VAMP3 and SNAP23 as Potential Targets for Preventing the Disturbed Flow-Accelerated Thrombus Formation.

Disturbed blood flow has been recognized to promote platelet aggregation and thrombosis via increasing accumulation of von Willebrand factor (VWF) at the arterial post-stenotic sites. The mechanism underlying the disturbed-flow regulated endothelial VWF production remains elusive. Here we described a mouse model, in which the left external carotid artery (LECA) is ligated to generate disturbed flow in the common carotid artery. Ligation of LECA increased VWF accumulation in the plasma. Carotid arterial thrombosis was induced by ferric chloride (FeCl3) application and the time to occlusion in the ligated vessels was reduced in comparison with the unligated vessels. In vitro, endothelial cells were subjected to oscillatory shear (OS, 0.5 ± 4 dynes/cm2) or pulsatile shear (PS, 12 ± 4 dynes/cm2). OS promoted VWF secretion as well as the cell conditioned media-induced platelet aggregation by regulating the intracellular localization of vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Disruption of vimentin intermediate filaments abolished the OS-induced translocation of SNAP23 to the cell membrane. Knockdown of VAMP3 and SNAP23 reduced the endothelial secretion of VWF. Systemic inhibition of VAMP3 and SNAP23 by treatment of mice with rapamycin significantly ameliorated the FeCl3-induced thrombogenesis, whereas intraluminal overexpression of VAMP3 and SNAP23 aggravated it. Our findings demonstrate VAMP3 and SNAP23 as potential targets for preventing the disturbed flow-accelerated thrombus formation.

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

A novel RARα/CAR-mediated mechanism for regulation of human organic solute transporter-ß gene expression.

The organic solute transporter-α/ß (OSTα/ß) is a heteromeric transporter that is essential for bile acid and sterol disposition and for the enterohepatic circulation. To better understand the mechanism underlying OST gene regulation, the effects of retinoic acid (RA) on OSTα/ß gene expression were investigated. The results show a dose-dependent induction of OSTß but not OSTα expression in both Huh7 and HepG2 cells by RA treatment. A novel functional RA receptor response element (RARE; so-called DR5) in the promoter of OSTß gene was identified. The interaction of RARα/RXRα with the RARE was verified by electrophoretic mobility shift and chromatin immunoprecipitation assays and its functional importance by hOSTß promoter activation in luciferase reporter assays. The studies demonstrated that the RARE is also a constitutive androstane receptor (CAR) binding site for OSTß gene regulation. These results suggest that OSTß is a target of both FXR-mediated (by binding to IR-1 element) and RARα- and CAR-mediated (by binding to DR5 element) gene regulation pathways. In summary, this study has uncovered a novel RARE (DR5) element in the promoter of OSTß that binds RARα or CAR heterodimerized with RXRα and appears to function synergistically with the IR-1 element to provide maximal induction of OSTß in response to RA. These findings demonstrate a role for RARα and CAR in controlling OSTß expression levels.

SUMOylation of the farnesoid X receptor (FXR) regulates the expression of FXR target genes.

BACKGROUND: Small ubiquitin-like modifiers (SUMO) are covalently conjugated to other proteins including nuclear receptors leading to modification of various cellular processes. RESULTS: Ligand-dependent SUMOylation of farnesoid X receptor (FXR) negatively regulates the expression of its target genes. CONCLUSION: SUMO modification attenuates the capacity of FXR to function as a transcriptional activator. SIGNIFICANCE: Defining post-translation modification of FXR bySUMOis important to understanding how this nuclear receptor functions in health and disease. The farnesoid X receptor (FXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metabolism including bile acid homeostasis. Here we show that FXR is covalently modified by the small ubiquitin-like modifier (Sumo1), an important regulator of cell signaling and transcription. Well conserved consensus sites at lysine 122 and 275 in the AF-1 and ligand binding domains, respectively, of FXR were subject to SUMOylation in vitro and in vivo. Chromatin immunoprecipitation (ChIP) analysis showed that Sumo1 was recruited to the bile salt export pump (BSEP), the small heterodimer partner (SHP), and the OSTα-OSTß organic solute transporter loci in a ligand-dependent fashion. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and Sumo1 to the BSEP and SHP promoters. Overexpression of Sumo1 markedly decreased binding and/or recruitment of FXR to the BSEP and SHP promoters on ChIP-ReChIP. SUMOylation did not have an apparent effect on nuclear localization of FXR. Expression of Sumo1 markedly inhibited the ligand-dependent, transactivation of BSEP and SHP promoters by FXR/retinoid X receptor α (RXRα) in HepG2 cells. In contrast, mutations that abolished SUMOylation of FXR or siRNA knockdown of Sumo1 expression augmented the transactivation of BSEP and SHP promoters by FXR. Pathways for SUMOylation were significantly altered during obstructive cholestasis with differential Sumo1 recruitment to the promoters of FXR target genes. In conclusion, FXR is subject to SUMOylation that regulates its capacity to transactivate its target genes in normal liver and during obstructive cholestasis.

