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Abstract

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We have generated a line of mutant mouse that lacks βKlotho, a protein that structurally resembles Klotho. The synthesis and excretion of bile acids were found to be dramatically elevated in these mutants, and the expression of 2 key bile acid synthase genes, cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1), was strongly upregulated. Nuclear receptor pathways and the enterohepatic circulation, which regulates bile acid synthesis, seemed to be largely intact; however, bile acid–dependent induction of the small heterodimer partner (SHP) NR0B2, a common negative regulator of Cyp7a1 and Cyp8b1, was significantly attenuated. The expression of Cyp7a1 and Cyp8b1 is known to be repressed by dietary bile acids via both SHP-dependent and -independent regulations. Interestingly, the suppression of Cyp7a1 expression by dietary bile acids was impaired, whereas that of Cyp8b1 expression was not substantially altered in βklotho−/− mice. Therefore, βKlotho may stand as a novel contributor to Cyp7a1-selective regulation. Additionally, βKlotho-knockout mice exhibit resistance to gallstone formation, which suggests the potential future clinical relevance of the βKlotho system.

Ito, S, Fujimori, T, Furuya, A, Satoh, J, Nabeshima, Y and Nabeshima, Yo-ichi. Impaired negative feedback suppression of bile acid synthesis in mice lacking βKlotho. Journal of Clinical Investigation 2005;115:2202–2208. (Reprinted with permission.)

Bile acids (BA) are considered key endobiotic molecules that perform multiple and crucial physiological functions. They are the main driving force for the generation of bile flow and biliary lipid secretion and assist in the absorption of dietary fat and fat-soluble vitamins.1 Moreover, BA may also act as signaling molecules by activating several nuclear receptors and regulating many pathways resulting in BA and cholesterol homeostasis. However, because of their inherent detergent and membrane disruptive properties, BA are intrinsically toxic and their intracellular accumulation may result in liver cell death. In fact, during conditions such as cholestasis BA-induced hepatotoxicity participates in both pathogenesis and perpetuation of liver injury.1 Therefore, hepatocellular BA levels are tightly maintained within a narrow concentration range. This task is accomplished through transcriptional control of genes involved in BA biosynthesis, detoxification, and transport. Several studies indicate that BA synthesis in particular is subjected to a fine and apparently redundant regulation by a variety of pathways.2 The findings by Ito et al.3 showing that βKlotho has a role as suppressor of BA synthesis in vivo adds a new and unexpected dimension to the list of regulators of BA metabolism.

βKlotho is a membrane protein with extensive similarity to Klotho, a protein that seems to function as an anti-aging hormone in mammals. This function is suggested by the phenotype of Klotho knockout mice that exhibit arteriosclerosis, neural degeneration, skin and gonadal atrophy and cognition impairment.4 Moreover, it has been shown recently that overexpression of Klotho extends life span in mice.5 Both βKlotho and Klotho have homology to family 1 glycosidases but little is known about the mechanisms by which both proteins act on distant targets. Whereas Klotho is expressed in brain, kidney, reproductive organs, pituitary gland, and parathyroid gland, βKlotho is predominantly expressed in liver and pancreas.3 In contrast to Klotho, no information on the function of βKlotho was available since its cloning 5 years ago.6 Ito and colleagues now show that disruption of the gene encoding βKlotho in mice results in marked increases in mRNA levels of cholesterol 7α-hydroxylase (CYP7A1), the first and rate-limiting enzyme in the BA biosynthetic pathway.2

One of the main mechanisms of controlling intracellular BA levels in the liver is through BA-mediated feedback regulation of BA synthesis, which is mainly achieved by downregulation of CYP7A1. This regulatory loop has turned out to be very complex (see Fig. 1) and seems to involve several nuclear receptors including the farnesoid x receptor (FXR, NR1H4), along with short heterodimer partner (SHP, NR0B2) and liver receptor-1 homolog (LRH-1, NR5A2). Once BAs reach the nucleus, they bind FXR, which dimerizes with RXR and activates SHP transcription. SHP in turn, inactivates LRH-1 and its binding to liver x receptor (LXR, NR1H3) and downregulates cyp7a1 transcription.2 Data showing increased BA synthesis in the fxr and shp-null mice are consistent with the central role of FXR in controlling BA homeostasis.7 FXR also induces FGF19 which has been shown to repress cyp7a1 in hepatocytes resulting in inhibition of BA biosynthesis.8 In addition, FGFR4, a receptor for FGF19, also participates in this pathway as fgfr4-null mice also show elevated BA levels in their sera.9 However, studies using shp10 and fgfr4-null mice have shown that BA can still suppress their own synthesis in these animals, suggesting that pathways other than SHP-mediated mechanisms are at play in BA-induced downregulation of cyp7a1. Among SHP-independent mechanisms of CYP7A1 repression that have been proposed are the activation of c-Jun NH2terminal kinases 1 and 2 (JNK1/2) and activation of two other nuclear receptors PXR and PPARα.2 Another regulatory step of BA biosynthesis is the transcriptional control of the enzyme sterol 12-hydroxylase (CYP8B1) that converts CDCA to CA and is strongly inhibited by BA. Regulation of this enzyme is important as it determines the hydrophobicity of bile thereby influencing cholesterol absorption. CYP8B1 gene promoter contains LRH-1 and HNF4 binding sites. SHP interaction with these transcription factors results in BA-induced repression of CYP8B1 transcription. Of note, in Cyp8b1-null mice feedback regulation of CYP7A1 is lost resulting in expansion of the bile acid pool and alterations in cholesterol metabolism.2

