Cellular regulation of hepatic bile acid transport in health and cholestasis

Vectorial transport of solutes from the sinusoidal space to the canaliculus provides the osmotic driving force for bile formation and is accomplished by various transporters located at the basolateral and canalicular membrane of hepatocytes and cholangiocytes.1-7 The term “cholestasis” is used to describe conditions associated with decreased bile formation. It is thus easy to appreciate the paradigm that cholestasis results when the ability of the liver to transport solutes into the canaliculus is compromised. Our present understanding of the pathogenesis of cholestasis is based on studies to define the physiologic regulation of various transporters (Fig. 1) and their deregulation in experimental models of cholestasis and patients with cholestatic disorders.8-11 In addition, studies on the expression of transporters in cholestatic diseases have provided valuable information on the role of specific transporters in the pathogenesis of some of these disorders. It is becoming clear that multiple pathways are involved in the regulation of hepatocellular solute transport and, hence, bile formation. Transporters involved in bile formation: hepatic uptake of bile acid (BA), organic anions (OA), and organic cations (OC) is mediated primarily by Na+/taurocholate cotransporting polypeptide (NTCP), the family of organic anion transporting proteins (OATPs), and organic cation transporters (OCTs), respectively. Na+/H+ exchanger (NHE1) and Na+/HCO cotransporter (NBC) at the sinusoidal membrane of hepatocytes and basolateral membrane of cholangiocytes are involved in intracellular pH regulation and HCO excretion. NHE3 present on the apical membrane is involved in fluid absorption, and Na+-K+-2Cl− (not shown) may be involved in fluid secretion in cholangiocytes. Multi-drug resistance proteins (MRP3 and MRP1) mediate sinusoidal efflux of organic anions, including toxic bile acids, while MRP2 and BSEP (bile salt export pump) mediate canalicular excretion of conjugated organic anions and bile acids, respectively. MDR1 and MDR2 (multidrug resistance gene products) are involved in biliary excretion of organic cations and phospholipids, respectively. Chloride/bicarbonate exchange is mediated by anion exchanger (AE) at the canalicular, as well as apical membrane, of cholangiocytes. Cystic fibrosis transmembrane conductance regulators (CFTR) act as chloride channels and reabsorption of conjugated bile acid from the biliary tree is mediated via Na+-dependent bile acid transporter (IBAT). NTCP, Na+/taurocholate cotransporting polypeptide; BSEP, bile salt export pump; MRP, multi-drug resistance protein; STAT, signal transducers and activators of transcription; cAmp, cyclic AMP; PKC, protein kinase C; PI3K, phosphatidylinositol-3-kinase; MAPK, mitogen activated protein kinases; NRs, nuclear receptors; RXR, retinoid X receptor; FXR, farnesol C receptor; SHP, small heterodimer partner; RAR, retinoic acid receptor; HNF1α, hepatocyte nuclear factor; OATP, organic anion transporting protein; PXR, pregnane X receptor; SXR, steroid and xenobiotic receptor; AV, arginine vasopressin; ERK, extracellular signal-regulated kinase; PP, protein phosphatase. Transport proteins, like other proteins, are synthesized in the endoplasmic reticulum, processed in the Golgi complex, and are then translocated to their intended site of action, the basolateral membrane for Na+/taurocholate cotransporting polypeptide (NTCP) and canalicular membrane for bile salt export pump (BSEP), for example. A transporter has to be inserted into the membrane for it to transport its solute across that membrane. This is a complex regulated process that requires the participation of various signaling molecules along with vesicles and cytoskeletons. A breakdown in this regulated process can lead to a decreased amount or an absence of a transport protein at its intended site, resulting in decreased or no transport function and, hence, cholestasis. In addition, the transporter activity may be decreased directly, leading to cholestasis. Indeed, cholestasis is associated with downregulation of NTCP and multi-drug resistance protein (MRP)212, 13 and upregulation of MRP3,14, 15 with a relatively preserved expression of BSEP.12 Mutations in a gene encoding for a particular transporter may result in a lack of important transport function leading to cholestasis. For example, mutations of BSEP, MDR3, and MRP2 (cMOAT, canalicular multispecific organic anion transporter) are implicated in type 2 progressive familial intrahepatic cholestasis (PFIC2), PFIC3, and Dubin-Johnson syndrome, respectively.16-18 The result of a point mutation may lead to loss of transport activity and/or inability to translocate to the plasma membrane, as recently demonstrated for BSEP.19 Apart from genetic defects, regulation of transporters at the level of transcription, translation, and