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1Department of Pharmacology and
2Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Abstract
Bile acids are natural detergents that assist in the absorption and digestion of fats in the intestine. In liver, the synthesis of bile acids from cholesterol is regulated by multiple signaling cascades that repress transcription of the gene encoding cholesterol 7-hydroxylase (CYP7A1), the rate-limiting enzyme in the classic bile acid synthesis pathway. In this issue of the JCI, Ito and coworkers demonstrate that mice lacking ?Klotho, a membrane protein with 2 putative glycosidase domains, have increased Cyp7a1 mRNA levels and bile acid concentrations. ?Klotho-KO mice also have small gallbladders and are resistant to cholesterol gallstone formation. These findings highlight the central role of ?Klotho in bile acid homeostasis and raise the possibility that this protein could be a pharmacologic target for the treatment of gallstones.
See the related article beginning on page 2202
Bile acids are cholesterol metabolites that are synthesized in the liver, stored in the gallbladder, and released during a meal into the small intestine, where they are crucial for the absorption of lipophilic nutrients and vitamins (1). Despite their importance in normal physiology, bile acids are strong detergents whose concentrations must be tightly controlled. Dysregulation of bile acid homeostasis is associated with a range of pathophysiological disorders including cholestatic liver disease and cholesterol gallstone formation. In this issue of the JCI, Ito et al. make an interesting and unexpected link between the protein ?Klotho and the regulation of bile acid synthesis (2).
Feedback repression of bile acid synthesis
Cholesterol 7-hydroxylase (CYP7A1), which is encoded by the CYP7A1 gene, catalyzes the rate-limiting step in the conversion of cholesterol to bile acids (Figure 1). Bile acids act via a feedback mechanism to repress CYP7A1 transcription. Work from a number of laboratories has revealed the complexity of this regulation (1, 3). One pathway through which CYP7A1 is repressed involves the nuclear bile acid receptor farnesoid X receptor (FXR). Activation of FXR represses CYP7A1 indirectly through induction of the orphan nuclear receptor small heterodimer partner (SHP), which then binds to another orphan nuclear receptor, liver receptor homolog–1, in the CYP7A1 gene promoter to repress gene transcription. Mice lacking FXR or SHP have increased Cyp7a1 expression and a corresponding increase in bile acid synthesis (4-7). FXR also induces the expression of FGF19 in human hepatocytes, which represses CYP7A1, presumably in an autocrine or paracrine fashion (8). Mice lacking fibroblast growth factor receptor 4 (FGFR4), a receptor for FGF19, have increased Cyp7a1 mRNA levels and a corresponding increase in bile acid synthesis (8). Thus, FXR contributes to the repression of Cyp7a1 through multiple mechanisms.
Figure 1
Shown are signaling pathways that repress transcription of the gene encoding CYP7A1, the rate-limiting enzyme in bile acid synthesis. Established pathways for the repression of Cyp7a1 in mice are indicated by solid lines. These include JNK and the nuclear bile acid receptor FXR. Activation of FXR represses CYP7A1 by induction of the orphan nuclear receptor SHP and FGF19, which acts through FGFR4. Activation of the nuclear receptors pregnane X receptor (PXR) and PPAR by xenobiotics and fatty acids, respectively, also represses CYP7A1 through unknown mechanisms. Speculative pathways are shown as dotted lines. In this issue, Ito and colleagues show that mice lacking ?Klotho have increased Cyp7a1 expression and bile acid synthesis and are resistant to the formation of gallstones (2). The similar phenotypes of ?Klotho-KO and FGFR4-KO mice (8), including increased bile acid synthesis and small gallbladders, suggest that these proteins may act through a common pathway.
However, FXR-mediated suppression does not constitute the whole story when it comes to feedback regulation of CYP7A1. Treatment of SHP-KO and FGFR4-KO mice with pharmacologic doses of bile acids still results in repression of Cyp7a1, which reveals the presence of additional regulatory mechanisms (5, 6, 9). One of the FXR/SHP-independent pathways involves activation of liver Kupffer cells, which secrete inflammatory cytokines such as TNF- (10). These cytokines repress Cyp7a1 in hepatocytes through a signaling pathway that involves JNK (11). The nuclear receptors pregnane X receptor and PPAR, which are activated by xenobiotics and fatty acids, respectively, also repress Cyp7a1 through mechanisms that do not appear to involve FXR or SHP (3). Thus, multiple regulatory pathways converge to repress Cyp7a1 (Figure 1).
