Cellular regulation of hepatic bile acid transport in health and cholestasis

Authors

  • M. Sawkat Anwer

    Corresponding author
    1. Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, N. Grafton, MA
    • Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, 200 Westboro Road, N. Grafton, MA 01536
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    • fax: 508-839-8787


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.

Figure 1.

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+/HCOmath image cotransporter (NBC) at the sinusoidal membrane of hepatocytes and basolateral membrane of cholangiocytes are involved in intracellular pH regulation and HCOmath image 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).

Abbreviations

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.

Regulation of Transporters

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

Figure 2.

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.

Role of Nuclear Receptors and STAT

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.

Figure 3.

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.

Role of cAMP

cAMP stimulates sinusoidal Na+/taurocholate (TC) cotransport, transcytotic vesicle trafficking, and canalicular secretion of bile acids, organic anions, and HCOmath image in hepatocytes.2, 34–36 Unlike cAMP, cGMP does not stimulate hepatobiliary bile acid transport, but stimulates bile formation by increasing biliary HCOmath image 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 Mrp2,40 Mdr2 and Mdr3,41 and Bsep42 in canalicular membranes. While the translocation to canalicular membrane is dependent on microtubules,41, 43 cAMP-mediated translocation to the basolateral membrane is dependent on microfilaments.39, 44 However, a recent study reported that while colchicine decreases basal level of Bsep in canalicular membranes, it does not inhibit cAMP-induced increases in Bsep translocation.45 Thus, the role of microtubules in cAMP-induced Bsep translocation remains unclear.

The effect of cAMP is believed to be mediated via cAMP-dependent kinase, also known as protein kinase A (PKA).2, 4 Recent studies, however, suggest that some effects of cAMP are PKA-independent and may be mediated via cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) or exchange factor directly activated by cAMP (Epac).46, 47 The cAMP/PKA pathway has been suggested in cAMP mediated increases in cytosolic Ca2+, stimulation of Na+/TC cotransport,36 and vesicle movement.48 The increase in cytosolic Ca2+ by cAMP involves both release of stored Ca2+ and influx of extracellular Ca2+;49 the release of stored Ca2+ is due to phosphorylation of the IP3 receptor with consequent increase in IP3 sensitivity.50, 51 Signaling pathway(s) downstream of cAMP leading to vesicle movement and consequent transporter translocation may involve activation of the PI3K signaling pathway and increases in cytosolic Ca2+ (see Figs. 4 and 5).

Figure 4.

Role of cAMP, Ca2+, and PKC in bile formation: cAMP stimulates translocation of Ntcp to the basolateral membrane and Bsep to the canalicular membrane. Increases in [Ca2+]i, due to influx and release of Ca2+ from intracellular store by PKA and IP3, activates Ca2+/calmodulin (Ca2+/CAM)-dependent kinases, which increase tight-junction (TJ) permeability via phosphorylation of myosin light-chain, and (Ca2+/CAM)-dependent PP2B, which facilitate Ntcp translocation by dephosphorylating Ntcp. PKC inhibits cAMP-mediated increases in bile acid uptake by diminishing PKA-mediated increases in [Ca2+]i, stimulates bile acid excretion probably by phosphorylating Bsep, and increases TJ permeability. nPKCϵ may produce cholestasis by retargeting Mrp2 to the basolateral membrane and cPKCα may stimulate Mrp2 translocation to the canalicular membrane. TLC (taurolithocholate) may produce cholestasis by activating nPKCϵ and inhibiting cPKCα. TUDC may reverse TLC-induced cholestasis by reversing the effect of TLC on cPKCα and nPKCϵ.

Figure 5.

Role of PI3K and MAPK signaling pathway in bile formation: Activation of PI3K by TC, cAMP, TUDC, and cell swelling leads to the formation of PIPs that activate the Ras/ERK pathway and the PKB/aPKCζ pathway. cAMP mediated activation of PKB/PKCζ may be involved in microfilament-dependent Ntcp translocation. TUDC and cell swelling induced microtubule-dependent Bsep translocation may involve PI3K/PIPs/Ras/Raf/Erk pathway. The effect of cAMP is not mediated via the ERK pathway; cAMP inhibits ERK1/2 by inhibiting Raf and activating MAPK phosphatase-1 (not shown). TUDC, cell swelling, and cAMP may stimulate Bsep translocation via P38 MAPK. ERK and p38 MAPK pathways may converge on MNK1/MSK1.