Identification of functionally relevant lysine residues that modulate human farnesoid X receptor activation.

Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-protein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR significantly impaired target gene [organic solute transporter α/ß and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (RXR) binding affinity but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by ∼21%. K460R decreased the FXR interaction with Set7/9 by ∼45% but had no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by ∼80 and ∼90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).

Finite element analysis of neodymium: yttrium-aluminum-garnet incisions for the prevention of anterior capsule contraction syndrome.

BACKGROUND: Anterior capsular contraction syndrome is a potential complication of continuous curvilinear capsulorhexis (CCC). Three neodymium: yttrium-aluminum-garnet (Nd:YAG) laser relaxing incisions decrease anterior capsular contraction but the mechanism is unknown. The present study analyzed the biomechanical mechanism of three Nd:YAG laser relaxing incisions made to reduce anterior capsular contraction. METHODS: A three-dimensional control model and a three-dimensional Nd:YAG model of the anterior capsule with an opening diameter of 6 mm were created. Three incisions of 1 mm in length were made centrifugally at intervals of 120° around the opening circle. The stress alterations of the anterior capsule after CCC with and without Nd:YAG relaxation were numerically simulated and compared. RESULTS: In the control model, the stress was axially uniform in the inner area and relatively high near the inner rim of the opening. Meanwhile, in the Nd:YAG model, the stress level was very low in the inner opening areas, especially near the three incisions. The relaxing incisions in the Nd:YAG model significantly released the relatively high stress on the anterior capsule. Additionally, there was a high stress gradient near the relaxing incisions. CONCLUSION: Biomechanical effects of stress release may be the preventive mechanism of Nd:YAG incision against anterior capsular contraction syndrome.

Human Organic Solute Transporter (hOST): protein interaction and membrane sorting process.

The human organic solute transporter (hOST) is a heterodimer composed of alpha and beta subunits. Physical association of hOSTα and ß subunits is essential for their polarized basolateral plasma membrane localization and function in the export of bile acids and steroids. To understand the role of carboxyl- and amino-tails of OSTß and mechanisms underlying membrane localization of hOST, the effects of tail deletion of the hOSTß subunit and biological reagents on membrane distribution and transport function of hOST were investigated in stably transfected MDCK cells. After deletion of 35 amino acids from the amino-tail of hOSTß, the efflux transport activity and polarized membrane distribution of the truncated hOSTß was abolished. A co-immunoprecipitation study verified that the amino-tail of hOSTß is essential for the association with hOSTα subunit. Treatments with acytochalasin D (interrupting ctin-filaments), bafilomycin A1 (inhibiting vacuolar H(+)-ATPase), brefeldin A (disrupting the Golgi complex), and calphostin C (inhibiting protein kinase C), significantly disrupted the polarized membrane distribution of hOST and markedly reduced transport activity in stably transfected MDCK cells. In summary, the 35 amino acid amino-terminal fragment of hOSTß contains critical information for interaction with the hOSTα subunit and subsequent trafficking to the plasma membrane. These studies suggest that the membrane sorting process of hOST is mediated by a bafilomycin A1-sensitive vesicular pathway that is associated with the actin-cytoskeleton network. The membrane localization of hOST is also partially mediated through a brefeldin A sensitive mechanism, which controls its transit from the ER to Golgi and is regulated by PKC.

The membrane protein ATPase class I type 8B member 1 signals through protein kinase C zeta to activate the farnesoid X receptor.

UNLABELLED: Prior loss-of-function analyses revealed that ATPase class I type 8B member 1 [familial intrahepatic cholestasis 1 (FIC1)] posttranslationally activated the farnesoid X receptor (FXR). Mechanisms underlying this regulation were examined by gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild-type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC zeta) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC zeta directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC zeta in Caco-2 cells resulted in activation of the human apical sodium-dependent bile acid transporter promoter. CONCLUSION: These results demonstrate that FIC1 signals to FXR via PKC zeta. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.

