M. Sawkat Anwer

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.

Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp

cAMP stimulates Na+-taurocholate (TC) cotransport by translocating the Na+-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70S6K activity. However, rapamycin, an inhibitor of p70S6K, failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70S6K. Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.cAMP stimulates Na(+)-taurocholate (TC) cotransport by translocating the Na(+)-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70(S6K) activity. However, rapamycin, an inhibitor of p70(S6K), failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70(S6K). Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.

Sodium-dependent bile salt transporters of the SLC10A transporter family: more than solute transporters

The SLC10A transporter gene family consists of seven members and substrates transported by three members (SLC10A1, SLC10A2 and SLC10A6) are Na+-dependent. SLC10A1 (sodium taurocholate cotransporting polypeptide [NTCP]) and SLC10A2 (apical sodium-dependent bile salt transporter [ASBT]) transport bile salts and play an important role in maintaining enterohepatic circulation of bile salts. Solutes other than bile salts are also transported by NTCP. However, ASBT has not been shown to be a transporter for non-bile salt substrates. While the transport function of NTCP can potentially be used as liver function test, interpretation of such a test may be complicated by altered expression of NTCP in diseases and presence of drugs that may inhibit NTCP function. Transport of bile salts by NTCP and ASBT is inhibited by a number of drugs and it appears that ASBT is more permissive to drug inhibition than NTCP. The clinical significance of this inhibition in drug disposition and drug–drug interaction remains to be determined. Both NCTP and ASBT undergo post-translational regulations that involve phosphorylation/dephosphorylation, translocation to and retrieval from the plasma membrane and degradation by the ubiquitin–proteasome system. These posttranslational regulations are mediated via signaling pathways involving cAMP, calcium, nitric oxide, phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) and protein phosphatases. There appears to be species difference in the substrate specificity and the regulation of plasma membrane localization of human and rodent NTCP. These differences should be taken into account when extrapolating rodent data for human clinical relevance and developing novel therapies. NTCP has recently been shown to play an important role in HBV and HDV infection by serving as a receptor for entry of these viruses into hepatocytes.

Cell Swelling-induced Translocation of Rat Liver Na+/Taurocholate Cotransport Polypeptide Is Mediated via the Phosphoinositide 3-Kinase Signaling Pathway

Cell swelling stimulates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) in hepatocytes, and the PI3K signaling pathway is involved in cAMP-mediated translocation of sinusoidal Na+/taurocholate (TC) cotransporter (Ntcp) to the plasma membrane. We determined whether cell swelling also stimulates TC uptake and Ntcp translocation via the PI3K and/or MAPK signaling pathway. All studies were conducted in isolated rat hepatocytes. Hepatocyte swelling induced by hypotonic media resulted in: 1) time- and medium osmolarity-dependent increases in TC uptake, 2) an increase in the V max of Na+/TC cotransport, and 3) wortmannin-sensitive increases in TC uptake and plasma membrane Ntcp mass. Hepatocyte swelling also induced wortmannin-sensitive activation of PI3K, protein kinase B, and p70S6K. Rapamycin, an inhibitor of p70S6K, inhibited cell swelling-induced activation of p70S6K but failed to inhibit cell swelling-induced stimulation of TC uptake. Because PD98095, an inhibitor of MAPK, did not inhibit cell swelling-induced increases in TC uptake, it is unlikely that the effect of cell swelling on TC uptake is mediated via the MAPK signaling pathway. Taken together, these results indicate that 1) cell swelling stimulates TC uptake by translocating Ntcp to the plasma membrane, 2) this effect is mediated via the PI3K, but not MAPK, signaling pathway, and 3) protein kinase B, but not p70S6K, is a likely downstream effector of PI3K.

PKCζ is required for microtubule-based motility of vesicles containing the ntcp transporter

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+‐taurocholate co‐transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCζ have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole‐cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin‐1 and dynein. These vesicles co‐localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCζ and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3‐kinase inhibitor. Specific inhibition of PKCζ blocks the motility of ntcp‐containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp‐GFP. These data indicate that PKCζ is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.

Protein Kinase B/Akt Mediates cAMP- and Cell Swelling-stimulated Na+/Taurocholate Cotransport and Ntcp Translocation

Cyclic AMP and cell swelling stimulate hepatic Na+/TC cotransport and Ntcp translocation via the phosphoinositide 3-kinase signaling pathway. To determine the downstream target of the phosphoinositide 3-kinase action, we examined the role of protein kinase B (PKB)/Akt using SB203580 in hepatocytes as well as by transfection with a dominant negative (DN-PKB) or a constitutively active (CA-PKB) form of PKB in HuH-Ntcp cells. Both cAMP and cell swelling stimulated p38 mitogen-activated protein (MAP) kinase as well as PKB activity. Although 100 μm SB203580 inhibited cell swelling- and 8-chlorophenylthio-cAMP-induced activation of both p38 MAP kinase and PKB, 1 μm SB203580 inhibited activation of p38 MAP kinase, but not of PKB, in hepatocytes. 100 μm, but not 1 μm SB203580, inhibited cell swelling- and cAMP-induced increases in taurocholate (TC) uptake and Ntcp translocation in hepatocytes. TC uptake in HuH-Ntcp cells was more than 90% dependent on extracellular Na+. Cyclic AMP and cell swelling increased TC uptake by 50–100% and PKB activity 2–4-fold in HuH-Ntcp cells transfected with the empty vector and failed to increase PKB activity, TC uptake, and Ntcp translocation in DN-PKB-transfected HuH-Ntcp cells. Transfection with CA-PKB increased PKB activity, TC uptake, and Ntcp translocation in HuH-Ntcp cells compared with cells transfected with the empty vector. In contrast, transfection with DN-PKB did not affect basal PKB activity, TC uptake, or Ntcp translocation. Taken together, these results strongly suggest that cell swelling and cAMP-mediated stimulation of hepatic Na+/TC cotransport and Ntcp translocation requires activation of PKB and is mediated at least in part via a phosphoinositide 3-kinase/PKB-signaling pathway.

