FXR Bile Acid Homeostasis Targets by PM5S

To confirm that the effects of the progesterone metabolites are mediated by FXR itself, we depleted primary human hepatocytes of FXR using small interfering RNA (siRNA). We were unable to test the FXR-dependent antagonistic effects of PM5S, as the depletion of FXR resulted in the complete blunting of the CDCA-mediated BSEP and SHP induction. We therefore examined the effects of 50 μM PM5S or CDCA on FXR target gene expression. FXR RNA levels were reduced by 80% compared with a nontargeting scrambled siRNA in all treatment groups (Fig. 6A). As expected, in the scrambled siRNA group, PM5S and CDCA significantly induced BSEP gene expression by 160% and 390%, respectively, but this induction was blunted in cells depleted of FXR to levels similar to those observed in the vehicle-treated group (Fig. 6B). Similarly, SHP gene expression was significantly induced in the siScrambled PM5S and CDCA-treated groups by 200% and 481% compared to the vehicle-treated control group but significantly blunted after FXR depletion (Fig. 6C). PM5S and CDCA treatment resulted in the repression of the indirect FXR target CYP7A1 expression by >90% in the siScramble group but was less marked following FXR depletion (Fig. 6D). Thus, PM5S influences FXR signaling directly and thereby modulates the expression of genes required for bile acid homeostasis.


Our results conclusively show that ICP is associated with supraphysiological levels of the 3β-sulfated progesterone metabolite epiallopregnanolone sulfate (PM5S) and demonstrate potential mechanisms to explain gestational hypercholanemia and cholestasis. The data also reveal a unique interaction between progesterone metabolites that are sulfated at the 3-carbon in the β-position, including PM5S at levels observed in ICP, and the hepatic bile acid nuclear receptor FXR, resulting in perturbed downstream FXR target gene expression and function. FXR plays a major role in the control of serum bile acid levels. It is activated by bile acids in an LBD-dependent manner31-33 and induces the expression of target genes, including BSEP, FGF19 and SHP, by binding to FXR-response elements.21,24,28 Reduced FXR function has been shown to be associated with high serum bile acid levels in FXR null mice,34 in pregnant wildtype mice,25 and in a subset of ICP patients with a mutation in the coding region of the FXR DNA-binding domain.4

We hypothesized that the cholestatic phenotype that presents as a defining feature of ICP may be due to impaired FXR function, resulting from the abnormally high levels of progesterone metabolites in ICP. Therefore, we investigated the impact of PM5S on FXR activity in the context of ICP.

It has been previously shown that 3α-sulfated progesterone metabolites are raised in ICP.15-18 In this report, we show for the first time that PM5S, a 3β-monosulfated progesterone metabolite, is significantly raised in women with ICP at concentrations greater than those of each individual sulfated progesterone metabolite species previously documented to be elevated.15,18 We induced hypercholanemia in mice, as normal murine pregnancy is hypercholanemic,25 and found that administering PM5S further increased serum bile acids. In parallel, cholic acid induction of bile acid-responsive genes was perturbed by PM5S, which is likely to explain the observed exacerbation of hypercholanemia and cholestasis. Furthermore, we demonstrate that PM5S found at levels associated with ICP can inhibit GW4064-mediated bile acid efflux by abrogating FXR induction of BSEP expression. PM5S inhibition of BSEP induction and function is likely to play a role in the pathogenesis of ICP, as BSEP activity is the rate-limiting step for hepatic bile acid clearance.35 Additionally, BSEP mutations have been reported in ICP cases and in individuals with the nongestational cholestatic conditions, progressive familial intrahepatic cholestasis and benign recurrent intrahepatic cholestasis type 2.5,6,36 These findings can also explain why cholestatic symptom severity in ICP is at its greatest in the third trimester, when progesterone levels are also at their highest.1 Mutations in MDR3 have been shown to be associated with ICP.9,10 Accordingly, we show that cholic acid/FXR-mediated Mdr2/MDR3 expression is impaired by PM5S. Additionally, FGF19 and SHP expression is perturbed by PM5S concentrations observed in our ICP patient cohort. The decrease in FGF19 expression together with the observed PM5S-mediated inhibition of FXR-induced SHP expression may result in elevated levels of CYP7A1, the rate-limiting enzyme in the bile acid biosynthetic pathway, as both SHP and FGF19 repress its expression.24,28 To date, there have been few reports of an endogenous non–bile acid ligand capable of inhibiting FXR activity. Several polyunsaturated fatty acid species have been shown to differentially modulate the downstream expression of FXR targets, enhancing and inhibiting agonist-induced BSEP and kininogen expression, respectively.37 We have demonstrated that bile acid-mediated FXR activity is reduced by sulfated progesterone metabolites, at levels consistent with those found in ICP, and that these compete with bile acids for the FXR-LBD, resulting in the inhibition of cofactor recruitment to the FXR-LBD. There are several coactivators that contain the LxxLL motif,38 which mediate the FXR signal to enhance transcription. Therefore, impaired recruitment of essential cofactors will in turn reduce FXR target gene expression. This mechanism could in part explain how the hormonal milieu during pregnancy unmasks the cholestatic phenotype of ICP in susceptible women with genetic variation in bile acid homeostasis genes such as ABCB11, ABCB4, and FXR.4-10

