FXR-dependent Rubicon induction impairs autophagy in models of human cholestasis

Autophagy is a basic physiological response that enables cells to quickly react to stimuli threatening cellular homeostasis.²x[2]Galluzzi, L., Pietrocola, F., Levine, B., and Kroemer, G. Metabolic control of autophagy. Cell. 2014;
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Elevated hydrophobic bile acids, a hallmark of cholestatic liver diseases, lead to organelle stress and damage.⁴x[4]Woolbright, B.L. and Jaeschke, H. Novel insight into mechanisms of cholestatic liver injury. World J Gastroenterol. 2012;
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Crossref | PubMed | Scopus (104) | Google ScholarSee all References From a teleological point of view increasing autophagy would be an appropriate response to rebalance cell homeostasis in cholestatic conditions. However, findings in cholestatic mice are conflicting and overall point towards impaired autophagic responses. One study in bile duct-ligated PIZZ mice reported that after a prolonged duration of cholestasis, LC3-II is increased and accumulated misfolded alpha-1-antitrypsin is reduced, suggestive of induced autophagy.²³x[23]Khan, Z., Yokota, S., Ono, Y., Bell, A.W., Oertel, M., Stolz, D.B. et al. Bile duct ligation induces ATZ globule clearance in a mouse model of alpha-1 antitrypsin deficiency. Gene Expr. 2017;
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Crossref | PubMed | Scopus (5) | Google ScholarSee all References In contrast, several others found increased accumulation of ubiquitinated proteins and p62 in bile duct-ligated mice and concluded that autophagy and protein degradation was actually impaired in cholestasis.⁵x[5]Kwak, B.J., Choi, H.J., Kim, O.H., Kim, K.H., You, Y.K., Lee, T.Y. et al. The role of phospho-c-Jun N-terminal kinase expression on hepatocyte necrosis and autophagy in the cholestatic liver. J Surg Res. 2019;
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Crossref | PubMed | Scopus (6) | Google ScholarSee all References In addition, mice with defective autophagy (i.e. Atg7 and Atg5 knockout mice or chloroquine inhibition of autophagy) have increased cholestatic liver injury.²⁵x[25]Khambu, B., Li, T., Yan, S., Yu, C., Chen, X., Goheen, M. et al. Hepatic autophagy deficiency compromises FXR functionality and causes cholestatic injury. Hepatology. 2018;
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Crossref | PubMed | Scopus (18) | Google ScholarSee all References In addition to these conflicting results in mice, there are significant species differences regarding bile acid composition between cholestatic mice and men. In cholestatic mice, muricholic acids, which are FXR antagonistic,²⁷x[27]Sayin, S.I., Wahlstrom, A., Felin, J., Jantti, S., Marschall, H.U., Bamberg, K. et al. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist. Cell Metab. 2013;
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Abstract | Full Text | Full Text PDF | PubMed | Scopus (216) | Google ScholarSee all References In contrast, in human cholestasis CDCA dominates the human bile acid pool, but is a strong FXR agonist.³x[3]Fickert, P. and Wagner, M. Biliary bile acids in hepatobiliary injury – what is the link?. J Hepatol. 2017;
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Here we report that in human cholestasis, a spectrum of mostly chronic liver diseases that cause elevated bile acids, autophagy is likely to be pathophysiologically impaired. In vitro data suggest that impairment of autophagy is due to FXR-dependent induction of Rubicon, an inhibitor of autophago-lysosomal maturation (Fig. 6Fig. 6). In line with our in vivo findings in cholestatic liver samples, cholestatic concentrations of CDCA, which is a natural FXR agonist ligand and the major retained bile acid during chronic cholestasis, as well as pharmacological concentrations of the selective FXR ligand OCA, impair autophagy in vitro. We did not observe effects on the mTOR kinase pathway and our autophagy flux experiments suggested blockage of autophagy downstream of the vesicle initiation and elongation process. LC3/LAMP1 co-staining experiments pointed to defective autophago-lysosomal fusion at the end of the autophagic road.¹⁵x[15]Shen, H.M. and Mizushima, N. At the end of the autophagic road: an emerging understanding of lysosomal functions in autophagy. Trends Biochem Sci. 2014;