post-translational modifications may be altered by chemicals/disease processes leading to decreased bile formation and, hence, cholestasis. Progress has been made in our understanding of the transcriptional and post-translational regulations of various transporters and how these regulations may be altered in cholestasis. It would appear that post-translational changes are early events, while the transcriptional changes are delayed effects of cholestasis. It is becoming evident that nuclear receptors20, 21 and STAT,2 a member of the signal transducers and activators of transcription, play an important role in the transcriptional regulation of various transporters, while the post-translational regulation is mediated via classical second messengers. It is now well established that activation of hepatocyte cell surface receptors results in the formation of cyclic AMP (cAMP), cGMP, increases in cytosolic Ca2+, and activation of kinases, such as protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K), and mitogen activated protein kinases (MAPK) (Fig. 2). These second messengers and kinases are involved in various aspects of bile formation like solute transport and vesicular trafficking. It should be noted that these signal transduction pathways are complex and involve a cascade of factors/enzymes, the details of which are still being worked out. Of all the solutes in bile, bile acids are the major determinant of bile formation,1, 3 and transhepatic transport of bile acids is accomplished by specific transporters22 located at the sinusoidal and canalicular membranes (see Fig. 1). The following is a summary of more recent studies as they relate to the role of nuclear receptors and second messengers in the regulation of hepatic bile acid transport under physiologic and cholestatic conditions. Role of other transporters and cholangiocytes not discussed here can be found in other excellent reviews.3, 4, 22-24 Major signal transduction pathways involved in bile formation: Interaction of glucagons/epinephrine and arginine vasopressin/phenylephrine with their receptors leads to the activation of adenylyl cyclase (AC) and phospholipase C (PLC), respectively. AC converts ATP into cAMP and PLC hydrolyzes phosphoinositide(4,5)-bisphosphate (PIP2) into inositol(1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG). IP3 and cAMP, acting via PKA, increase [Ca2+]i by releasing Ca2+ from its cytoplasmic store, and DAG activates novel and conventional PKCs (nPKCs and cPKCs). Insulin and hepatocyte growth factor (HGF), acting via receptor tyrosine kinase (RTK), activate PI3K and MAPK pathways. TC and cAMP also activate the PI3K pathway and cell swelling and TUDC activates the PI3K and MAPK pathways. BAs, by binding to NRs, affect transcriptional regulation of transporter genes. Some of the effects mediated via these signaling pathways are listed in respective boxes. BA = bile acid, OA = organic anion, OC = organic cation, TUDC = tauroursodeoxycholate, NR = nuclear receptor. Expression of NTCP and OATP-C is downregulated in patients with cholestasis,25, 26 and nuclear receptors (NRs) are suggested to play a role in the mechanism of this downregulation. Nuclear receptors are involved in transcriptional regulation of organic anion transporters, bile acid synthesis, and other hepatic functions.20, 21 Nuclear receptors, by responding to intracellular changes in sensitive ligands, alter the expression of target genes. There are over 150 members of the NR family, and those shown to affect bile acid transporters are mentioned here. These NRs receptors (class II NR) need to form a heterodimer with retinoid X receptor (RXR) in order to bind to response elements in the promoter region of the target gene and to regulate the initiation of transcription. Farnesol X receptor (FXR), one of the RXR partner NRs, binds bile acids with high affinity. Studies with a cholic acid feeding model showed that FXR knockout mice, compared to wild-type mice, express low level of Bsep and are unable to downregulate Ntcp and cholesterol-7-hydroxylase expression. Thus, FXR acts as a sensor of intracellular bile acid levels and is involved in the coordinate regulation of bile acid uptake, synthesis, and expression. FXR has been suggested to affect Ntcp expression by a complex mechanism (Fig. 3) involving small heterodimer partner (SHP).20, 21 The expression of Ntcp is activated by retinoic acid receptor (RAR) as a heterodimer with RXR (RAR:RXR). Increased intracellular bile acid in cholestasis activates FXR response element in the SHP promoter leading to the expression of SHP, which in turn downregulates RXR:RAR activation of Ntcp. In addition, a recent study27 suggests that bile acid-induced suppression of hepatocyte nuclear factor (HNF1α), via activation of HNF4, is involved in cholestasis-induced downregulation of NTCP and OATP-C, both of which are transcriptionally activated by HNF1α. Bile acids can also suppress