In common with many well-documented competitive inhibitors,30,39 we ascertained that sulfated progesterone metabolites that are able to competitively inhibit bile acid-mediated FXR activation also possess mild agonistic activity. A screen of progesterone-based compounds specifically revealed that those that possess a sulfate group at the 3-carbon in the β-position of the steroid ring are able to transactivate FXR in a dose-dependent manner. This is intriguing, as bile acids that modulate FXR have 3-carbon hydroxyl groups, whereas PM5S, EPAS, and EPS have 3-carbon sulfate groups. Moreover, it has been proposed that the human FXR-ligand binding pocket has evolved to accommodate bile salts that possess the cis conformation, i.e., the 5-carbon hydrogen in the β-position,40 generating an overall bent conformation. On the other hand, PM5S and EPAS possess a hydrogen at the 5-carbon in the α-position (trans), giving the overall steroid structure a planar conformation. The mode of interaction between the sulfated progesterone metabolites and the FXR-LBD may lead to the formation of an AF2 surface that is incapable of interacting with coactivators that rely on LxxLL motifs. Accordingly, sulfated progesterone metabolites identified as activators of FXR were unable to recruit the SRC-1 LxxLL motif to the FXR-LBD. Presumably this altered conformation does not prevent the recruitment of coactivators elsewhere on the receptor or alternatively is capable of binding to novel proteins/coactivators which may not normally be reported to interact with the AF2 domain. Similarly, polyunsaturated fatty acids and catechin, a tea-derived compound, were also shown to activate FXR, while unable to potentiate the recruitment of a peptide containing the LxxLL motif to the FXR-LBD.37,41 The data presented demonstrate that FXR is sensitive to sulfated progesterone metabolites with an atypical steroid backbone structure and sidechain group and that the interaction between the two is capable of perturbing bile acid homeostasis and thus contribute to the etiology of ICP.

It is not known why sulfated progesterone metabolite levels are found at supraphysiological concentrations in ICP, but it has been suggested that excretion of sulfated progesterone metabolites may be impaired due to aberrant hepatobiliary excretion and or dysregulated phase II metabolism of progesterone.17 Ursodeoxycholic acid is commonly used to treat ICP and in the majority of cases it ameliorates symptoms and in parallel reduces serum and urinary levels of sulfated progesterone metabolites and bile acids.42,43 This suggests that sulfated progesterone metabolites and bile acids share common ursodeoxycholic acid-mediated pathways of metabolism or excretion.

Women with ICP have an increased risk of developing gallstones.13 Given that women with ICP are hypercholesterolemic44 and that the type of gallstones associated with ICP are cholesterol-rich,45 it is conceivable that raised serum levels of sulfated progesterone metabolites enhance the formation of gallstones by disrupting cholesterol and bile acid metabolism pathways modulated by FXR. Consistent with this, it has been shown that FXR activation in the mouse liver ameliorates cholesterol gallstones.46

The fetal complications of ICP are more prevalent in pregnancies with higher maternal serum bile acid levels,3 and any therapeutic intervention that reduces maternal bile acids is likely to reduce the risk of preterm labor, fetal hypoxia, and intrauterine death in affected pregnancies. In this study, we identify PM5S, a progesterone metabolite, as being supraphysiologically raised in women with ICP. We also show that levels of PM5S found in ICP are capable of reducing the function of the bile acid nuclear receptor FXR and therefore impact the downstream FXR bile acid homeostasis target function. This has important ramifications for the etiology of ICP, as it pairs the endocrine environment of pregnancy to the dysregulation of bile acid homeostasis in susceptible women. This highlights a novel FXR interaction as being a potential therapeutic target for the treatment of cholestasis. By implication, this may also improve the fetal outcome in ICP pregnancies by reducing maternal serum bile acid levels.

