Ca2+ signaling, TRP channels, and endothelial permeability

Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors trigger increased endothelial permeability by increasing the intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i activates key signaling pathways that mediate cytoskeletal reorganization (through myosin-light-chain-dependent contraction) and the disassembly of VE-cadherin at the adherens junctions. The Ca2+-dependent protein kinase C (PKC) isoform PKCalpha plays a crucial role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca2+]i induced by inflammatory agonists such as thrombin and histamine is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3-receptors, release of stored intracellular Ca2+, and Ca2+ entry through plasma membrane channels. IP3-sensitive Ca2+-store depletion activates plasma membrane cation channels (i.e., store-operated cation channels [SOCs] or Ca2+ release-activated channels [CRACs]) to cause Ca2+ influx into endothelial cells. Recent studies have identified members of Drosophila transient receptor potential (TRP) gene family of channels that encode functional SOCs in endothelial cells. These studies also suggest that the canonical TRPC homologue TRPC1 is the predominant isoform expressed in human vascular endothelial cells, and is the essential component of the SOC in this cell type. Further, evidence suggests that the inflammatory cytokine tumor necrosis factor-alpha can induce the expression of TRPC1 in human vascular endothelial cells signaling via the nuclear factor-kappaB pathway. Increased expression of TRPC1 augments Ca2+ influx via SOCs and potentiates the thrombin-induced increase in permeability in human vascular endothelial cells. Deletion of the canonical TRPC homologue in mouse, TRPC4, caused impairment in store-operated Ca2+ current and Ca2+-store release-activated Ca2+ influx in aortic and lung endothelial cells. In TRPC4 knockout (TRPC4-/-) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4-/- mouse-lung endothelial cells exhibited lack of actin-stress fiber formation and cell retraction in response to thrombin activation of protease-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to PAR-1 activation was inhibited in TRPC4-/- mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling agonist-induced increases in endothelial permeability.     

Cigarette smoke disrupts the integrity of airway adherens junctions through the aberrant interaction of p120‐catenin with the cytoplasmic tail of MUC1

Adherens junctions (AJs) containing epithelial cadherin (E‐cad) bound to p120‐catenin (p120ctn) and β‐catenin (β‐ctn) play a crucial role in regulating cell–cell adhesion. Cigarette smoke abrogates cell–cell adhesion between epithelial cells by disrupting E‐cad, a hallmark of epithelial–mesenchymal transition (EMT), yet the underlying mechanism remains unknown. We used an organotypic culture of primary human bronchial epithelial (HBE) cells treated with smoke‐concentrated medium (Smk) to establish an essential role for the interaction between p120ctn and the cytoplasmic tail of MUC1 (MUC1‐CT) in regulating E‐cad disruption. Within the first 4 h of smoke exposure, apical MUC1‐CT repositioned to the basolateral membrane of pseudo‐stratified HBE cells, where it interacted with p120ctn. A time‐dependent increase in MUC1‐CT/p120ctn complexes occurred in conjunction with a time‐dependent dissociation of p120ctn/E‐cad/β‐ctn complexes, as well as the coordinated degradation of p120ctn and E‐cad. Interestingly, Smk induced a similar interaction between MUC1‐CT and β‐ctn, but this occurred 44 h after MUC1‐CT's initial interaction with p120ctn, and well after the AJs were destroyed. Blocking MUC1‐CT's interaction with p120ctn using a MUC1‐CT dominant‐negative peptide, PMIP, successfully abolished Smk's disruptive effects on AJs and recovered apical‐basolateral polarity of HBE cells. The MUC1‐CT/p120ctn interaction was highly dependent on EGFR/Src/Jnk‐mediated tyrosine phosphorylation (TyrP) of MUC1‐CT. Accordingly, EGFR, Src or Jnk inhibitors (AG1478, PP2, SP600125, respectively) abrogated Smk‐induced MUC1‐CT‐TyrP, MUC1‐CT/p120ctn interaction, AJ disruption, and loss of cellular polarity. Our work identified MUC1‐CT and p120ctn as important regulators of epithelial polarity and cell‐cell adhesion during a smoke‐induced EMT‐like process. Novel therapeutics designed to inhibit MUC1‐CT/p120ctn complex formation may prevent EMT in the smoker's airway. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Reduced N-cadherin expression is associated with metastatic potential and poor surgical outcomes of hepatocellular carcinoma

Background and Aim: N‐cadherin (N‐cad), one of the classic cadherins, has been reported to be involved in tumor metastasis in some types of tumors. This study aims to investigate the expression status of N‐cad in hepatocellular carcinoma (HCC) and the correlation between N‐cad expression and metastatic potential, as well as the surgical outcomes of HCC. Methods: N‐cad expression in HCC and adjacent liver tissues, as well as normal liver tissues, was studied by immunohistochemistry and Western blot, and the relationship between N‐cad expression and the clinicopathological features of HCC was evaluated. By using RNA interference technique, the correlation of N‐cad expression and metastatic potential was investigated by downregulating N‐cad expression in HCCLM3 cells, and the effects of N‐cad downregulation on cell aggregation, migration, and invasion were then analyzed. Furthermore, the correlation between N‐cad expression and the surgical outcomes of a cohort of HCC patients was analyzed. Results: In liver tissues, N‐cad was strongly expressed on cell–cell boundaries, whereas various reduced‐expression patterns were observed in tumors. Of 64 HCC, 34 (53%) tumors showed reduced N‐cad expression, compared with their adjacent liver tissues. The decreased expression of N‐cad was significantly correlated with poorer tumor differentiation (P = 0.001) and vascular invasion (P = 0.003). N‐cad knockdown in HCCLM3 cells resulted in decreased cell aggregation and increased cell migration and invasion. The decreased expression of N‐cad in HCC was significantly associated with shorter postoperative disease‐free survival (P = 0.039). Conclusions: N‐cad expression is decreased in HCC, and the downregulation of N‐cad is associated with the metastatic potential of HCC and poorer surgical prognosis.     