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Figure 1. Schematic representation of pathways involved in feedback inhibition of bile acid synthesis involving cyp7a1 and the role of βklotho: Feedback inhibition of bile acid production is primarily achieved by transcriptional control of CYP7A1. Bile acid activation of FXR represses cyp7a1 via increasing the expression of SHP, a non-DNA binding protein which then interacts with LRH-1, an obligate factor required for LXR induction of cyp7a1. FXR also induces FGF19 which represses cyp7a1 probably through FGFR4, a receptor for FGF19. “FXR/SHP-independent” mechanisms include the activation of JNK1/2 and activation of two other nuclear receptors PXR and PPARα. PXR interferes with HNF4a signaling by targeting its coactivator PGC-1α and PPARα and its agonists inhibit the CYP7A1 gene by inhibiting HNF4a production and activation of CYP7A1 transcription. βKlotho participates in cyp7a1 transcriptional control by as yet undefined mechanisms. Data from null mice indicate βklotho may participate in the FXR/SHP cascade. βklotho may also interfere with the FGF19 and FGFR4-mediated suppression of cyp7a1. Finally, βKlotho seems to control the expression of CYP8B1, the key enzyme involved in synthesis of CA from CDCA since βKlotho-null mice exhibit increased CYP8B1 levels. BSEP, Bile salt export pump; CYP7A1, Cholesterol 7 α-hydroxylase; FXR, Farnesoid X receptor; SHP, Small heterodimer partner; LRH-1, Liver receptor homolog; LXR, Liver X receptor; FGF19, Fibroblast growth factor 19; FGFR4, Fibroblast growth factor receptor 4; IBABP-Ileal bile acid binding protein; JNK, c-jun NH2-terminal kinases 1 and 2 (JNK1/2); PXR, Pregnane X receptor; HNF4α, Hepatocyte nuclear factor-α; PPAR-α, Peroxisome proliferator activating receptor alpha; PGC-1, Peroxisome proliferator activating receptor-coactivator-1; CYP8B1, sterol 12-α hydroxylase.

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The main finding of Ito et al. is that βklotho-null mice exhibit pronounced alterations of BA homeostasis characterized by elevated basal cyp7a1 and cyp8b1 mRNA levels leading to increased BA synthesis and fecal excretion. Moreover, an impaired negative feedback suppression of cyp7a1, but not cyp8b1, by dietary BA was also observed in the mutant mice. Although the authors assessed the expression levels of a number of genes in both basal conditions or under dietary challenge with BA, the precise underlying mechanisms of the impaired regulation of cyp7a1 remain elusive. Whereas basal levels shp mRNA were not different between wild-type and knockout mice, BA-induction of SHP was blunted in null mice indicating that βklotho may participate in the FXR/SHP cascade. It is also likely that βklotho may somehow participate in the FGF19 and FGFR4-mediated suppression of cyp7a1 since glycosaminoglycans have been shown to participate in FGF signaling via FGFR and βklotho resembles glycosidases in its structure. Further studies are needed to pinpoint how and if these proteins talk to each other. The potential roles of βklotho in controlling Cyp7a1 expression are depicted in the figure.

Another interesting observation of this study is that mice lacking βklotho are resistant to cholesterol gallstone formation when fed a lithogenic diet. Gallstones are formed when the ratio of cholesterol to BA and/or phospholipids is increased resulting in cholesterol supersaturation of bile. Since assessment of hepatic, fecal, and serum cholesterol levels as well as expression of the dimeric canalicular cholesterol transporters abcg5 and abcg8 showed no differences between null mice and control animals, resistance of the βklotho-null mice to develop gallstones was attributed to increased secretion of BA into bile. Although this indeed may be the case, the fact that the secretion of biliary lipids was not assessed in these studies prevent further interpretation of their data. In fact, the sole overexpression of the bile salt export pump (thus resulting in increased BA secretion) by itself failed to prevent gallstone formation in a recent study.11 It remains plausible that βklotho may influence biliary phospholipid output and BA-independent bile flow thus lowering the cholesterol saturation index by these complementary mechanisms on a lithogenic diet. Interestingly, βklotho–null mice have small gallbladders compared to wild-type under lithogenic diet, which may suggest a role for an enhanced gallbladder motility as a protective factor on gallstones. Finally, it may be of interest to examine the BA pool composition and dynamics of the enterohepatic circulation in βKlotho-knockout mice since a more hydrophilic and frequent cycling of BA pool may also confer protection to gallstone development.

In summary, this paper by Ito et al. adds βklotho as yet another regulator to the complex orchestration of feedback repression of BA biosynthesis. Although the modus operandi by which βklotho exerts its effect is unknown at the present time, these findings open new avenues of investigation to hepatobiliary researchers. The fact that βklotho may also play a role in lithogenesis suggests potential for development of candidate drugs aimed at inhibiting βklotho that might be beneficial to gallstone patients.

References

  1. Top of page
  2. Abstract
  3. References