HNF1α via SHP. Thus, the down regulation of Ntcp may also be mediated via suppression of HNF1α. The down regulation of Ntcp in pregnancy and by endotoxin may also be due to suppression of HNF1α and RXR:RAR.28, 29 Proinflammatory cytokines have also been shown to suppress RXR and may be involved in the loss of RXR, RARα, and SHP in bile duct ligated rats.3 Although Bsep expression is decreased in FXR knockout mice and FXR has been shown to be a potent activator of the Bsep promoter,30 an increased bile acid flux, induced by bile acid feeding, decreases Ntcp expression and increases Bsep expression without affecting the level of FXR.31 Thus, additional factors beyond FXR may be involved in the transcriptional regulation of Bsep. Transcriptional regulation of organic anion transporters by nuclear receptors: BAs stimulate expression of BSEP and SHP by activating FXR response elements in the BSEP and SHP promoters. SHP, in turn interferes with RXR:RAR transactivation of NTCP promoter, resulting in decreased expression of NTCP. HNF1α transcriptionally activates NTCP and OATP-C. BA decreases NTCP and OATP-C expression by suppressing HNF1α via its inhibitory effect on HNF4α. SHP also inhibits HNF4α-mediated transactivation of the HNF1α promoter and autoregulates its own promoter by repressing LRH-1 (liver receptor homologue). Above mentioned transcriptional regulation of transporters is a consequence of cholestasis as the effects are initiated by an increase in the intracellular bile acid concentration. These effects are thus considered to be cellular responses initiated to decrease intracellular bile acid levels and thereby limit or minimize bile acid-induced cellular toxicity. Another nuclear receptor, like pregnane X receptor (PXR) in the rodent, and steroid and xenobiotic receptor (SXR) in the human, is an activator of OATP2, MDR1, and MRP2 genes. However, any role PXR/SXR may play in steroid-induced cholestasis has not been determined. Glucocorticoids, which upregulate intestinal bile acid transporter, do not affect hepatic taurocholate uptake,32 indicating a lack of regulation of Ntcp by glucocorticoid-responsive element. Prolactin transcriptionally upregulates Ntcp in rats33 and this effect is mediated via STAT5 binding to Ntcp-interferon gamma-activated sequence-like elements (GLEs). Interestingly, reduced Ntcp expression in pregnant rats in late gestation was associated with an increased level of STAT5,28 an effect thought to be due to increased level of prolactin. Because STAT5 is known to upregulate Ntcp, it would appear that pregnancy is associated with generation of signals that can downregulate as well as upregulate Ntcp. It remains to be clarified whether these opposite effects on Ntcp expression are mediated via different hormones of pregnancy. cAMP stimulates sinusoidal Na+/taurocholate (TC) cotransport, transcytotic vesicle trafficking, and canalicular secretion of bile acids, organic anions, and HCO in hepatocytes.2, 34-36 Unlike cAMP, cGMP does not stimulate hepatobiliary bile acid transport, but stimulates bile formation by increasing biliary HCO excretion.37 The ability of cAMP to stimulate microtubule-dependent vesicle trafficking in hepatocytes34 led to the suggestion that cAMP may stimulate various solute transport by translocating the transporters to the plasma membrane. Indeed, cAMP increases Ntcp38, 39 in sinusoidal membranes and and and in canalicular the to canalicular membrane is on to the basolateral membrane is on However, a recent that while decreases level of Bsep in canalicular it does not increases in Bsep Thus, the role of in Bsep remains The effect of cAMP is to be mediated via also known as protein kinase A that some effects of cAMP are and may be mediated via exchange factor or exchange factor activated by cAMP The pathway has been suggested in cAMP mediated increases in cytosolic Ca2+, of and vesicle The increase in cytosolic Ca2+ by cAMP both of Ca2+ and of extracellular the of Ca2+ is due to of the IP3 receptor with increase in IP3 of cAMP leading to vesicle and transporter may involve activation of the PI3K signaling pathway and increases in cytosolic Ca2+ (see and Role of cAMP, Ca2+, and in bile formation: cAMP stimulates of Ntcp to the basolateral membrane and Bsep to the canalicular membrane. in due to and of Ca2+ from intracellular by and activates kinases, which increase via of and which Ntcp by Ntcp. inhibits increases in bile acid uptake by increases in stimulates bile acid excretion by and increases may cholestasis by to the basolateral membrane and may stimulate to the canalicular membrane. may cholestasis by activating and TUDC may cholestasis by the effect of on and Role of PI3K and MAPK signaling pathway in bile formation: of PI3K by cAMP, and cell swelling leads to the formation of that activate the pathway and the cAMP mediated activation of may be involved in Ntcp TUDC and cell swelling induced microtubule-dependent