Protein kinase Cδ mediates cyclic adenosine monophosphate–stimulated translocation of sodium taurocholate cotransporting polypeptide and multidrug resistant associated protein 2 in rat hepatocytes†

Cyclic adenosine monophosphate (cAMP) stimulates translocation of Na+‐taurocholate (TC) cotransporting polypeptide (Ntcp) and multidrug resistant associated protein 2 (Mrp2) to the plasma membrane. Because cAMP activates phosphoinositide‐3‐kinase (PI3K) and protein kinase C (PKC) activation is PI3K‐dependent, the aim of the current study was to determine whether cAMP activates conventional and novel PKCs in hepatocytes and whether such activation plays a role in cAMP‐stimulated Ntcp and Mrp2 translocation. The effect of cAMP on PKCs, TC uptake, and Ntcp and Mrp2 translocation was studied in isolated rat hepatocytes using a cell‐permeable cAMP analog, CPT‐cAMP. The activity of PKCs was assessed from membrane translocation of individual PKCs, and phospho‐specific antibodies were used to determine PKCδ phosphorylation. TC uptake was determined from time‐dependent uptake of 14C‐TC, and a cell surface biotinylation method was used to determine Ntcp and Mrp2 translocation. CPT‐cAMP stimulated nPKCδ but not cPKCα or nPKCϵ, and induced PI3K‐dependent phosphorylation of nPKCδ at Thr505. Rottlerin, an inhibitor of nPKCδ, inhibited cAMP‐induced nPKCδ translocation, TC uptake, and Ntcp and Mrp2 translocation. Bistratene A, an activator of nPKCδ, stimulated nPKCδ translocation, TC uptake, and Ntcp and Mrp2 translocation. The effects of cAMP and bistratene A on TC uptake and Ntcp and Mrp2 translocation were not additive. Conclusion: These results suggest that cAMP stimulates Ntcp and Mrp2 translocation, at least in part, by activating nPKCδ via PI3K‐dependent phosphorylation at Thr505. (HEPATOLOGY 2008.)

Role of protein kinase C isoforms in bile formation and cholestasis.

Transhepatic solute transport provides the osmotic driving force for canalicular bile formation. Choleretic and cholestatic agents affect bile formation, in part, by altering plasma membrane localizations of transporters involved in bile formation. These short‐term dynamic changes in transporter location are highly regulated posttranslational events requiring various cellular signaling pathways. Interestingly, both choleretic and cholestatic agents activate the same intracellular signaling kinases, such as phosphoinositide‐3‐kinase (PI3K), protein kinase C (PKC), and mitogen‐activated protein kinase (MAPK). An emerging theme is that choleretic and cholestatic effects may be mediated by different isoforms of these kinases. This is most evident for PKC‐mediated regulation of plasma membrane localization of Na+‐taurocholate cotransporting polypeptide (NTCP) and multidrug resistance‐associated protein 2 (MRP2) by conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ). aPKCζ may mediate choleretic effects by inserting NTCP into the plasma membrane, and nPKCε may mediate cholestatic effects by retrieving MRP2 from the plasma membrane. On the other hand, cPKCα and nPKCδ may be involved in choleretic, cholestatic, and anticholestatic effects by inserting, retrieving, and inhibiting retrieval of transporters, respectively. The effects of PKC isoforms may be mediated by phosphorylation of the transporters, actin binding proteins (radixin and myristoylated alanine‐rich C kinase substrate), and Rab proteins. Human NTCP plays an important role in the entry of hepatitis B and D viruses into hepatocytes and consequent infection. Thus, PKCs, by regulating NTCP trafficking, may also play an important role in hepatic viral infections. (Hepatology 2014;60:1090–1097)

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells†‡

Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug‐resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)–MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine‐rich C kinase substrate (MARCKS) is a membrane‐bound F‐actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine‐rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC‐induced MRP2 retrieval involves PKCϵ‐mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH‐NTCP cells) and in rat hepatocytes. TLC increased PM–PKCϵ and decreased PM‐MRP2 in both HuH‐NTCP cells and hepatocytes. cAMP did not affect PM‐PKCϵ and increased PM‐MRP2 in these cells. In HuH‐NTCP cells, dominant‐negative (DN) PKCϵ reversed TLC‐induced decreases in PM‐MRP2 without affecting cAMP‐induced increases in PM‐MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH‐NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM‐MARCKS in HuH‐NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH‐NTCP cells transfected with DN‐PKCϵ, and this suggested PKCϵ‐mediated phosphorylation of MARCKS by TLC. In HuH‐NTCP cells transfected with phosphorylation‐deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM‐MRP2. Conclusion: Taken together, these results support the hypothesis that TLC‐induced MRP2 retrieval involves TLC‐mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane. (HEPATOLOGY 2013;)

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane.

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.