Circulating Anti-cytolethal Distending Toxin B and Anti-vinculin Antibodies as Biomarkers in Community and Healthcare Populations With Functional Dyspepsia and Irritable Bowel Syndrome.

OBJECTIVES: Anti-cytolethal distending toxin B (CdtB) and anti-vinculin antibodies have been proposed as biomarkers that discriminate irritable bowel syndrome (IBS) diarrhea from inflammatory bowel disease; however, it is unknown whether they can also discriminate patients with IBS and IBS subtypes and functional dyspepsia (FD) from healthy individuals in the general population. We aimed to determine whether anti-CdtB and anti-vinculin can discriminate IBS and FD from health and from organic gastrointestinal (GI) disease. METHODS: Adults were enrolled from 2 Australian studies: (i) a random, population-based study (n = 331) with subjects diagnosed with IBS (n = 63) or FD (n = 61) by modified Rome III criteria or healthy control subjects (n = 246) who did not meet criteria for IBS and/or FD and (ii) an outpatient-based study with subjects diagnosed with IBS (n = 256) and/or FD (n = 55) or organic GI disease (n = 182) by an independent clinician. Serum levels of anti-CdtB/anti-vinculin antibodies were determined by enzyme-linked immunosorbent assay. RESULTS: There was a significantly higher mean value of anti-CdtB in FD vs healthy controls (mean = 2.46 [SD = 0.72] vs mean = 2.14 [SD = 0.77]; P = 0.005) and IBS/FD overlap vs healthy controls (mean = 2.47 [SD = 0.78] vs mean = 2.14 [SD = 0.77]; P = 0.02). There were no significant differences in anti-CdtB in IBS and FD outpatients or IBS/FD subgroups compared with patients with organic GI disease. In terms of anti-vinculin, there were no significant differences between IBS and FD and healthy controls or between IBS and FD and organic GI disease controls. DISCUSSION: We did not confirm that anti-CdtB/anti-vinculin discriminated IBS diarrhea from organic GI disease in Australian subjects. However, we did find higher anti-CdtB in FD and IBS/FD overlap vs healthy controls. Postinfectious FD may be more common than currently recognized.

Binding of Vinculin to Lipid Membranes in Its Inhibited and Activated States.

Phosphoinositols are an important class of phospholipids that are involved in a myriad of cellular processes, from cell signaling to motility and adhesion. Vinculin (Vn) is a major adaptor protein that regulates focal adhesions in conjunction with PIP2 in lipid membranes and other cytoskeletal components. The binding and unbinding transitions of Vn at the membrane interface are an important link to understanding the coordination of cell signaling and motility. Using different biophysical tools, including atomic force microscopy combined with confocal fluorescence microscopy and Fourier transform infrared spectroscopy, we studied the nanoscopic interactions of activated and autoinhibited states of Vn with lipid membranes. We hypothesize that a weak interaction occurs between Vn and lipid membranes, which leads to binding of autoinhibited Vn to supported lipid bilayers, and to unbinding in freestanding lipid vesicles. Likely driving forces may include tethering of the C-terminus to the lipid membrane, as well as hydrophobic helix-membrane interactions. Conversely, activated Vn binds strongly to membranes through specific interactions with clusters of PIP2 embedded in lipid membranes. Activated Vn harbored on PIP2 clusters may form small oligomeric interaction platforms for further interaction partners, which is necessary for the proper function of focal adhesion points.

Clinical experience with the use of anti-CdtB and anti-vinculin antibodies in patients with diarrhea in Mexico. / Experiencia clínica con el uso de los anticuerpos anti-CdtB y anti-vinculina en pacientes con diarrea en México.

INTRODUCTION: Circulating anti-CdtB/anti-vinculin antibodies have been validated as biomarkers to distinguish IBS-D from IBD, but there is no experience with them in Latin America. MATERIALS AND METHODS: The analysis was carried out on patients seen at a FGIDs/motility clinic over the last 7 months for diarrhea with abdominal pain and/or bloating who were tested for these antibodies. The patients were diagnosed according to the Rome III criteria or with organic disorders, and those presenting with IBS were further classified as post-infectious (PI) or non-PI-IBS. RESULTS: Thirty patients were studied. Positive biomarkers were found in IBS-D and IBS-D Overlap (58.8%) and IBS-M (33.3%), with no differences between PI-IBS (71.4%) vs. non-PI-IBS (41.7%) subjects (P=.21). There was no positivity in patients with other FGIDs or organic diarrhea, except for one with small intestinal bacterial overgrowth (SIBO). CONCLUSIONS: Our findings support the use of this test as a first-line diagnostic tool to confirm the presence of IBS-D/IBS-M according to the Rome III criteria.