Bsep may involve The effect of cAMP is not mediated via the cAMP inhibits by and activating MAPK (not cell and cAMP may stimulate Bsep via and MAPK pathways may on There is to that the cAMP signaling pathway may be altered in cholestasis. cholestasis induced by bile duct and endotoxin is associated with decreased expression of Ntcp, and 13 These decreases are for Ntcp and Bsep. Bile duct also decreases the ability of to increase cAMP in and this to be due to decreased expression of of as well as inhibitory the receptor to adenylyl cAMP has also been to and decreases in canalicular membrane Bsep in these studies that the cAMP signaling pathway may be downregulated in cholestasis. It may be noted that a more decrease in bile acid uptake by Ntcp compared to bile acid secretion by Bsep may a hepatocellular mechanism of intracellular bile acids and, hence, the of cholestasis. This is by the that the downregulation of canalicular is associated with an upregulation of which bile acids across the sinusoidal in extracellular as well as intracellular Ca2+ have been shown to affect bile formation. A decrease in extracellular Ca2+ is associated with a decrease in bile formation. This is due to an increase in resulting in of and is not due to a effect on hepatic uptake or biliary excretion of in [Ca2+]i on the other affect hepatic transport of bile The effect of [Ca2+]i has been like arginine like or and intracellular like or of [Ca2+]i by or decreases of in Thus, [Ca2+]i an important role in acid Interestingly, is also in response to increases in [Ca2+]i by and in but not in This may in the regulation of acid by have been discussed in a recent In to its effect on uptake, increases bile acid excretion in and bile acid efflux in hepatocytes by for the these effects are due to increases in [Ca2+]i or activation of (see Fig. was not Ca2+ also a role in bile acid uptake in that this effect of cAMP is on its ability to increase [Ca2+]i from the The signal is via to kinases and that effects by and other proteins, In effects of on of cotransporter are mediated via (Fig. of is mediated via the of the protein activated by increases in The role of the signaling pathway in hepatic bile formation and cholestasis is not in [Ca2+]i have been associated with as well as cholestasis. For example, cholestatic as well as bile acid increase and TUDC can cholestasis by Ca2+ has also been shown to increase canalicular by of and and thereby Some studies would that increases in [Ca2+]i may lead to increased and and this effect may be mediated via TUDC may stimulate of vesicular by Ca2+ into which may be in However, increases in [Ca2+]i do not in Studies to define the role of [Ca2+]i have been by the that some used to increase [Ca2+]i also activate (Fig. 2). In addition, increases in [Ca2+]i by different may lead to activation of different kinases, including and resulting in different effects in and cholestatic and this may be regulated by and changes in [Ca2+]i induced by different is a family of at These conventional and novel and and and and These in their on Ca2+ and phospholipids, such that are on Ca2+ and diacylglycerol are and are of both Ca2+ and PKCs shown to be present in hepatocytes and with the of being The role of PKCs in bile formation has been known activators and of These studies that known to activate PKCs and bile acid and stimulate biliary excretion of bile PKCs bile acid uptake, at in by the ability of cAMP to increase increases bile acid efflux by and Thus, the effect of is mediated via may stimulate bile acid excretion by Bsep and thereby (Fig. of PKCs has been implicated in bile acid-induced cholestasis and requires activation of and but not and the effect of is due to activation of the other activates in and inhibits in The effect of on to be mediated via the PI3K signaling TUDC may cholestasis by activating and/or activation of may be involved in the of to the canalicular membrane and of organic anion excretion in cholestatic A recent that PKCs not canalicular to the basolateral membrane and this may to It is that the may be mediated via (Fig. has also been suggested to cholestasis by increasing These studies that bile formation and cholestasis can be mediated via but the role of as well as the of have not been PI3K is one of the kinases that the at the 3 The resulting acting in with kinases are involved in the activation of kinases, such as and These kinases are involved in vesicle trafficking, cell cell cell and transport of and bile A of bile acids, including and and cholestatic and bile acids, have been shown to activate A role of PI3K in bile formation is evident from a that a specific of PI3K, bile bile acid and vesicle trafficking in then studies have provided a role for PI3K in the regulation of hepatobiliary PI3K is involved in 1) cell swelling and of transport of bile acids and other organic anions across the canalicular 3) of Bsep and to the canalicular