We thank all of the women who donated samples for this study. We thank CisBio for their expert assistance in HTRF and Mr. Bilal for helpful discussion.

1. Geenes V,Williamson C. Intrahepatic cholestasis of pregnancyWorld J GastroenterolYear: 2009152049206619418576
2. Fisk NM,Storey GN. Fetal outcome in obstetric cholestasisBr J Obstet GynaecolYear: 198895113711433207643
3. Glantz A,Marschall HU,Mattsson LA. Intrahepatic cholestasis of pregnancy: relationships between bile acid levels and fetal complication ratesHEPATOLOGYYear: 20044046747415368452
4. Van Mil SW,Milona A,Dixon PH,Mullenbach R,Geenes VL,Chambers J,et al. Functional variants of the central bile acid sensor FXR identified in intrahepatic cholestasis of pregnancyGastroenterologyYear: 200713350751617681172
5. Pauli-Magnus C,Lang T,Meier Y,Zodan-Marin T,Jung D,Breymann C,et al. Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancyPharmacogeneticsYear: 2004149110215077010
6. Dixon PH,Van Mil SW,Chambers J,Strautnieks S,Thompson RJ,Lammert F,et al. Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancyGutYear: 20095853754418987030
7. de Vree JM,Jacquemin E,Sturm E,Cresteil D,Bosma PJ,Aten J,et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasisProc Natl Acad Sci U S AYear: 1998952822879419367
8. Jacquemin E,Cresteil D,Manouvrier S,Boute O,Hadchouel M. Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancyLancetYear: 19993532102119923886
9. Dixon PH,Weerasekera N,Linton KJ,Donaldson O,Chambers J,Egginton E,et al. Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein traffickingHum Mol GenetYear: 200091209121710767346
10. Mullenbach R,Linton KJ,Wiltshire S,Weerasekera N,Chambers J,Elias E,et al. ABCB4 gene sequence variation in women with intrahepatic cholestasis of pregnancyJ Med GenetYear: 200340e7012746424
11. Sookoian S,Castano G,Burgueno A,Gianotti TF,Pirola CJ. Association of the multidrug-resistance-associated protein gene (ABCC2) variants with intrahepatic cholestasis of pregnancyJ HepatolYear: 20084812513217997497
12. Kreek MJ,Weser E,Sleisenger MH,Jeffries GH. Idiopathic cholestasis of pregnancy. The response to challenge with the synthetic estrogen, ethinyl estradiolN Engl J MedYear: 1967277139113956081143
13. Williamson C,Hems LM,Goulis DG,Walker I,Chambers J,Donaldson O,et al. Clinical outcome in a series of cases of obstetric cholestasis identified via a patient support groupBJOGYear: 200411167668115198757
14. Sjovall K,Sjovall J. Serum bile acid levels in pregnancy with pruritus (bile acids and steroids 158)Clin Chim ActaYear: 1966132072115941738
15. Meng LJ,Reyes H,Palma J,Hernandez I,Ribalta J,Sjovall J. Profiles of bile acids and progesterone metabolites in the urine and serum of women with intrahepatic cholestasis of pregnancyJ HepatolYear: 1997273463579288610
16. Meng LJ,Reyes H,Axelson M,Palma J,Hernandez I,Ribalta J,et al. Progesterone metabolites and bile acids in serum of patients with intrahepatic cholestasis of pregnancy: effect of ursodeoxycholic acid therapyHEPATOLOGYYear: 199726157315799398000
17. Reyes H,Sjovall J. Bile acids and progesterone metabolites in intrahepatic cholestasis of pregnancyAnn MedYear: 2000329410610766400
18. Sjovall J,Sjovall K. Steroid sulphates in plasma from pregnant women with pruritus and elevated plasma bile acid levelsAnn Clin ResYear: 197023213375493462
19. Abu-Hayyeh S,Martinez-Becerra P,Sheikh Abdul Kadir SH,Selden C,Romero MR,Rees M,et al. Inhibition of Na+-taurocholate Co-transporting polypeptide-mediated bile acid transport by cholestatic sulfated progesterone metabolitesJ Biol ChemYear: 2010285165041651220177056
20. Vallejo M,Briz O,Serrano MA,Monte MJ,Marin JJ. Potential role of trans-inhibition of the bile salt export pump by progesterone metabolites in the etiopathogenesis of intrahepatic cholestasis of pregnancyJ HepatolYear: 2006441150115716458994
21. Ananthanarayanan M,Balasubramanian N,Makishima M,Mangelsdorf DJ,Suchy FJ. Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptorJ Biol ChemYear: 2001276288572886511387316
22. Huang L,Zhao A,Lew JL,Zhang T,Hrywna Y,Thompson JR,et al. Farnesoid X receptor activates transcription of the phospholipid pump MDR3J Biol ChemYear: 2003278510855109014527955
