LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.
ABSTRACT: Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.
Project description:This study describes a unique function of taurocholate in bile canalicular formation involving signaling through a cAMP-Epac-MEK-Rap1-LKB1-AMPK pathway. In rat hepatocyte sandwich cultures, polarization was manifested by sequential progression of bile canaliculi from small structures to a fully branched network. Taurocholate accelerated canalicular network formation and concomitantly increased cAMP, which were prevented by adenyl cyclase inhibitor. The cAMP-dependent PKA inhibitor did not prevent the taurocholate effect. In contrast, activation of Epac, another cAMP downstream kinase, accelerated canalicular network formation similar to the effect of taurocholate. Inhibition of Epac downstream targets, Rap1 and MEK, blocked the taurocholate effect. Taurocholate rapidly activated MEK, LKB1, and AMPK, which were prevented by inhibition of adenyl cyclase or MEK. Our previous study showed that activated-LKB1 and AMPK participate in canalicular network formation. Linkage between bile acid synthesis, hepatocyte polarization, and regulation of energy metabolism is likely important in normal hepatocyte development and disease.
Project description:<h4>Unlabelled</h4>Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus.<h4>Conclusion</h4>LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317-1329).
Project description:The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.
Project description:AMP-activated protein kinase (AMPK), a cellular metabolic sensor, is essential in energy regulation and metabolism. Hepatocyte polarization during liver development and regeneration parallels increased metabolism. The current study investigates the effects of AMPK and its upstream activator LKB1 on polarity and bile canalicular network formation and maintenance in collagen sandwich cultures of rat hepatocytes. Immunostaining for the apical protein ABCB1 and the tight junction marker occludin demonstrated that canalicular network formation is sequential and is associated with activation of AMPK and LKB1. AMPK and LKB1 activators accelerated canalicular network formation. Inhibition of AMPK or LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs blocked canalicular network formation. AICAR and 2-deoxyglucose, which activate AMPK, circumvented the inhibitory effect of kinase-dead LKB1 on canalicular formation, indicating that AMPK directly affects canalicular network formation. After the canalicular network was formed, inhibition of AMPK and LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs resulted in loss of canalicular network, indicating that AMPK and LKB1 also participate in network maintenance. In addition, activation of AMPK and LKB1 prevented low-Ca(2+)-mediated disruption of the canalicular network and tight junctions. These studies reveal that AMPK and its upstream kinase, LKB1, regulate canalicular network formation and maintenance.
Project description:UNLABELLED: Estradiol 17ß-D-glucuronide (E17G) induces acute cholestasis in rat with endocytic internalization of the canalicular transporters bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). Classical protein kinase C (cPKC) and PI3K pathways play complementary roles in E17G cholestasis. Since non-conjugated estradiol is capable of activating these pathways via estrogen receptor alpha (ER?), we assessed the participation of this receptor in the cholestatic manifestations of estradiol glucuronidated-metabolite E17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHC). In both models, E17G activated ER?. In PRL, E17G maximally decreased bile flow, and the excretions of dinitrophenyl-glutathione, and taurocholate (Abcc2 and Abcb11 substrates, respectively) by 60% approximately; preadministration of ICI 182,780 (ICI, ER? inhibitor) almost totally prevented these decreases. In IRHC, E17G decreased the canalicular vacuolar accumulation of cholyl-glycylamido-fluorescein (Abcb11 substrate) with an IC50 of 91±1 µM. ICI increased the IC50 to 184±1 µM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Abcc2 substrate, glutathione-methylfluorescein. ICI also completely prevented E17G-induced delocalization of Abcb11 and Abcc2 from the canalicular membrane, both in PRL and IRHC. The role of ER? in canalicular transporter internalization induced by E17G was confirmed in ER?-knocked-down hepatocytes cultured in collagen sandwich. In IRHC, the protection of ICI was additive to that produced by PI3K inhibitor wortmannin but not with that produced by cPKC inhibitor Gö6976, suggesting that ER? shared the signaling pathway of cPKC but not that of PI3K. Further analysis of ER? and cPKC activations induced by E17G, demonstrated that ICI did not affect cPKC activation whereas Gö6976 prevented that of ER?, indicating that cPKC activation precedes that of ER?. CONCLUSION: ER? is involved in the biliary secretory failure induced by E17G and its activation follows that of cPKC.
Project description:Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na(+)-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid transport individually or in combination. [(3)H]-Taurocholate served as the NTCP/BSEP probe substrate. Individually, cyclosporin A and rifampin decreased taurocholate in vitro biliary clearance (Cl(biliary)) and biliary excretion index (BEI) by more than 20% in rat SCH, suggesting that these drugs primarily inhibited canalicular efflux. In contrast, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, carbamazepine, pioglitazone, and troglitazone decreased the in vitro Cl(biliary) by more than 20% with no notable change in BEI, suggesting that these drugs primarily inhibited taurocholate uptake. Cassette dosing (n=2-4 compounds per cassette) in rat SCH yielded similar findings, and results in human SCH were consistent with rat SCH. In summary, cassette dosing in SCH is a useful in vitro approach to identify compounds that inhibit the hepatic uptake and/or excretion of bile acids, which may cause DILI.