Membrane trafficking of the human organic anion-transporting polypeptide C (hOATPC).

INTRODUCTION: The human organic anion transporting polypeptide C (OATPC) is one of the major transport proteins involved in the enterohepatic circulation of bile salts and plays an important role in vectorial transport of organic anions and drugs across hepatocytes. MATERIALS AND METHODS: In this study, the effects of biological reagents on the membrane localization of OATPC were investigated by confocal microscopy and estrone-3-sulfate transport. RESULTS: Our results demonstrated that the functional membrane expression of fluorescent chimera OATPC-GFP was achieved in non-polarized (COS7 and HEK293) and polarized (MDCK) cells. Both brefeldin A (a Golgi complex disruptor) and bafilomycin A1 (an inhibitor of vacuolar H+-ATPase) treatment significantly decreased the polarized membrane trafficking and markedly reduced the uptake of estrone-3-sulfate ( approximately 40-90%) in OATPC-GFP transfected cells, suggesting that membrane sorting of hOATPC-GFP was mediated by Golgi complex and vacuolar H+-ATPase-related vesicle transport pathways. Treatment with 8-Br-cAMP (a cAMP analog) stimulated OATPC-GFP membrane localization and enhanced estrone-3-sulfate uptake by approximately 20%. The protein kinase A (PKA) inhibitors (H89 and KT5720), but not a PKG inhibitor, blocked the polarized membrane expression of OATPC-GFP and reduced estrone-3-sulfate transport activity. The simultaneous treatment of cells with PKA activator/inhibitor and bafilomycin A1 demonstrated that bafilomycin A1 did not change the effects of 8-Br-cAMP and H89 on the membrane localization of OATPC-GFP compared with the use of 8-Br-cAMP and H89 alone. DISCUSSION: These data suggest that a cAMP-PKA sensitive membrane sorting pathway for OATPC-GFP is independent of the vacuolar H+-ATPase associated (bafilomycin A1 sensitive) vesicle mediated membrane sorting pathway. In contrast, with combined treatment with brefeldin A, neither the PKA-activator (8-Br-cAMP) nor the inhibitor (H89) further altered the plasma membrane expression and transport activity of OATPC-GFP compared with brefeldin A treatment alone. These data suggest that the cAMP-PKA regulation of OATPC membrane expression involves the Golgi complex. When the Golgi apparatus was disrupted by brefeldin A treatment, the effects of cAMP-PKA on the Golgi-to-basolateral surface sorting process of OATPC was also diminished. In summary, the plasma membrane localization of human OATPC is mediated by Golgi complex and vacuolar H+-ATPase vesicle mediated membrane sorting pathways. cAMP-PKA regulates sorting process through the Golgi complex but not the vacuolar H+-ATPase associated vesicular pathway.

Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter.

High-affinity, sodium-dependent dicarboxylate transporter (NaDC3) is responsible for transport of Krebs cycle intermediates and may involve in regulation of aging and life span. Hydropathy analysis predicts that NaDC3 contains 11 or 12 hydrophobic transmembrane (TM) domains. However, the actual membrane topological structure of NaDC3 remains unknown. In this study, confocal immunofluorescence microscopy and membrane biotinylation of epitope-tagged N and C termini of NaDC3 provide evidence of an extracellular C terminus and an intracellular N terminus, indicating an odd number of transmembrane regions. The position of hydrophilic loops within NaDC3 was identified with antibodies against the loops domains combined with cysteine accessibility methods. A confocal image of membrane localization and transport activity assay of the cysteine insertion mutants show behavior similar to that of wild-type NaDC3 in transfected HEK293 cells, suggesting that these mutants retain a native protein configuration. We find that NaDC3 contains 11 transmembrane helices. The loops 1, 3, 5, 7, and 9 face the extracellular side, and loops 2, 4, 6, and 10 face the cytoplasmic side. A re-entrant loop-like structure between TM8 and TM9 may protrude into the membrane. Our results support the topography of 11 transmembrane domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model for NaDC3.

Protein-protein interactions and membrane localization of the human organic solute transporter.

