Mutations in the WNK4 gene cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder of hyperkalemia and hypertension. The target molecules of this putative kinase and the molecular mechanisms by which the mutations cause the phenotypes are currently unknown. Although recent reports found that expression of WNK4 in Xenopus oocytes causes inhibition of the thiazide-sensitive NaCl cotransporter and the renal K channel ROMK, there may be additional targets of WNK4. For example, an increase in paracellular chloride permeability has been postulated to be a mediator of PHAII pathogenesis, a possibility supported by the localization of WNK4 at tight junctions in vivo. To determine the validity of this hypothesis, we measured transepithelial Na and Cl permeability in Madin-Darby canine kidney II cells stably expressing wild-type or a pathogenic mutant of WNK4. We found that transepithelial paracellular Cl permeability was increased in cells expressing a disease-causing mutant WNK4 (D564A) but that Na permeability was decreased slightly. Furthermore, WNK4 bound and phosphorylated claudins 1-4, major tight-junction membrane proteins known to be involved in the regulation of paracellular ion permeability. The increases in phosphorylation of claudins were greater in cells expressing the mutant WNK4 than in cells expressing wild-type protein. These results clearly indicate that the pathogenic WNK4 mutant possesses a gain-of-function activity and that the claudins may be important molecular targets of WNK4 kinase. The increased paracellular ''chloride shunt'' caused by the mutant WNK4 could be the pathogenic mechanism of PHAII.

BACKGROUND & AIMS: Long-term outcomes of patients with branch-duct intraductal papillary mucinous neoplasms (IPMNs), particularly those after 5 years of surveillance, have not been fully evaluated in large studies. We analyzed incidences of IPMN-derived carcinoma and concomitant ductal adenocarcinoma (pancreatic ductal adenocarcinoma [PDAC]) over 20 years in a large population of patients. METHODS: We identified 1404 consecutive patients (52% women; mean age, 67.5 years) with a diagnosis of branch-duct IPMN, from 1994 through 2017, at the University of Tokyo in Japan. Using a competing risk analysis, we estimated cumulative incidence of pancreatic carcinoma, overall and by carcinoma type. We used competing risks proportional hazards models to estimate subdistribution hazard ratios (SHRs) for incidences of carcinomas. To differentiate IPMN-derived and concomitant carcinomas, we collected genomic DNA from available paired samples of IPMNs and carcinomas and detected mutations in GNAS and KRAS by polymerase chain reaction and pyrosequencing. RESULTS: During 9231 personyears of follow-up, we identified 68 patients with pancreatic carcinomas (38 patients with IPMN-derived carcinomas and 30 patients with concomitant PDACs); the overall incidence rates were 3.3%, 6.6%, and 15.0% at 5, 10, and 15 years, respectively. Among 804 patients followed more than 5 years, overall cumulative incidence rates of pancreatic carcinoma were 3.5% at 10 years and 12.0% at 15 years from the initial diagnosis. The size of the IPMN and the diameter of the main pancreatic duct associated with incidence of IPMNderived carcinoma (SHR 1.85; 95% confidence interval 1.38-2.48 for a 10-mm increase in the IPMN size and SHR 1.56; 95% confidence interval 1.33-1.83 for a 1-mm increase in the main pancreatic duct diameter) but not with incidence of concomitant PDAC. CONCLUSIONS: In a large long-term study of patients with branch-duct IPMNs, we found the 5year incidence rate of pancreatic malignancy to be 3.3%, reaching 15.0% at 15 years after IPMN diagnosis. We observed heterogeneous risk factor profiles between IPMNderived and concomitant carcinomas.