23. Denson LA,Sturm E,Echevarria W,Zimmerman TL,Makishima M,Mangelsdorf DJ,et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcpGastroenterologyYear: 200112114014711438503
24. Goodwin B,Jones SA,Price RR,Watson MA,McKee DD,Moore LB,et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesisMol CellYear: 2000651752611030332
25. Milona A,Owen BM,Cobbold JF,Willemsen EC,Cox IJ,Boudjelal M,et al. Raised hepatic bile acid concentrations during pregnancy in mice are associated with reduced farnesoid X receptor functionHEPATOLOGYYear: 2010521341134920842631
26. Mullenbach R,Bennett A,Tetlow N,Patel N,Hamilton G,Cheng F,et al. ATP8B1 mutations in British cases with intrahepatic cholestasis of pregnancyGutYear: 20055482983415888793
27. Kullak-Ublick GA,Beuers U,Paumgartner G. Hepatobiliary transportJ HepatolYear: 20003231810728790
28. Song KH,Li T,Owsley E,Strom S,Chiang JY. Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7alpha-hydroxylase gene expressionHEPATOLOGYYear: 20094929730519085950
29. Heery DM,Kalkhoven E,Hoare S,Parker MG. A signature motif in transcriptional co-activators mediates binding to nuclear receptorsNatureYear: 19973877337369192902
30. Yu J,Lo JL,Huang L,Zhao A,Metzger E,Adams A,et al. Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activityJ Biol ChemYear: 2002277314413144712052824
31. Makishima M,Okamoto AY,Repa JJ,Tu H,Learned RM,Luk A,et al. Identification of a nuclear receptor for bile acidsScienceYear: 19992841362136510334992
32. Parks DJ,Blanchard SG,Bledsoe RK,Chandra G,Consler TG,Kliewer SA,et al. Bile acids: natural ligands for an orphan nuclear receptorScienceYear: 19992841365136810334993
33. Wang H,Chen J,Hollister K,Sowers LC,Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BARMol CellYear: 1999354355310360171
34. Sinal CJ,Tohkin M,Miyata M,Ward JM,Lambert G,Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasisCellYear: 200010273174411030617
35. Gerloff T,Stieger B,Hagenbuch B,Madon J,Landmann L,Roth J,et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liverJ Biol ChemYear: 199827310046100509545351
36. Strautnieks SS,Bull LN,Knisely AS,Kocoshis SA,Dahl N,Arnell H,et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasisNat GenetYear: 1998202332389806540
37. Zhao A,Yu J,Lew JL,Huang L,Wright SD,Cui J. Polyunsaturated fatty acids are FXR ligands and differentially regulate expression of FXR targetsDNA Cell BiolYear: 20042351952615307955
38. Kemper JK. Regulation of FXR transcriptional activity in health and disease: emerging roles of FXR cofactors and post-translational modificationsBiochim Biophys ActaYear: 2011181284285021130162
39. Kaimal R,Song X,Yan B,King R,Deng R. Differential modulation of farnesoid X receptor signaling pathway by the thiazolidinedionesJ Pharmacol Exp TherYear: 200933012513419369578
40. Reschly EJ,Ai N,Ekins S,Welsh WJ,Hagey LR,Hofmann AF,et al. Evolution of the bile salt nuclear receptor FXR in vertebratesJ Lipid ResYear: 2008491577158718362391
41. Li G,Lin W,Araya JJ,Chen T,Timmermann BN,Guo GL. A tea catechin, epigallocatechin-3-gallate, is a unique modulator of the farnesoid X receptorToxicol Appl PharmacolYear: 201225826827422178739
42. Meng LJ,Reyes H,Palma J,Hernandez I,Ribalta J,Sjovall J. Effects of ursodeoxycholic acid on conjugated bile acids and progesterone metabolites in serum and urine of patients with intrahepatic cholestasis of pregnancyJ HepatolYear: 199727102910409453429
43. Glantz A,Reilly SJ,Benthin L,Lammert F,Mattsson LA,Marschall HU. Intrahepatic cholestasis of pregnancy: amelioration of pruritus by UDCA is associated with decreased progesterone disulphates in urineHEPATOLOGYYear: 20084754455117968976
44. Dann AT,Kenyon AP,Wierzbicki AS,Seed PT,Shennan AH,Tribe RM. Plasma lipid profiles of women with intrahepatic cholestasis of pregnancyObstet GynecolYear: 200610710611416394047
45. Lammert F,Marschall HU,Glantz A,Matern S. Intrahepatic cholestasis of pregnancy: molecular pathogenesis, diagnosis and managementJ HepatolYear: 2000331012102111131439
46. Moschetta A,Bookout AL,Mangelsdorf DJ. Prevention of cholesterol gallstone disease by FXR agonists in a mouse modelNat MedYear: 2004101352135815558057