?Klotho regulates bile acid homeostasis
Klotho made a dramatic entrance several years ago when it was shown that mice with reduced levels of this protein have a premature aging phenotype that includes dysregulation of calcium and phosphorus homeostasis (12). Recently, a close relative, termed ?Klotho, was shown to be expressed in liver, pancreas, and fat (13). Both Klotho and ?Klotho are membrane proteins containing 2 regions in the extracellular domain with homology to those in family 1 glycosidases, which hydrolyze glycosidic bonds. To gain insight into ?Klotho’s function, Ito and colleagues disrupted the gene encoding ?Klotho in mice (2). Notably, ?Klotho-KO mice have pronounced alterations in bile acid metabolism, including marked increases in Cyp7a1 mRNA levels and a corresponding increase in bile acid excretion in the feces. These changes are consistent with disruption of the normal bile acid feedback regulatory loop (Figure 1).
It is not clear how ?Klotho contributes to the repression of Cyp7a1. Although bile acid–mediated induction of hepatic Shp mRNA was reduced in the ?Klotho-KO mice, there was no significant difference in basal Shp mRNA levels in ?Klotho KO and wild-type mice (2). These data suggest that ?Klotho-mediated repression of Cyp7a1 does not involve an increase in SHP concentrations. Studies with selective FXR agonists in the ?Klotho-KO mice are needed to clarify whether ?Klotho is required for FXR to repress Cyp7a1.
One intriguing possibility is that ?Klotho works in concert with FGFR4. Both are highly expressed in liver, and the ?Klotho-KO and FGFR4-KO mice have remarkably similar phenotypes, including elevated Cyp7a1 mRNA levels and bile acid synthesis as well as a small gallbladder (2, 8). Interestingly, heparin/heparan sulphate–like glycosaminoglycans serve as cofactors for FGF signaling through FGFRs, which raises the possibility that ?Klotho, with its 2 putative glycosidase domains, might regulate the concentrations or activity of a cofactor required for FGFR4 signaling.
Absence of ?Klotho protects against gallstones
What are the potential clinical implications of this work? Ito et al. (2) show that ?Klotho-KO mice are resistant to the formation of gallstones, which develop when the ratio of cholesterol to bile acids and phospholipids in the bile increases to the point that cholesterol precipitates (14). In mice, cholesterol gallstone formation can be induced by a lithogenic diet that includes high cholesterol and cholic acid concentrations (15). The authors suggest that the lack of gallstone formation in ?Klotho-KO mice could be due to the increase in hepatic bile acid synthesis, which may alter the cholesterol/bile acid ratio in bile to reduce gallstone formation. However, the lipid composition of the bile in the ?Klotho-KO mice was not tested directly nor were the hepatic protein and mRNA levels determined for bile salt export pump (BSEP; also known as ABCB11) and multidrug-resistance protein 2 (also known as ABCB4), which transport bile acids and phosphatidylcholine into the bile (16, 17). It is noteworthy that an increase in biliary bile acid secretion was not sufficient to prevent cholesterol gallstone formation in transgenic mice overexpressing the bile acid transporter BSEP (18). Thus, the resistance of ?Klotho-KO mice to gallstones is likely to be a consequence of more than just increased bile acid concentrations in the bile.
One possibility is that the small size of the gallbladder in the ?Klotho-KO mice reflects changes that contribute to the prevention of gallstone formation. An increase in gallbladder contraction and emptying would be expected to reduce the time that cholesterol has to precipitate and form stones in the bile (19). Indeed, gallbladder stasis is associated with gallstone formation in both humans and rodents (20, 21). It will be interesting to learn whether ?Klotho is present in the gallbladder and how it affects gallbladder function. Moreover, it will be interesting to see whether FGFR4-KO mice, which also have small gallbladders, are resistant to gallstones.
In summary, the work of Ito and colleagues (2) adds ?Klotho to the complex tapestry of signaling networks that regulate bile acid homeostasis. Precisely how ?Klotho integrates with these other regulatory pathways remains to be determined.
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