R. Brian Doctor

Evidence for Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) Self-association through PDZ-PDZ Interactions

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a versatile membrane-cytoskeleton linking protein that binds to the COOH-tail of specific integral membrane proteins through its two PDZ domains. These EBP50 binding interactions have been implicated in sequestering interactive sets of proteins into common microdomains, regulating the activity of interacting proteins, and modulating membrane protein trafficking. With only two PDZ domains, it is unclear how EBP50 forms multiprotein complexes. Other PDZ proteins increase their breadth and diversity of protein interactions through oligomerization. Hypothesizing that EBP50 self-associates to amplify its functional capacity, far-Western blotting of cholangiocyte epithelial cell proteins with EBP50 fusion protein revealed that EBP50 binds to a 50-kDa protein. Far-Western blotting of EBP50 isolated by two-dimensional gel electrophoresis or immunoprecipitation demonstrates that the 50-kDa binding partner is itself EBP50. Further, co-transfection/co-precipitation studies show the self-association can occur in an intracellular environment. In vitro analysis of the EBP50-EBP50 binding interaction indicates it is both saturable and of relatively high affinity. Analysis of truncated EBP50 proteins indicates EBP50 self-association is mediated through its PDZ domains. The ability to self-associate provides a mechanism for EBP50 to expand its capacity to form multiprotein complexes and regulate membrane transport events.

Polycystic liver: clinical characteristics of patients with isolated polycystic liver disease compared with patients with polycystic liver and autosomal dominant polycystic kidney disease

Aim: The goal of this study was to compare the clinical features of patients with isolated polycystic liver disease (PCLD) with those of patients with polycystic liver and autosomal dominant polycystic kidney disease (ADPKD).

The lipid products of phosphoinositide 3-kinase contribute to regulation of cholangiocyte ATP and chloride transport.

ATP stimulates Cl− secretion and bile formation by activation of purinergic receptors in the apical membrane of cholangiocytes. The purpose of these studies was to determine the cellular origin of biliary ATP and to assess the regulatory pathways involved in its release. In Mz-Cha-1 human cholangiocarcinoma cells, increases in cell volume were followed by increases in phophoinositide (PI) 3-kinase activity, ATP release, and membrane Cl− permeability. PI 3-kinase signaling appears to play a regulatory role because ATP release was inhibited by wortmannin or LY294002 and because volume-sensitive current activation was inhibited by intracellular dialysis with antibodies to the 110 kDa-subunit of PI 3-kinase. Similarly, in intact normal rat cholangiocyte monolayers, increases in cell volume stimulated luminal Cl− secretion through a wortmannin-sensitive pathway. To assess the role of PI 3-kinase more directly, cells were dialyzed with the synthetic lipid products of PI 3-kinase. Intracellular delivery of phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate activated Cl− currents analogous to those observed following cell swelling. Taken together, these findings indicate that volume-sensitive activation of PI 3-kinase and the generation of lipid messengers modulate cholangiocyte ATP release, Cl− secretion, and, hence, bile formation.

Bicarbonate‐rich choleresis induced by secretin in normal rat is taurocholate‐dependent and involves AE2 anion exchanger

Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR)–dependent Cl− efflux and subsequent biliary HCO3− secretion, possibly via Cl−/HCO3− anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl−/HCO3− exchange in secretin‐stimulated HCO3− secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3− and Cl− excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl− channel inhibitor 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB) or the Cl−/HCO3− exchange inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3− and Cl−. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin‐induced increases in bile flow and HCO3− excretion but not the increased Cl− excretion, revealing a role of biliary Cl−/HCO3− exchange in secretin‐induced, bicarbonate‐rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+‐independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin‐stimulated Cl−/HCO3− exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate‐rich choleresis in the normal rat and that this occurs via a chloride–bicarbonate exchange process consistent with AE2 function. (HEPATOLOGY 2006;43:266–275.)

Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid

The principal extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) involves formation of liver cysts derived from intrahepatic bile ducts. Autocrine and paracrine factors secreted into the cyst would be positioned to modulate the rate of hepatic cyst growth. The aim of this study was to identify potential growth factors present in human ADPKD liver cyst fluid. Cytokine array and enzyme‐linked immunosorbent assay analysis of human ADPKD liver cyst fluid detected epithelial neutrophil attractant 78, interleukin (IL)‐6 (503 ± 121 pg/mL); and IL‐8 (4,488 ± 355 pg/mL); and elevated levels of vascular endothelial growth factor compared with non‐ADPKD bile (849 ± 144 pg/mL vs. 270 pg/mL maximum concentration). ADPKD liver cyst cell cultures also released IL‐8 and vascular endothelial growth factor, suggesting that cystic epithelial cells themselves are capable of secreting these factors. Western blotting of cultured cyst cells and immunostaining of intact cysts demonstrate that cysteine‐X‐cysteine receptor 2, an epithelial neutrophil attractant 78 and IL‐8 receptor, is expressed at the apical domain of cyst lining epithelial cells. Suggesting the cystic epithelial cells may exist in hypoxic conditions, electron microscopy of the ADPKD liver cyst epithelium revealed morphological features similar to those observed in ischemic bile ducts. These features include elongation, altered structure, and diminished abundance of apical microvilli. In conclusion, IL‐8, epithelial neutrophil attractant 78, IL‐6, and vascular endothelial growth factor may serve as autocrine and paracrine factors to direct errant growth of ADPKD liver cyst epithelia. Interruption of these signaling pathways may provide therapeutic targets for inhibiting liver cyst expansion. (Hepatology 2004;40:836–846).