Project description:Estrogen-induced cholestasis occurs in many women who are susceptible due to pregnancy or hormone replacement therapy for postmenopausal syndrome. 17?-Ethinylestradiol (EE), as a synthetic estrogen, has been widely used to study the underlying mechanisms of estrogen-induced cholestasis. Recent studies have also reported that liver kinase B1 (LKB1)-mediated activation of AMP-activated protein kinase (AMPK) plays a critical role in the regulation of canalicular network formation. However, the role of AMPK in EE-induced cholestasis remains to be determined. In this study, the effects of EE (1-100 µM) on AMPK activation and the expression of farnesoid X receptor (FXR) and hepatic bile acid transporters were examined in in vitro using 3D-cultured rat primary hepatocytes and in in vivo using rat cholestasis models. We also used specific chemical agonist and antagonist of AMPK, AMPK subunit-specific antibodies and lentiviral shRNAs for AMPK?1 and AMPK?2 to delineate the role of AMPK in EE-induced cholestasis and potential cellular mechanisms. We found that EE-induced phosphorylation of AMPK?1 via extracellular signal-regulated kinases-LKB1-mediated signaling pathways and subsequent nuclear translocation accounted for the down-regulation of FXR and bile acid transporters and disruption of bile acid homeostasis. Inhibition of AMPK activation using an AMPK antagonist Compound C (2 µM) or down-regulation of AMPK?1 using gene-specific shRNA attenuated EE-induced cholestasis both in in vitro and in in vivo. In conclusion, these results revealed that activation of cAMP-ERK-LKB1-AMPK?1 signaling pathway plays a critical role in EE-mediated dysregulation of the expression of FXR and bile acid transporters. AMPK?1 may represent an important therapeutic target for estrogen-induced cholestasis.
Project description:The bile salt export pump (BSEP, ABCB11) plays an essential role in the formation of bile. In hepatocytes, BSEP is localized within the apical (canalicular) membrane and a deficiency of canalicular BSEP function is associated with severe forms of cholestasis. Regulation of correct trafficking to the canalicular membrane and of activity is essential to ensure BSEP functionality and thus normal bile flow. However, little is known about the identity of interaction partners regulating function and localization of BSEP. In our study, interaction partners of BSEP were identified in a complementary approach: Firstly, BSEP interaction partners were co-immunoprecipitated from human liver samples and identified by mass spectrometry (MS). Secondly, a membrane yeast two-hybrid (MYTH) assay was used to determine protein interaction partners using a human liver cDNA library. A selection of interaction partners identified both by MYTH and MS were verified by in vitro interaction studies using purified proteins. By these complementary approaches, a set of ten novel BSEP interaction partners was identified. With the exception of radixin, all other interaction partners were integral or membrane-associated proteins including proteins of the early secretory pathway and the bile acyl-CoA synthetase, the second to last, ER-associated enzyme of bile salt synthesis.
Project description:In estradiol 17?-d-glucuronide (E17G)-induced cholestasis, the canalicular hepatocellular transporters bile salt export pump (Abcb11) and multidrug-resistance associated protein 2 (Abcc2) undergo endocytic internalization. cAMP stimulates the trafficking of transporter-containing vesicles to the apical membrane and is able to prevent internalization of these transporters in estrogen-induced cholestasis. Hepatocyte levels of cAMP are regulated by hormones such as glucagon and adrenaline (via the ?2 receptor). We analyzed the effects of glucagon and salbutamol (a ?2 adrenergic agonist) on function and localization of Abcb11 and Abcc2 in isolated rat hepatocyte couplets exposed to E17G and compared the mechanistic bases of their effects. Glucagon and salbutamol partially prevented the impairment in Abcb11 and Abcc2 transport capacity. E17G also induced endocytic internalization of Abcb11 and Abcc2, which partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained internalized and mainly colocalizing with Rab11a in the perinuclear region after incubation with glucagon. Glucagon prevention was dependent on cAMP-dependent protein kinase (PKA) and independent of exchange proteins activated directly by cAMP (Epac) and microtubules. In contrast, salbutamol prevention was PKA independent and Epac/MEK and microtubule dependent. Anticholestatic effects of glucagon and salbutamol were additive in nature. Our results show that increases in cAMP could activate different anticholestatic signaling pathways, depending on the hormonal mediator involved.
Project description:Hepatocytes polarize by forming functionally distinct sinusoidal (basolateral) and canalicular (apical) plasma membrane domains. Two distinct routes are used for delivery of membrane proteins to the canaliculus. Proteins having glycosylphosphatidylinositol anchors or single transmembrane domains are targeted to the sinusoidal plasma membrane from where they transcytose to the canalicular domain. In contrast, apical ATP-binding-cassette (ABC) transporters, which are required for energy-dependent biliary secretion of bile acids (ABCB11), phospholipids (ABCB4), and nonbile acid organic anions (ABCC2), lack initial residence in the basolateral plasma membrane and traffic directly from Golgi membranes to the canalicular membrane. While investigating mechanisms of apical targeting in WIF-B9 cells, a polarized hepatic epithelial cell line, we observed that rab11a is required for canalicular formation. Knockdown of rab11a or overexpression of the rab11a-GDP locked form prevented canalicular formation as did overexpression of the myosin Vb motorless tail domain. In WIF-B9 cells, which lack bile canaliculi, apical ABC transporters colocalized with transcytotic membrane proteins in rab11a-containing endosomes and, unlike the transcytotic markers, did not distribute to the plasma membrane. We propose that polarization of hepatocytes (i.e., canalicular biogenesis) requires recruitment of rab11a and myosin Vb to intracellular membranes that contain apical ABC transporters and transcytotic markers, permitting their targeting to the plasma membrane. In this model, polarization is initiated upon delivery of rab11a-myosin Vb-containing membranes to the surface, which causes plasma membrane at the site of delivery to differentiate into apical domain (bile canaliculus).