贲门腺癌组织中RKIP、E-cadherin的表达变化及相关性

目的 观察Raf激酶抑制蛋白（RKIP）、上皮型钙黏蛋白（E-cadherin）在贲门腺癌组织中的表达变化,并探讨其相关性.方法 应用免疫组化SP法检测160例贲门腺癌组织及癌旁非肿瘤组织中RKIP、E-cadherin的蛋白表达情况,分析其表达与临床病理参数的关系以及两者表达的相关性.结果 RKIP和E-cadherin在贲门腺癌组织中表达明显低于癌旁组织（P均＜0.05）.RKIP和E-cadherin在高分化、中分化和低分化的贲门腺癌组织中的表达差异有统计学意义（P均＜0.01）,在有淋巴结转移的贲门腺癌组织中表达低于无淋巴结转移的组织（P均＜0.05）.RKIP和E-cadherin在贲门腺癌组织中的表达呈正相关（r=0.388,P＜0.05）.结论 RKIP和E-cadherin 在贲门腺癌组织中表达降低,且两者表达呈正相关.     

Human oral keratinocyte E‐cadherin degradation by Candida albicans and Candida glabrata

J Oral Pathol Med (2010) 39 : 275–278 Background: E‐cadherin (E‐Cad) is a 120‐kDa adhesive protein found in adherens junctions of the digestive tract epithelium. We tested the ability of two Candida strains to degrade human E‐Cad in the Candida virulence factor perspective. Materials and methods: We set out to study oral mucosal E‐Cad degradation by clinical and reference strains of Candida albicans and Candida glabrata. We also included hyphal and secreted aspartic proteinase (Sap) mutants of C. albicans to test the effect of yeast/hyphal transition on the ability to degrade E‐Cad. The tests were performed at pH 4 and pH 6 to determine the effect of local tissue acidity on the activation of Saps. The C. albicans strains used were: CCUG 32723; clinical strain SC5314 which is known to be strongly invasive; hyphal mutants of SC5314: HLC52 (efg1/efg1), HLC54 (cph1/cph1 efg1/efg1) and JKC19 (cph1/cph1); clinical strain B1134; Sap 1–3 and Sap 4–6 mutants of SC5314. The C. glabrata strains used were ATCC 90030, and the clinical strains 5WT and G212. Results: The sonicated yeast cells of C. albicans JKC19 and SC5314, both in hyphal form, degraded E‐Cad at pH 4. The 10× concentrated growth media of the strains HLC‐52, HLC‐54, 32723 and B1134; all in yeast form, caused degradation at pH 4, HLC‐52 and HLC‐54 also at pH 6. The C. glabrata strains did not degrade E‐Cad. Conclusions: pH is a strain dependent triggering factor in activating yeast or hyphal form related Candida Saps in degrading epithelial cell associated E‐Cads.     

Regulation of cellular invasion and matrix metalloproteinase activity in HepG2 cell by connexin 26 transfection.

We previously reported that connexin (Cx) 26 expression is involved in negative growth control of HepG2 cells established from a human hepatoma. We also found that induction of E-cadherin and subsequent formation of a cell adhesion complex were induced in HepG2 cells by Cx 26 expression. To examine the exact role of Cx 26-induced E-cadherin junctions in regulating appearance of malignant phenotypes of HepG2 cells, we expressed a Cx 26 antisense oligodeoxynucleotide (AS-ODN) in an established HepG2 cell clone that has stable expression of Cx 26 genes. We investigated changes in the expression of E-cadherin, the localization of beta-catenin, and some malignant phenotypes of HepG2 clone after the suppression of Cx 26 expression by AS-ODN treatment. The AS-ODN treatment prevented the expression of Cx 26 and E-cadherin, and the localization of beta-catenin was changed from cytoplasmic membrane to the cytoplasm. In parallel, a morphological change from a monolayer of polygonal cells to multilayered colonies was induced by the treatment, indicating a change of a malignant phenotype of HepG2 cells. The activity of matrix metalloproteinase 9 (MMP-9) was elevated by the AS-ODN treatment. A concomitant increase in invasiveness of the Cx 26-expressing cells by the treatment was also observed in an in vitro assay with Matrigel matrix. These results suggest that the induction of E-cadherin and formation of the cell adhesion complex by Cx 26 expression contribute to the reversal of some malignant phenotypes of HepG2 cells. Furthermore, the Cx 26-dependent expression of E-cadherin leads to reduction of the invasiveness of the cells through suppression of MMP-9 activity.