Characterization of ionotrophic purinergic receptors in hepatocytes

Ionotrophic purinergic (P2X) receptors function as receptor‐gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na+ and Ca2+. Based on evidence that extracellular adenosine 5′‐triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP‐dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 2′3′‐O‐(4‐benzoyl‐benzoyl)‐adenosine 5′‐triphosphate (BzATP; 10 μM) caused a rapid increase in membrane Na+ conductance. Similarly, with Fluo‐3 fluorescence, BzATP induced an increase in intracellular [Ca2+]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu2+, and the P2X4 modulators Zn2+ and ivermectin (0.3‐3 μM) each increased intracellular [Ca2+]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen‐rich liver samples. Conclusion: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na+ and Ca2+ transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism. (HEPATOLOGY 2007.)

Characterization of an ankyrin repeat-containing Shank2 isoform (Shank2E) in liver epithelial cells.

Shank proteins are a family of multidomain scaffolding proteins best known for their role in organizing the postsynaptic density region in neurons. Unlike Shank1 and Shank3, Shank2 [also known as Pro-SAP1 (proline-rich synapse-associated protein 1), CortBP1 (cortactin binding protein 1) or Spank-3] has been described as a truncated family member without an N-terminal ankyrin repeat domain. The present study utilized bioinformatics to demonstrate the presence of exons encoding ankyrin repeats in the region preceding the previously described Shank2 gene. cDNA sequencing of mRNA from epithelial cells revealed a novel spliceoform of Shank2, termed Shank2E, that encodes a predicted 200 kDa protein with six N-terminal ankyrin repeats. Shank2 mRNA from epithelial tissues was larger than transcripts in brain. Likewise, the apparent mass of Shank2 protein was larger in epithelial tissues (230 kDa) when compared with brain (165/180 kDa). Immunofluorescence and membrane fractionation found Shank2E concentrated at the apical membrane of liver epithelial cells. In cultured cholangiocytes, co-immunoprecipitation and detergent solubility studies revealed Shank2E complexed with actin and co-distributed with actin in detergent-insoluble lipid rafts. These findings indicate epithelial cells express an ankyrin repeat-containing Shank2 isoform, termed Shank2E, that is poised to co-ordinate actin-dependent events at the apical membrane.

Human podocytes perform polarized, caveolae-dependent albumin endocytosis

The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes.

Distribution of epithelial ankyrin (Ank3) spliceoforms in renal proximal and distal tubules.

In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3(200/215). To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3(200/215) but no Ank3(170) or Ank3(120). Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3(200/215). These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3(200/215) and Na-K-ATPase abundance measured a lower Ank3(200/215)-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1 cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3(200/215) suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.

Extracellular nucleotides stimulate Cl− currents in biliary epithelia through receptor-mediated IP3 and Ca2+ release

Extracellular ATP regulates bile formation by binding to P2 receptors on cholangiocytes and stimulating transepithelial Cl(-) secretion. However, the specific signaling pathways linking receptor binding to Cl(-) channel activation are not known. Consequently, the aim of these studies in human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers was to assess the intracellular pathways responsible for ATP-stimulated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and membrane Cl(-) permeability. Exposure of cells to ATP resulted in a rapid increase in [Ca(2+)](i) and activation of membrane Cl(-) currents; both responses were abolished by prior depletion of intracellular Ca(2+). ATP-stimulated Cl(-) currents demonstrated mild outward rectification, reversal at E(Cl(-)), and a single-channel conductance of approximately 17 pS, where E is the equilibrium potential. The conductance response to ATP was inhibited by the Cl(-) channel inhibitors NPPB and DIDS but not the CFTR inhibitor CFTR(inh)-172. Both ATP-stimulated increases in [Ca(2+)](i) and Cl(-) channel activity were inhibited by the P2Y receptor antagonist suramin. The PLC inhibitor U73122 and the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB both blocked the ATP-stimulated increase in [Ca(2+)](i) and membrane Cl(-) currents. Intracellular dialysis with purified IP3 activated Cl(-) currents with identical properties to those activated by ATP. Exposure of normal rat cholangiocyte monolayers to ATP increased short-circuit currents (I(sc)), reflecting transepithelial secretion. The I(sc) was unaffected by CFTR(inh)-172 but was significantly inhibited by U73122 or 2-APB. In summary, these findings indicate that the apical P2Y-IP3 receptor signaling complex is a dominant pathway mediating biliary epithelial Cl(-) transport and, therefore, may represent a potential target for increasing secretion in the treatment of cholestatic liver disease.