There is evidence to suggest that the cAMP signaling pathway may be altered in cholestasis. Experimental cholestasis induced by bile duct ligation and endotoxin is associated with decreased expression of Ntcp, Mrp2, and Bsep.12, 13 These decreases are much pronounced for Ntcp and Mrp2 than Bsep. Bile duct ligation also decreases the ability of glucagon to increase cAMP in hamster hepatocytes,52 and this appears to be due to decreased expression of α-subunit(s) of stimulatory as well as inhibitory G proteins53 coupling the receptor to adenylyl cyclase. cAMP has also been reported to reverse estradiol-17β-glucuronide- and taurolithocholate-induced decreases in canalicular membrane Bsep in rats.54, 55 Taken together, these studies suggest that the cAMP signaling pathway may be downregulated in cholestasis. It may be noted that a more pronounced decrease in bile acid uptake by Ntcp compared to bile acid secretion by Bsep may represent a hepatocellular defense mechanism against accumulation of intracellular bile acids and, hence, the exacerbation of cholestasis. This is further supported by the finding that the downregulation of canalicular Mrp2 is associated with an upregulation of Mrp3,15 which exports bile acids across the sinusoidal membrane.56

Role of Calcium

Changes in extracellular as well as intracellular Ca2+ ([Ca2+]i) have been shown to affect bile formation. A decrease in extracellular Ca2+ below 50 μM is associated with a decrease in bile formation. This is due to an increase in tight-junction permeability resulting in reflux of secreted solutes, and is not due to a direct effect on either hepatic uptake or biliary excretion of TC.57, 58 Changes in [Ca2+]i on the other hand, affect hepatic transport of bile acids.

The effect of [Ca2+]i has been studied using calcium mobilizing agents, like arginine vasopressin (AV), calcium ionophores, like A23187 or ionomycin, and intracellular calcium chelators, like MAPTA or BAPTA. Chelation of [Ca2+]i by MAPTA, BAPTA, or EDTA decreases basal rate of Na+/TC cotransport in hepatocytes.36, 59 Thus, resting [Ca2+]i plays an important role in maintaining basal Na+/bile acid cotransport. Interestingly, basal Na+/TC cotransport is also inhibited in response to increases in [Ca2+]i produced by calcium ionophores and AV in hamster hepatocytes,59 but not in rat hepatocytes.36, 60 This may suggest species differences in the regulation of Na+/bile acid cotransport by [Ca2+]i. Other potential mechanisms have been discussed in a recent review.23

In contrast to its effect on uptake, AV increases bile acid excretion transiently in perfused rat livers61 and bile acid efflux in isolated rat hepatocytes by decreasing substrate affinity for the transporter.60 Whether these effects are due to increases in [Ca2+]i or activation of PKC (see Fig. 2) was not investigated. Intracellular Ca2+ also plays a role in cAMP-stimulated bile acid uptake in that this effect of cAMP is dependent on its ability to increase [Ca2+]i from the IP3-sensitive pool.36

The calcium signal is transduced via calmodulin to calmodulin-dependent kinases and phosphatases that produce biologic effects by phosphorylating and dephosphorylating other proteins, respectively.62 In hepatocytes,36, 63 effects of calcium on cAMP-induced stimulation of Na+/TC cotransporter are mediated via calmodulin (Fig. 4). cAMP-induced dephosphorylation of Ntcp64 is mediated via the action of the calmodulin-dependent phosphatase, protein phosphatase 2B, activated by cAMP-induced increases in [Ca2+]i.65