Integrin-mediated internalization of Staphylococcus aureus does not require vinculin.

BACKGROUND: Disease manifestations of Staphylococcus aureus are connected to the fibronectin (Fn)-binding capacity of these Gram-positive pathogens. Fn deposition on the surface of S. aureus allows engagement of α5ß1 integrins and triggers uptake by host cells. For several integrin- and actin-associated cytoplasmic proteins, including FAK, Src, N-WASP, tensin and cortactin, a functional role during bacterial invasion has been demonstrated. As reorganization of the actin cytoskeleton is critical for bacterial entry, we investigated whether vinculin, an essential protein linking integrins with the actin cytoskeleton, may contribute to the integrin-mediated internalization of S. aureus. RESULTS: Complementation of vinculin in vinculin -/- cells, vinculin overexpression, as well as shRNA-mediated vinculin knock-down in different eukaryotic cell types demonstrate, that vinculin does not have a functional role during the integrin-mediated uptake of S. aureus. CONCLUSIONS: Our results suggest that vinculin is insignificant for the integrin-mediated uptake of S. aureus despite the critical role of vinculin as a linker between integrins and F-actin.

Gene expression profiling and pathway analysis identify the integrin signaling pathway to be altered by IL-1ß in human pancreatic cancer cells: role of JNK.

The pro-inflammatory cytokine, IL-1ß, is a critical component of the persistent inflammatory milieu that pancreatic cancer cells frequently encounter. Although several studies report diverse mechanisms responsible for this association, yet a comprehensive global analysis of the effect of IL-1ß in these cells is not clearly evident. In this study, we performed whole genome transcriptome analysis of control and IL-1ß treated human pancreatic MIA PaCa-2 cells, validated the most targeted pathway and evaluated the role of JNK therein. 225 Genes were up-regulated and 1215 were down-regulated and these were categorized into biological processes and cellular pathways using the PANTHER classification system. The altered genes categorized into significant biological processes that included those of cell cycle, mitosis, transport and intracellular protein trafficking. The integrin signaling pathway emerged as harboring the maximum number of differentially expressed genes. Two important genes of this pathway, namely vinculin and α5-integrin were validated and both depicted significant inhibition by IL-1ß that was prevented in the presence of JNK siRNA. In a wound healing assay, IL-1ß increased the migratory rate of MIA PaCa-2 and Panc-1 cells that was abrogated by JNK inhibition. Additionally, vinculin and α-integrin siRNAs also increased the migration of these cells along the wound edge. These results suggest that in these pancreatic cancer cells, IL-1ß inhibits components of the integrin signaling pathway in a JNK dependent manner that contributes to their increased migratory potential. Therefore, JNK might be potentially targeted to prevent the migration and invasion of pancreatic cancer cells.

Myosin II activity dependent and independent vinculin recruitment to the sites of E-cadherin-mediated cell-cell adhesion.

BACKGROUND: Maintaining proper adhesion between neighboring cells depends on the ability of cells to mechanically respond to tension at cell-cell junctions through the actin cytoskeleton. Thus, identifying the molecules involved in responding to cell tension would provide insight into the maintenance, regulation, and breakdown of cell-cell junctions during various biological processes. Vinculin, an actin-binding protein that associates with the cadherin complex, is recruited to cell-cell contacts under increased tension in a myosin II-dependent manner. However, the precise role of vinculin at force-bearing cell-cell junctions and how myosin II activity alters the recruitment of vinculin at quiescent cell-cell contacts have not been demonstrated. RESULTS: We generated vinculin knockdown cells using shRNA specific to vinculin and MDCK epithelial cells. These vinculin-deficient MDCK cells form smaller cell clusters in a suspension than wild-type cells. In wound healing assays, GFP-vinculin accumulated at cell-cell junctions along the wound edge while vinculin-deficient cells displayed a slower wound closure rate compared to vinculin-expressing cells. In the presence of blebbistatin (myosin II inhibitor), vinculin localization at quiescent cell-cell contacts was unaffected while in the presence of jasplakinolide (F-actin stabilizer), vinculin recruitment increased in mature MDCK cell monolayers. CONCLUSION: These results demonstrate that vinculin plays an active role at adherens junctions under increased tension at cell-cell contacts where vinculin recruitment occurs in a myosin II activity-dependent manner, whereas vinculin recruitment to the quiescent cell-cell junctions depends on F-actin stabilization.