BSEP bile salt export pump
CA cholic acid
CDCA chenodeoxycholic acid
FGF19 fibroblast growth factor 19
FXR farnesoid X receptor
ICP intrahepatic cholestasis of pregnancy
LBD ligand binding domain
MDR3/Mdr2 multidrug resistance protein 3/2
PM5S epiallopregnanolone sulfate
Supplementary material

Additional Supporting Information may be found in the online version of this article.


[Figure ID: fig01]
Fig. 1 

Levels of epiallopregnanolone-sulfate (PM5S) are supraphysiologically raised in ICP. Serum concentrations of PM5S in women with ICP and uncomplicated pregnancies at 33-38 weeks of gestation. Black line represents mean serum concentrations of PM5S. Measurements were carried out by UPLC/MSMS on a minimum of n = 12 samples. *P < 0.05 for control pregnant versus ICP serum samples as determined by Student's t test.

[Figure ID: fig02]
Fig. 2 

PM5S exacerbates hypercholanemia in the mouse. (A) UPLC/MSMS-derived serum bile acid concentrations represented in two graphs according to the range of their concentrations and (B) hepatic gene expression levels of Bsep, Mdr2, Ost-β, and Sult2a1 in mice gavaged with vehicle, cholic acid, or cholic acid and PM5S at four timepoints over 2 days. *P < 0.05 for vehicle or cholic acid-gavaged group versus cholic acid and PM5S co-gavaged group. #P < 0.05 for vehicle versus cholic acid-gavaged group as determined by one-way analysis of variance (ANOVA). Values represent mean ± standard error of the mean (SEM) of n = 6. CA, cholic acid; T-CA, taurocholic acid; G-CA, glycocholic acid; CDCA, chenodeoxycholic acid; T-CDCA, taurochenodeoxycholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; T-UDCA, tauroursodeoxycholic acid; T-LCA, taurolithocholic acid; T-αMCA, tauro-α muricholic acid; T-βMCA, tauro-β muricholic acid.

[Figure ID: fig03]
Fig. 3 

PM5S inhibits function and expression of FXR target genes. (A) Huh7 cells were treated overnight with vehicle or 0.5 μM GW4064 ± 25-50 μM PM5S, following which cells were washed for 1.5 hours and allowed to efflux3HTC for 5 minutes. (B) Representative western blot and graphical representation of normalized BSEP to GAPDH levels in Huh7 cells treated with vehicle or 0.5 μM GW4064 ± 0-100 μM PM5S for 24 hours. (C) Huh7 cells were treated with vehicle or 0.5 μM GW4064 / 50 μM CDCA ±0-100 μM PM5S for 24 hours, after which cells were analyzed with qPCR for relative BSEP gene expression. (D) GW4064-mediated secretion of FGF19 is inhibited by PM5S. Secreted FGF19 was assayed in cell culture media taken from Huh7 cells incubated with 0.5 μM GW4064 ±0-100 μM PM5S for 24 hours. (E) GW4064-mediated FGF19 gene expression is inhibited by PM5S. Experiments were performed as in (C). (F) GW4064-mediated SHP and MDR3 gene expression is inhibited by PM5S. Experiments were performed as in (C). *P < 0.05 for 0.5 μM GW4064/50 μM CDCA versus vehicle control. #P < 0.05 for 0.5 μM GW4064/50 μM CDCA versus cotreatment group as determined by one-way ANOVA. Values represent mean ± SEM of n = 3.