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We initially speculated that Rab7 might be involved in inhibition of autophago-lysosomal fusion, since bile acids, autophago-lysosomal fusion defects and Rab7 have been linked before.⁹x[9]Manley, S., Ni, H.M., Kong, B., Apte, U., Guo, G., and Ding, W.X. Suppression of autophagic flux by bile acids in hepatocytes. Toxicol Sci. 2014;
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Crossref | PubMed | Scopus (31) | Google ScholarSee all References However, we did not detect any differences in human Rab7 at the protein or mRNA levels (not shown). Instead, we found a clear FXR-dependent role for Rubicon in mediating the inhibiting effects of bile acids on autophagic flux. Rubicon is localized to lysosomes as well as to both autophagosomes, and endosomes and suppresses autophagy as well as endocytic trafficking by interacting with the UVRAG-Beclin1-PI3KC3 complex.¹⁸x[18]Matsunaga, K., Saitoh, T., Tabata, K., Omori, H., Satoh, T., Kurotori, N. et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat Cell Biol. 2009;
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Crossref | PubMed | Scopus (571) | Google ScholarSee all References It should be noted that autophagosomes not only directly fuse with lysosomes to build autophagolysosomes but may also first fuse with endosomes to form amphisomes which later eventually fuse with lysosomes.²⁹x[29]Lin, M.G. and Zhong, Q. Interaction between small GTPase Rab7 and PI3KC3 links autophagy and endocytosis: a new Rab7 effector protein sheds light on membrane trafficking pathways. Small GTPases. 2011;
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Rubicon is also significantly overexpressed in patients with non-alcoholic fatty liver disease.³¹x[31]Tanaka, S., Hikita, H., Tatsumi, T., Sakamori, R., Nozaki, Y., Sakane, S. et al. Rubicon inhibits autophagy and accelerates hepatocyte apoptosis and lipid accumulation in nonalcoholic fatty liver disease in mice. Hepatology. 2016;
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Crossref | PubMed | Scopus (72) | Google ScholarSee all References There, Rubicon overexpression not only impaired autophagic flux but was also associated with the detrimental effects of lipotoxicity. However, in that particular study post-translational events triggered by certain fatty acids were suggested as the basis for increased Rubicon expression.³¹x[31]Tanaka, S., Hikita, H., Tatsumi, T., Sakamori, R., Nozaki, Y., Sakane, S. et al. Rubicon inhibits autophagy and accelerates hepatocyte apoptosis and lipid accumulation in nonalcoholic fatty liver disease in mice. Hepatology. 2016;
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Crossref | PubMed | Scopus (72) | Google ScholarSee all References Most recently, Rubicon was linked to aging and shown to increase with age in worms, flies and mice. Knockout of Rubicon increased longevity and ameliorated age-associated phenotypes in mice.³²x[32]Nakamura, S., Oba, M., Suzuki, M., Takahashi, A., Yamamuro, T., Fujiwara, M. et al. Suppression of autophagic activity by Rubicon is a signature of aging. Nat Commun. 2019;
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Crossref | PubMed | Scopus (12) | Google ScholarSee all References Our experiments show that Rubicon knockout releases bile acid-induced impairment of autophagy processing and unleashes a massive counteractive autophagic (“adaptive”) response resulting in almost complete loss of accumulated P62 and LC3. Overall, we think that Rubicon is a relevant player in chronic metabolic liver diseases, including cholestasis, through its ability to tune autophagic responses; thus, it represents an interesting therapeutic target.