The exact role of the calcium-dependent signaling pathway in hepatic bile formation and cholestasis is not clearly understood. Increases in [Ca2+]i have been associated with choleresis as well as cholestasis. For example, cholestatic (TLC) as well as choleretic (TUDC) bile acid increase [Ca2+]i,66, 67 and TUDC can reverse cholestasis produced by TLC.68 Cytosolic Ca2+ has also been shown to increase canalicular peristalsis by stimulating contraction of pericanalicular actin and myosin and thereby facilitate choleresis.69 Some studies would suggest that increases in [Ca2+]i may lead to increased tight-junction permeability and hence cholestasis70, 71 and this effect may be mediated via myosin light chain phosphorylation.72 TUDC may stimulate Ca2+-dependent stimulation of vesicular exocytosis by enhancing Ca2+ entry into hepatocytes, which may be compromised in cholestasis.73 However, increases in [Ca2+]i do not influence exocytosis in normal hepatocytes.74 Studies to define the role of [Ca2+]i have been complicated by the fact that some agents used to increase [Ca2+]i also activate PKC (Fig. 2). In addition, increases in [Ca2+]i by different agents may lead to activation of different downstream kinases, including PI3K75 and PKB,76 resulting in different effects in normal and cholestatic hepatocytes, and this may be regulated by temporal and spatial changes in [Ca2+]i induced by different agents.77, 78

Role of PKC

PKC is a family of at least 12 isozymes.79 These include conventional (cPKCα, β, βI, βII and γ), novel (nPKCδ, ϵ, η and θ), and atypical (aPKCζ and λ) isoforms, and PKCμ. These isoforms differ in their dependency on Ca2+ and phospholipids, such that cPKCs are dependent on Ca2+ and diacylglycerol (DAG), nPKCs are Ca2+-independent, and aPKCs are independent of both Ca2+ and DAG. PKCs shown to be present in rat hepatocytes include cPKCα, nPKCδ, nPKCϵ, and aPKCζ, with the presence of cPKCβII being controversial.80–82

The role of PKCs in bile formation has been studied using known activators (phorbol esters) and inhibitors of PKCs. These studies indicate that agents known to activate PKCs produce cholestasis,83 inhibit basal and cAMP-stimulated bile acid uptake,36, 59 and stimulate biliary excretion of bile acids.61 PKCs inhibit cAMP-stimulated bile acid uptake, at least in part, by inhibiting the ability of cAMP to increase [Ca2+]i.36 AV increases bile acid efflux by decreasing Km60 and cPKCα phosphorylates Bsep.84 Thus, if the effect of AV is mediated via PKCs, cPKCα may stimulate bile acid excretion by phosphorylating Bsep and thereby enhancing substrate affinity (Fig. 4).

Activation of PKCs has been implicated in bile acid-induced cholestasis and apoptosis. GCDC-induced apoptosis requires activation of cPKCα and nPKCδ, but not nPKCϵ,81 and the non-apoptotic effect of TCDC is due to activation of aPKCζ.85 On the other hand, TLC activates nPKCϵ in isolated hepatocytes80 and inhibits cPKCα in isolated perfused rat livers.86 The effect of TLC on nPKCϵ appears to be mediated via the PI3K signaling pathway.87 TUDC may reverse TLC-induced cholestasis by activating cPKCα and/or inhibiting nPKCϵ.86, 88 TUDC-induced activation of cPKCα may be involved in the translocation of Mrp2 to the canalicular membrane and consequent restoration of organic anion excretion in cholestatic livers.86 A recent study shows that PKCs (isoforms not studied) retargets canalicular Mrp2 to the basolateral membrane and this may contribute to cholestasis.89 It is possible that the retargeting may be mediated via nPKCϵ (Fig. 4). PKC has also been suggested to produce cholestasis by increasing tight-junction permeability.70 These studies suggest that bile formation and cholestasis can be mediated via PKC isoform-dependent processes, but the role of PKC isoforms, as well as the downstream targets of PKC isoforms, have not been clearly established.