[Figure ID: fig04]
Fig. 4 

PM5S reduces ligand-mediated FXR activity. (A) Huh7 cells were transfected with the human expression constructs RXR, FXRα2 / empty vector, FXR-luciferase reporter and renilla construct for 24 hours. Transfected cells were cotreated with 100 μM CDCA and increasing doses of PM5S. RLU, relative light units. n = 3 ± SEM. (B) PM5S inhibits ligand activated FXR recruitment of SRC-1. GST-tagged FXR-LBD and biotinylated LxxLL SRC-1 peptide incubated in the presence of 9 or 100 μM CDCA and 0-600 μM PM5S and HTRF measured after 1 hour incubation at room temperature with orbital shaking. n = 3 ± standard deviation (SD). (C) PM5S exhibits dose-dependent mild FXR agonistic characteristics. Transfection experiments were performed as in (A). Huh7 cells were treated with 0-100 μM PM5S. RLU, relative light units. n = 3 ± SEM. (D) PM5S is unable to recruit the LxxLL motif to the FXR-LBD. Experiments were performed as in (B). HTRF was measured following a 1-hour incubation of HTRF mixture and increasing CDCA/PM5S doses. n = 3 ± SD.

[Figure ID: fig05]
Fig. 5 

3β-Sulfated progesterone-based compounds modulate FXR activity. (A) Huh7 cells were transfected with the human expression constructs for RXR, FXRα2 / empty vector, FXR-luciferase reporter, and renilla construct for 24 hours. Fifty μM of compound or 1 μM GW4064 was used to treat the transfected cells for 24 hours. *P < 0.05 for treatment group versus vehicle control. RLU, relative light units. n = 3 ± SEM. Dose response curves of 0-100 μM EPAS and EPS, which were identified in the compound screen. RLU, relative light units. n = 3 ± SEM. (B) EPAS and EPS are unable to recruit the LxxLL motif to the FXR-LBD. GST-FXR-LBD and biotinylated LxxLL SRC-1 peptide were incubated in the presence of increasing EPAS, EPS, and CDCA concentrations and HTRF measured after 1-hour incubation at room temperature with orbital shaking. n = 3 ± SD. (C) Huh7 cells were transfected as in (A) and cotreated with 100 μM CDCA and increasing doses of EPAS and EPS. n = 3 ± SEM. (D) EPAS and EPS inhibit ligand-activated FXR recruitment of SRC-1. HTRF experiments were performed as in (B). HTRF reaction mix was incubated in the presence of 9 or 100 μM CDCA and 0-600 μM EPAS/EPS. n = 3 ± SD.

[Figure ID: fig06]
Fig. 6 

Depletion of FXR in primary human hepatocytes blunts the response to PM5S and CDCA. Primary human hepatocytes were transfected with siRNA against FXR (hatched bar) or scrambled siRNA (black bar) for 6 hours, following which cells were allowed to recover overnight and then treated with 50 μM PM5S or 50 μM CDCA for 24 hours. Relative messenger RNA (mRNA) expression levels are shown for the genes (A) FXR, (B) BSEP, (C) SHP, and (D) CYP7A1. *P < 0.05 for treatment group versus vehicle siScrambled vehicle control as determined by one-way ANOVA. Values represent mean ± SEM of n = 3.

[TableWrap ID: tbl1] Table 1 

PM5S Concentrations Observed in Mice Gavaged with Vehicle, Cholic Acid or Cholic Acid and PM5S

Mean Concentration (μM) ± SEM

Serum Compound Vehicle Cholic Acid Cholic Acid + PM5S
PM5S 4.9 ± 3.9 10.7 ± 9.6 3208.6 ± 1847.1*

*P < 0.05 for cholic acid versus cholic acid + PM5S, as determined by one-way ANOVA. Values represent mean ± SEM of n ≥ 6.

Article Categories:
  • Autoimmune, Cholestatic and Biliary Disease

Previous Document:  Unlike ?? T cells, ?? T cells, LTi cells and NKT cells do not require IRF4 for the production of IL-...
Next Document:  2,4,6-Trinitrotoluene: A Surprisingly Insensitive Energetic Fuel and Binder in Melt-Cast Decoy Flare...