Treatment options for cholestatic liver diseases are limited. UDCA is the baseline treatment for many cholestatic diseases and is effective in halting disease progression in up to 65% of patients with primary biliary cholangitis. The exact mode of action of UDCA is not known but potential contributions include choleretic effects, modulation of bile acid pool composition and anti-apoptotic properties.³³x[33]Beuers, U. Drug insight: mechanisms and sites of action of ursodeoxycholic acid in cholestasis. Nat Clin Pract Gastroenterol Hepatol. 2006;
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Crossref | PubMed | Scopus (275) | Google ScholarSee all References Here, we add another potential mechanism of UDCA as an enhancer of autophagic flux. Since autophagy is impaired in cholestatic liver diseases, UDCA treatment to rebalance autophagic responses may contribute to its beneficial anti-cholestatic properties. Most importantly, since UDCA has only mild side-effects, its use as an autophagy enhancer for other liver diseases, where induction of autophagy is clinically appropriate (e.g. alpha-1-antitrypsin deficiency), may be therapeutically very relevant.³⁴x[34]Lykavieris, P., Ducot, B., Lachaux, A., Dabadie, A., Broue, P., Sarles, J. et al. Liver disease associated with ZZ alpha1-antitrypsin deficiency and ursodeoxycholic acid therapy in children. J Pediatr Gastroenterol Nutr. 2008;
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Crossref | PubMed | Scopus (22) | Google ScholarSee all References Currently, we do not understand how exactly UDCA stimulates autophagy. UDCA is regarded to act as an FXR antagonist,²¹x[21]Mueller, M., Thorell, A., Claudel, T., Jha, P., Koefeler, H., Lackner, C. et al. Ursodeoxycholic acid exerts farnesoid X receptor-antagonistic effects on bile acid and lipid metabolism in morbid obesity. J Hepatol. 2015;
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Abstract | Full Text | Full Text PDF | PubMed | Scopus (91) | Google ScholarSee all References which would be consistent with our observation that UDCA decreases Rubicon.

OCA is a second-line option for patients with primary biliary cholangitis, who do not respond or tolerate UDCA.³⁵x[35]Nevens, F., Andreone, P., Mazzella, G., Strasser, S.I., Bowlus, C., Invernizzi, P. et al. A placebo-controlled trial of obeticholic acid in primary biliary cholangitis. N Engl J Med. 2016;
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Crossref | PubMed | Scopus (334) | Google ScholarSee all References OCA is a potent semi-synthetic FXR agonist with several anti-cholestatic qualities such as repression of endogenous bile acid synthesis and modulation of hepatocellular bile acid transporter systems.³⁶x[36]Wagner, M., Zollner, G., and Trauner, M. Nuclear receptors in liver disease. Hepatology. 2011;
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Crossref | PubMed | Scopus (172) | Google ScholarSee all References We found that OCA impairs autophagic flux in vitro and also shows a similar trend in vivo. In context with the rodent data, which show that cholestasis progresses when autophagy is blocked,²⁵x[25]Khambu, B., Li, T., Yan, S., Yu, C., Chen, X., Goheen, M. et al. Hepatic autophagy deficiency compromises FXR functionality and causes cholestatic injury. Hepatology. 2018;
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Crossref | Scopus (8) | Google ScholarSee all References^,26x[26]Gao, L., Lv, G., Guo, X., Jing, Y., Han, Z., Zhang, S. et al. Activation of autophagy protects against cholestasis-induced hepatic injury. Cell Biosci. 2014;
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Crossref | PubMed | Scopus (18) | Google ScholarSee all References our findings would argue against OCA in the treatment of cholestasis. However, the multiple anti-cholestatic properties of OCA on bile acid production are apparently potent enough and overcome the potential negative effects of reduced autophagy.³⁵x[35]Nevens, F., Andreone, P., Mazzella, G., Strasser, S.I., Bowlus, C., Invernizzi, P. et al. A placebo-controlled trial of obeticholic acid in primary biliary cholangitis. N Engl J Med. 2016;
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In summary, we provide evidence that impaired autophagic processing is a common feature in models of human cholestasis and therefore may represent a common novel drug target in cholestatic liver diseases. In fact, stimulation of autophagy with the mTOR inhibitor rapamycin reduces injury in bile duct-ligated mice.²⁶x[26]Gao, L., Lv, G., Guo, X., Jing, Y., Han, Z., Zhang, S. et al. Activation of autophagy protects against cholestasis-induced hepatic injury. Cell Biosci. 2014;
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Crossref | PubMed | Scopus (18) | Google ScholarSee all References Mechanistically, we show that bile acids prevent proper fusion of autophagosomes with lysosomes in an FXR-dependent manner via induction of Rubicon. Since Rubicon is also generally involved in endosomal transport, we cannot exclude additional broader effects on vesicle trafficking. Conceptually, this study also suggests that Rubicon could be a potential new molecular target in the treatment of cholestatic liver diseases. UDCA, which has been established in the treatment of various liver diseases for more than forty years, has significant pro-autophagic effects along with reduction of Rubicon, which may add to the beneficial anti-cholestatic effects of this drug. However, more specific and more potent small molecule inhibitors of Rubicon, which are not yet available, could be an interesting future approach in cholestasis.