Two proteins that mediate bile acid export from the ileal enterocyte, organic solute transporter (OST)-alpha and -beta, have recently been identified. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interactions, transport functions, and membrane localization of human (h)OST-alpha and -beta proteins were examined. The results demonstrated that coexpression of hOST-alpha and -beta in transfected cells resulted in a three- to fivefold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and nontransfected cells. Confocal microscopy demonstrated plasma membrane colocalization of hOST-alpha and -beta proteins in cells cotransfected with hOST-alpha and -beta cDNAs. Protein-protein interactions between hOST-alpha and -beta were demonstrated by mammalian two-hybrid and coimmunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-alpha abolished its interaction with hOST-beta and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-alpha still interacted with hOST-beta, and majority of this cytoplasmic tail-truncated protein was expressed on the basolateral membrane when it was stably cotransfected with hOST-beta protein in Madin-Darby canine kidney cells. In summary, hOST-alpha and -beta proteins are physically associated. The intracellular carboxyl-terminal domain of hOST-alpha is not essential for this interaction with hOST-beta. The extracellular amino-terminal fragment of hOST-alpha may contain important information for the assembly of the heterodimer and trafficking to the plasma membrane.

Functional role of the C terminus of human organic anion transporter hOAT1.

Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536-550) or the last 30 amino acids (residues 521-550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506-550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506-520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.

Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter.

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).

Association of the 16-kDa subunit c of vacuolar proton pump with the ileal Na+-dependent bile acid transporter: protein-protein interaction and intracellular trafficking.

The rat ileal apical sodium-dependent bile acid transporter (Asbt) transports conjugated bile acids in a Na+-dependent fashion and localizes specifically to the apical surface of ileal enterocytes. The mechanisms that target organic anion transporters to different domains of the ileal enterocyte plasma membrane have not been well defined. Previous studies (Sung, A.-Q., Arresa, M. A., Zeng, L., Swaby, I'K., Zhou, M. M., and Suchy, F. J. (2001) J. Biol. Chem. 276, 6825-6833) from our laboratory demonstrated that rat Asbt follows an apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift. Furthermore, a 14-mer signal sequence that adopts a beta-turn conformation is required for apical localization of rat Asbt. In this study, a vacuolar proton pump subunit (VPP-c, the 16-kDa subunit c of vacuolar H+-ATPase) has been identified as an interacting partner of Asbt by a bacterial two-hybrid screen. A direct protein-protein interaction between Asbt and VPP-c was confirmed in an in vitro pull-down assay and in an in vivo mammalian two-hybrid analysis. Indirect immunofluorescence confocal microscopy demonstrated that the Asbt and VPP-c colocalized in transfected COS-7 and MDCK cells. Moreover, bafilomycin A1 (a specific inhibitor of VPP) interrupted the colocalization of Asbt and VPP-c. A taurocholate influx assay and membrane biotinylation analysis showed that treatment with bafilomycin A1 resulted in a significant decrease in bile acid transport activity and the apical membrane localization of Asbt in transfected cells. Thus, these results suggest that the apical membrane localization of Asbt is mediated in part by the vacuolar proton pump associated apical sorting machinery.

A 14-amino acid sequence with a beta-turn structure is required for apical membrane sorting of the rat ileal bile acid transporter.

The rat ileal sodium-dependent bile acid transporter (Asbt) is a polytopic membrane glycoprotein, which is specifically expressed on the apical domain of the ileal brush-border membrane. In the present study, an essential 14-amino acid (aa 335-348) sorting signal was defined on the cytoplasmic tail of Asbt with two potential phosphorylation sites motifs for casein kinase II ((335)SFQE) and protein kinase C (PKC) ((339)TNK). Two-dimension NMR spectra analysis demonstrated that a tetramer, (340)NKGF, which overlaps with the potential PKC site within the 14-mer signal sequence, adopts a type I beta-turn conformation. Replacement of the potential phosphorylation residue Ser(335) and Thr(339) with alanine or deletion of either the 4 ((335)SFQE) or 10 aa (338-348, containing (339)TNKGF) from the C terminus of Asbt resulted in a significantly decreased initial bile acid transport activity and increased the basolateral distribution of the mutants by 2-3-fold compared with that of wild type Asbt. Deletion of the entire last 14 amino acids (335-348) from the C terminus of Asbt abolished the apical expression of the truncated Asbt. Moreover, replacement of the cytoplasmic tail of the liver basolateral membrane protein, Na(+)/taurocholate cotransporting polypeptide, with the 14-mer peptide tail of Asbt redirected the chimera to the apical domain. In contrast, a chimera consisting of the 14-mer peptide of Asbt fused with green fluorescent protein was expressed in an intracellular transport vesicle-like distribution in transfected Madin-Darby canine kidney and COS 7 cells. This suggests that the apical localization of the 14-mer peptide requires a membrane anchor to support proper targeting. The results from biological reagent treatment and low temperature shift (20 degrees C) suggests that Asbt follows a transport vesicle-mediated apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift.