and increases in bile acid Interestingly, of Bsep and to the canalicular membrane is not on swelling also stimulates biliary bile acid of Bsep and to the canalicular and activates Thus, PI3K may also be involved in cell swelling induced of Bsep and However, other have been (see studies into the PI3K signaling pathway involved in the regulation of transporters involved in bile formation (Fig. swelling and cAMP activate and but is not involved in the of and Ntcp The that the signaling pathway is involved is by results that the of activation decreases cell swelling and of and Ntcp of transporter to be mediated via the as well as the signaling studies that the signaling pathway may also be involved in Ntcp of these signaling pathways is also involved in PI3K mediated of canalicular transporters has not been but is by which the of hepatobiliary transporters is mediated via the PI3K signaling pathway are canalicular transporters, like Bsep and the receptor on the and PI3K like and are involved in vesicle It can thus be that the signaling pathway stimulates by increasing vesicle trafficking along the This can lead to the of intracellular vesicles to the plasma membrane leading to plasma membrane of various transporters in the intracellular In addition, activation of the MAPK may also be involved (Fig. The a of protein kinases that are activated by a of have 2 major of MAPK 1) extracellular signal-regulated kinase activated in response to activation of receptor tyrosine kinase by growth and activated protein kinases activated by and of protein 2 kinases and These mediate signal transduction from the cell surface to the are involved in cell and under activation and of these requires involving various The MAPK signaling pathways involving and have also been implicated in biliary excretion of bile cell swelling increases the of biliary TC excretion and this effect requires a and tyrosine activation of but is of increases in bile acid excretion are on this activation is not on tyrosine or In addition, cAMP, shown to in activation of and increases in bile acid The of by cAMP may be due to of and activation of The effect of TUDC to be mediated via a activation of and increases in bile acid secretion and Bsep to the canalicular membrane also activation of activation of In to bile acid cell of hepatic uptake of bile acid is not on These studies that of Bsep and the increase in bile acid excretion can be mediated via activation of as well as MAPK pathway (Fig. Because and MAPK can activate the like MAPK kinase and and protein kinase it is that Bsep along the is mediated via one of these The role of PI3K and MAPK pathways in cholestasis has not been However, their role in bile these pathways are to be altered in cholestasis. the other that stimulate these pathways should as is by the effects of Interestingly, a recent that the cholestatic effect of may be mediated via activation of Thus, the PI3K signaling pathway may be involved in as well as cholestatic with the by the kinase being of cellular processes protein by kinases and by a of studies the role of various kinases in bile studies to the role of protein are Studies with of protein and such as acid and that microtubule-dependent vesicle in acid, which inhibits in the ability of cAMP to stimulate TC uptake, translocate and Ntcp, and increase [Ca2+]i in The ability of cAMP to Ntcp is by a AMP activates known as Ntcp and an of inhibits and of these results that both and are involved in of Ntcp. A mechanism may be as cAMP, in a increases which activates and in turn Ntcp its to the plasma membrane (Fig. of by extracellular ATP and acid decreases its activity without affecting its a role for in the regulation of These studies that protein such as and may regulate vesicular transport and organic anion uptake in In of it would of to whether protein are also involved in the regulation of other transporters and are altered in cholestasis. Our understanding of bile acid transporters and their and has increased as has our understanding of the cellular mechanism these It is becoming more evident that and cholestatic the function of these transporters various signal transduction pathways. Nuclear receptors regulate of various transporter genes. cAMP stimulates transhepatic transport of bile acids by translocating Ntcp and Bsep to the sinusoidal and the canalicular membrane, respectively. The PI3K signaling pathway is involved in biliary excretion of bile acids and activation of stimulates biliary bile acid excretion via the MAPK signaling pathway and cholestasis by of to the canalicular membrane. acting via of Ntcp. stimulates bile acid by Bsep. However, the role of specific involved in bile formation and cholestasis is still by which PI3K and MAPK signaling pathways stimulate transporter along the have not been Our of the role of protein and nuclear receptors in bile formation and cholestasis is It that understanding in these be in the