Role of PI3K Signaling Pathway

PI3K is one of the PI kinases that phosphorylate the inositol ring at the 3 position. The resulting phosphorylated PIs (PIPs), acting in concert with phosphoinositide kinases (PDKs), are involved in the activation of downstream kinases, such as aPKCζ/λ, PKB/Akt, and p70S6K. These kinases are involved in vesicle trafficking, cell survival, cell proliferation, cell migration, and transport of glucose and bile acids.2, 90, 91 A number of bile acids, including choleretic (TC and TUDC) and cholestatic (TLC and TCDC) bile acids, have been shown to activate PI3K.85, 87, 92, 93 A role of PI3K in bile formation is evident from a study94 showing that wortmannin, a specific inhibitor of PI3K, inhibited bile formation, bile acid secretion, and vesicle trafficking in isolated perfused rat liver. Since then various, studies have provided evidence supporting a role for PI3K in the regulation of hepatobiliary transporters. PI3K is involved in 1) cell swelling and cAMP-mediated translocation of Ntcp,44, 95 2) ATP-dependent transport of bile acids and other organic anions across the canalicular membrane,96 3) TC-induced translocation of Bsep and Mrp2 to the canalicular membrane,92 and 4) TUDC-induced increases in bile acid secretion.93 Interestingly, cAMP-induced translocation of Bsep and Mrp2 to the canalicular membrane is not dependent on PI3K.45 Cell swelling also stimulates biliary bile acid excretion, translocation of Bsep and Mrp2 to the canalicular membrane,97, 98 and activates PI3K.95, 99 Thus, PI3K may also be involved in cell swelling induced translocation of Bsep and Mrp2. However, other mechanisms have been proposed (see below).

Recent studies provide further insight into the PI3K signaling pathway involved in the regulation of transporters involved in bile formation (Fig. 5). Cell swelling and cAMP activate wortmannin-sensitive PKB and p70S6K, but p70S6K is not involved in the stimulation of Na+/TC cotransport and Ntcp translocation.44, 95 The possibility that the PI3K/PKB signaling pathway is involved is supported by results showing that the inhibition of PKB activation decreases cell swelling and cAMP-mediated stimulation of Na+/TC cotransport and Ntcp translocation.100 Translocation of glucose transporter appears to be mediated via the P13K/PKB as well as the PI3K/aPKCζ signaling pathway.101, 102 Preliminary studies suggest that the PI3K/aPKCζ signaling pathway may also be involved in Ntcp translocation (unpublished data). Whether either of these signaling pathways is also involved in PI3K mediated translocation of canalicular transporters has not yet been studied, but is likely.

Mechanisms by which the translocation of hepatobiliary transporters is mediated via the PI3K signaling pathway are unclear. Several canalicular transporters, like Bsep and Mrp2, the polymeric IgA receptor traffic on the same vesicle,103 and PI3K products, like PIP, PIP2, and PIP3, are involved in vesicle trafficking.90, 104 It can thus be speculated that the PI3K/PKB(aPKCζ) signaling pathway stimulates exocytosis by increasing vesicle trafficking along the cytoskeleton. This can lead to the fusion of intracellular vesicles to the plasma membrane leading to plasma membrane translocation of various transporters stored in the intracellular vesicles. In addition, PI3K-mediated activation of the MAPK cascade91 may also be involved (Fig. 5).

Role of MAPK Signaling Pathway

The MAPKs include a group of protein kinases that are activated by a variety of signals.105–107 Mammalian cells have 2 major types of MAPK cascade: 1) extracellular signal-regulated kinase (ERK1/ERK2) activated in response to activation of receptor tyrosine kinase by growth factors, and 2) stress activated protein kinases (SAPKs) activated by ultraviolet radiation, inflammatory cytokines, DNA damaging agents, and inhibitors of protein synthesis. SAPKs include 2 distinct subfamilies: c-Jun amino-terminal kinases (JNKs) and p38 MAPK. These MAPKs mediate signal transduction from the cell surface to the nucleus. They are involved in cell proliferation and differentiation under controlled activation and oncogenesis during uncontrolled activation. Activation of these MAPKs requires sequential phosphorylation involving various kinases.107

The MAPK signaling pathways involving ERK1/2 and p38 have also been implicated in biliary excretion of bile acids. Hypoosmotic cell swelling increases the capacity of biliary TC excretion and this effect requires a G-protein- and tyrosine kinase-dependent activation of ERK1/2, but is independent of PKCs.108 Similarly, TUDC-induced increases in bile acid excretion are dependent on ERK1/2 activation, except this activation is not dependent on G-protein, tyrosine kinase, or PKCs.109 In addition, cAMP, shown to inhibit ERK1/2 in hepatocytes,44 inhibited TUDC-induced activation of ERK1/2 and increases in bile acid excretion.109 The inhibition of ERK1/2 by cAMP may be due to inhibition of Raf110 and activation of MAPK-phosphatase-1.111 The effect of TUDC appears to be mediated via a PI3K-dependent activation of Ras/ERK pathway.93 Cell swelling- and TUDC-induced increases in bile acid secretion and Bsep translocation to the canalicular membrane also require activation of PI3K-independent activation of p38 MAPK.112 In contrast to bile acid secretion, cell swelling-induced stimulation of hepatic uptake of bile acid is not dependent on ERK1/2.95 These studies indicate that stimulation of Bsep translocation and the consequent increase in bile acid excretion can be mediated via activation of ERK1/2 as well as p38 MAPK pathway (Fig. 5). Because ERK1/2 and p38 MAPK can activate the downstream kinases,106 like MAPK signal-integrating kinase 1 (MNK1) and mitogen- and stress-activated protein kinase 1 (MSK1), it is possible that Bsep translocation along the cytoskeleton is mediated via one of these kinases.