Inflammatory-mediated repression of the rat ileal sodium-dependent bile acid transporter by c-fos nuclear translocation.

BACKGROUND & AIMS: Ileal malabsorption of bile salts is observed in Crohn's ileitis. We define the transcriptional mechanisms involved in cytokine-mediated repression of the rat apical sodium-dependent bile acid transporter (ASBT). METHODS: ASBT regulation was studied in IL-1beta-treated IEC-6 and Caco-2 cells and in indomethacin-treated rats. RESULTS: Indomethacin-induced ileitis in Lewis rats leads to specific reductions in ileal ASBT messenger RNA and protein levels, whereas c-jun and c-fos are induced. The proinflammatory cytokines interleukin-1beta and tumor necrosis factor repress the activity of the ASBT promoter in Caco-2 and intestinal epithelial cell-6 cells. This effect is blocked by the proteasome inhibitor, MG-132, or by the phosphatidyl inositol 3-kinase inhibitor, wortmannin. Indomethacin (in vivo) or proinflammatory cytokine (in vitro) treatment leads to serine phosphorylation and nuclear translocation of c-fos. Mutation of a 5' activated protein (AP)-1 site inactivates the ASBT promoter, whereas mutation of the 3' site abrogates the proinflammatory cytokine-mediated repression. The 5' site binds a c-jun homodimer, whereas the 3' site binds a c-jun/c-fos heterodimer. c-Jun overexpression enhances ASBT promoter activity, whereas a dominant negative c-jun construct inactivates the promoter. c-Fos overexpression represses promoter activity. A 27 base pair cis-element from the 3' site in the ASBT promoter imparts cytokine-mediated down-regulation to a heterologous SV40 promoter construct. CONCLUSIONS: The ASBT promoter contains 2 distinct cis AP-1 elements; the 5' element binds homodimeric c-jun and mediates basal transcription. Inflammation is associated with up-regulation, phosphorylation, and nuclear translocation of c-fos, which then represses ASBT promoter activity via binding of the 3' AP-1 element by a c-fos/c-jun heterodimer.

Transporter-mediated bile acid uptake causes Ca2+-dependent cell death in rat pancreatic acinar cells.

BACKGROUND & AIMS: The mechanism by which cholelithiasis increases the risk of acute pancreatitis remains obscure. Because bile acids can enter the pancreas either by luminal diffusion or by interstitial leakage during gallstone impaction and pancreatitis is associated with impaired Ca(2+) signaling, we examined the effect of bile acids on pancreatic acinar cell signaling and the associated intracellular events. METHODS: Rat pancreatic acinar cells were isolated by collagenase digestion and the effects of bile acids on [Ca(2+)](i) signaling, cell survival, inflammatory signals, and the molecular and functional expressions of bile uptake transporters were analyzed. RESULTS: Bile acids specifically inhibited the sarco/endoplasmic reticulum Ca(2+) ATPase pump to chronically deplete part of the Ca(2+) stored in the endoplasmic reticulum. This in turn led to the activation of capacitative Ca(2+) entry and a chronic [Ca(2+)](i) load. The increase in [Ca(2+)](i) and Ca(2+) load activated the inflammation-associated signals of c-Jun amino-terminal kinases and NF-kappaB and led to cell death, which was inhibited by buffering [Ca(2+)](i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid. A comprehensive molecular analysis of bile acid transporters revealed that pancreatic acinar cells express the bile uptake transporters Na(+)-taurocholate co-transporting polypeptide and organic anion transporting polypeptide in the luminal and basolateral membranes, respectively. Bile acid uptake into acinar cells was in part Na(+)-dependent and in part Na(+)-independent, suggesting that both transporters contribute to bile acid influx into acinar cells. CONCLUSIONS: These results suggest that bile acids can be transported into pancreatic acinar cells through specific membrane transporters and induce cell death by impairing cellular Ca(2+) signaling.