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Bone marrow (BM)-derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)؊2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E 2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3 ؊/؊ mice and EP4 ؊/؊ mice, in which stromal expression of CXCL12/ CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4 Recent advances in tumor biology have identified the stroma as an important regulator of carcinogenesis and a potentially valuable therapeutic target. Although interactions between the epithelium and stroma have long been considered to be important in tumor progression, the efficacy of targeting stromal components as a therapeutic strategy has not been established, because the specific regulators of such interactions remain unclear. In addition to endothelial cells, macrophages and fibroblasts 1 are the major stromal components of the tumor microenvironment, and they play key roles in the enhancement of angiogenesis. It has recently been established that bone marrow (BM)-derived hematopoietic cells are the major components of the stroma of tumors, and that they determine the tumor microenvironment [2][3][4][5][6][7][8] ; however, the specific factors that enhance the functions of BM-derived precursor cells, and the mechanism of recruitment of these cells during tumor angiogenesis, are not fully understood. Tumor-associated angiogenesis in the tumor stroma is a prerequisite for invasive growth of a tumor larger than 2 to 3 mm in diameter, and then metas-

Background & AimsHepatitis B virus (HBV) infection is a major health concern worldwide. Although currently used nucleos(t)ide analogs efficiently inhibit viral replication, viral proteins transcribed from the episomal viral covalently closed circular DNA (cccDNA) minichromosome continue to be expressed long-term. Because high viral RNA or antigen loads may play a biological role during this chronicity, the elimination of viral products is an ultimate goal of HBV treatment. HBV regulatory protein X (HBx) was recently found to promote transcription of cccDNA with degradation of Smc5/6 through the interaction of HBx with the host protein DDB1. Here, this protein–protein interaction was considered as a new molecular target of HBV treatment.MethodsTo identify candidate compounds that target the HBx–DDB1 interaction, a newly constructed split luciferase assay system was applied to comprehensive compound screening. The effects of the identified compounds on HBV transcription and cccDNA maintenance were determined using HBV minicircle DNA, which mimics HBV cccDNA, and the natural HBV infection model of human primary hepatocytes.ResultsWe show that nitazoxanide (NTZ), a thiazolide anti-infective agent that has been approved by the FDA for protozoan enteritis, efficiently inhibits the HBx–DDB1 protein interaction. NTZ significantly restores Smc5 protein levels and suppresses viral transcription and viral protein production in the HBV minicircle system and in human primary hepatocytes naturally infected with HBV.ConclusionsThese results indicate that NTZ, which targets an HBV-related viral–host protein interaction, may be a promising new therapeutic agent and a step toward a functional HBV cure.

ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.

Vascular endothelial growth factor (VEGF) and its receptors promote liver regeneration. The objective of the present study was to examine the role of VEGF receptor 1 (VEGFR1) signaling in hepatic tissue repair after acetaminophen (N-acetyl-para-aminophenol) (APAP)-induced liver injury. To do this, we treated VEGFR1 tyrosine kinase knockout (VEGFR1 TK(-/-)) and wild-type (WT) mice with APAP (300 mg/kg, ip). In WT mice, serum levels of alanine aminotransferase (ALT) and the necrotic area peaked between 8 and 24 h and then declined. In VEGFR1 TK(-/-) mice, ALT levels remained high at 48 h and extensive hepatic necrosis and hemorrhage were observed, as well as high mortality. Downregulation of hepatic messenger RNA expression of VEGFR1 and VEGFR2 was also noted in VEGFR1 TK(-/-) mice. VEGFR1 TK(-/-) mice displayed lower expression of proliferating cell nuclear antigen and of growth factors including hepatocyte growth factor, CD31, and basic fibroblast growth factor than WT. The hepatic microvasculature in VEGFR1 TK(-/-) was compromised as evidenced by impaired sinusoidal perfusion, suppressed endocytosis in liver sinusoidal endothelial cells (LSECs), and the formation of large gaps in LSECs. In WT mice, immunofluorescence revealed that recruited VEGFR1(+) cells in the necrotic area were positive for CD11b. VEGFR1 TK(-/-) exhibited fewer VEGFR1(+) and VEGFR2(+) cells. These results suggest that VEGFR1 signaling facilitates liver recovery from APAP hepatotoxicity by preventing excessive hemorrhage and reconstituting the sinusoids through recruitment of VEGFR1-expressing macrophages to the injured area and also through affecting expression of genes including hepatotrophic and pro-angiogenic growth factors.

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