The role of PI3K and MAPK pathways in cholestasis has not been directly evaluated. However, considering their role in bile formation, these pathways are likely to be altered in cholestasis. On the other hand, agents that stimulate these pathways should produce anticholestatic effects, as is indicated by the anticholestatic effects of ursodeoxycholate.113 Interestingly, a recent study reported that the cholestatic effect of TLC may be mediated via PI3K-dependent activation of nPKCϵ.87 Thus, the PI3K signaling pathway may be involved in choleretic as well as cholestatic effects, with the final outcome determined by the terminal kinase being activated.

Role of Protein Phosphatases

Regulation of cellular processes involves protein phosphorylation by kinases and dephosphorylation by phosphatases. While a number of studies investigated the role of various kinases in bile formation, studies to determine the role of protein phosphatases are limited. Studies with inhibitors of protein phosphatase 1 and 2A (PP1/2A), such as okadaic acid and microcystin, suggest that PP2A regulates microtubule-dependent vesicle movement in hepatocytes.114 Okadaic acid, which inhibits PP2A in hepatocytes, blocks the ability of cAMP to stimulate TC uptake, translocate and dephosphorylate Ntcp, and increase [Ca2+]i in hepatocytes.115 The ability of cAMP to dephosphorylate Ntcp is inhibited by a calcium chelator.64 Cyclic AMP activates Ca2+/calmodulin-dependent PP2B (also known as calcineurin), PP2B directly dephosphorylates Ntcp and an inhibitor of PP2B inhibits cAMP-induced dephosphorylation and translocation of Ntcp.65 Taken together, these results suggest that both PP2A and 2B are involved in cAMP-induced translocation of Ntcp. A likely mechanism may be as follows: cAMP, in a PP2A dependent manner, increases [Ca2+]i, which activates PP2B, and PP2B in turn dephosphorylates Ntcp facilitating its translocation to the plasma membrane (Fig. 4). Phosphorylation of Oatp1 by extracellular ATP and okadaic acid decreases its activity without affecting its distribution, suggesting a role for PP2A in the regulation of Oatp1.116 These studies suggest that protein phosphatases, such as PP2A and PP2B, may regulate vesicular transport and organic anion uptake in hepatocytes. In view of this, it would of interest to determine whether protein phosphatases are also involved in the regulation of other transporters and are altered in cholestasis.

Summary

Our understanding of bile acid transporters and their substrates and locations has increased steadily as has our understanding of the cellular mechanism regulating these transporters. It is becoming more evident that choleretic and cholestatic agents modify the function of these transporters through various signal transduction pathways. Nuclear receptors regulate transcription 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 taurolithocholate-induced activation of PKCϵ. Tauroursodeoxycholate stimulates biliary bile acid excretion via the P38 MAPK signaling pathway and reverses TLC cholestasis by stimulating PKC-mediated translocation of Mrp2 to the canalicular membrane. Calcium, acting via Ca2+/calmodulin dependent kinases/phosphatases, augments cAMP-mediated translocation of Ntcp. PKC stimulates bile acid secretion, most likely by phosphorylating Bsep. However, the role of specific PKC isoforms involved in bile formation and cholestasis is still unclear. Molecular mechanisms by which PI3K and MAPK signaling pathways stimulate transporter translocation along the cytoskeleton have not been elucidated. Our knowledge of the role of protein phosphatases and nuclear receptors in bile formation and cholestasis is rather limited. It is, however, anticipated that further understanding in these areas will be forthcoming in the near future.

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