Vinculin regulates cell-surface E-cadherin expression by binding to beta-catenin.

Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Deletion studies are consistent with the idea that vinculin is important for the organization of cell-cell junctions. However, this approach removes vinculin from both cell-matrix and cell-cell adhesions, making it impossible to distinguish its contribution at each site. To define the role of vinculin in cell-cell junctions, we established a powerful short hairpin-RNA-based knockdown/substitution model system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cadherin-dependent adhesion was reduced. These defects resulted from impaired E-cadherin cell-surface expression. We have investigated the mechanism for the effects of vinculin and found that the reduced surface E-cadherin expression could be rescued by introduction of vinculin, but not of a vinculin A50I substitution mutant that is defective for beta-catenin binding. These findings suggest that an interaction between beta-catenin and vinculin is crucial for stabilizing E-cadherin at the cell surface. This was confirmed by analyzing a beta-catenin mutant that fails to bind vinculin. Thus, our study identifies vinculin as a novel regulator of E-cadherin function and provides important new insight into the dynamic regulation of adherens junctions.

Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells.

BACKGROUND: CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions. METHODS: Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells. RESULTS: Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression. CONCLUSION: CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

Coincidence of actin filaments and talin is required to activate vinculin.

Vinculin regulates cell adhesion by strengthening contacts between extracellular matrix and the cytoskeleton. Binding of the integrin ligand, talin, to the head domain of vinculin and F-actin to its tail domain is a potential mechanism for this function, but vinculin is autoinhibited by intramolecular interactions between its head and tail domain and must be activated to bind talin and actin. Because autoinhibition of vinculin occurs by synergism between two head and tail interfaces, one hypothesis is that activation could occur by two ligands that coordinately disrupt both interfaces. To test this idea we use a fluorescence resonance energy transfer probe that reports directly on activation of vinculin. Neither talin rod, VBS3 (a talin peptide that mimics a postulated activated state of talin), nor F-actin alone can activate vinculin. But in the presence of F-actin either talin rod or VBS3 induces dose-dependent activation of vinculin. The activation data are supported by solution phase binding studies, which show that talin rod or VBS3 fails to bind vinculin, whereas the same two ligands bind tightly to vinculin head domain (K(d) approximately 100 nM). These data strongly support a combinatorial mechanism of vinculin activation; moreover, they are inconsistent with a model in which talin or activated talin is sufficient to activate vinculin. Combinatorial activation implies that at cell adhesion sites vinculin is a coincidence detector awaiting simultaneous signals from talin and actin polymerization to unleash its scaffolding activity.

Heterozygous inactivation of the vinculin gene predisposes to stress-induced cardiomyopathy.

Vinculin and its muscle splice variant metavinculin link focal adhesions and cell-to-cell contact sites to the actin cytoskeleton. We hypothesized that normal expression of vinculin isoforms would be essential for integrity of cardiomyocytes and preservation of normal cardiac function. We studied heterozygous vinculin knockout mice (Vin+/-) that develop and breed normally. The Vin+/- mice displayed: 1) a 58% reduction of vinculin and a 63% reduction of metavinculin protein levels versus wild-type littermates; 2) normal basal cardiac function and histology but abnormal electrocardiograms, intercalated disks, and ICD-related protein distribution; 3) increased mortality following acute hemodynamic stress imposed by transverse aortic constriction (TAC); 4) cardiac dysfunction by 6 weeks post-TAC; and 5) misalignment of alpha-actinin containing Z-lines and abnormal myocardial ultrastructure despite preserved cardiac function. Decreased expression of vinculin/metavinculin leads to abnormal myocyte structure without baseline physiological evidence of cardiac dysfunction. These structural changes predispose to stress-induced cardiomyopathy.

Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion.

Cell migration involves many steps, including membrane protrusion and the development of new adhesions. Here we have investigated whether there is a link between actin polymerization and integrin engagement. In response to signals that trigger membrane protrusion, the actin-related protein (Arp)2/3 complex transiently binds to vinculin, an integrin-associated protein. The interaction is regulated, requiring phosphatidylinositol-4,5-bisphosphate and Rac1 activation, and is sufficient to recruit the Arp2/3 complex to new sites of integrin aggregation. Binding of the Arp2/3 complex to vinculin is direct and does not depend on the ability of vinculin to associate with actin. We have mapped the binding site for the Arp2/3 complex to the hinge region of vinculin, and a point mutation in this region selectively blocks binding to the Arp2/3 complex. Compared with WT vinculin, expression of this mutant in vinculin-null cells results in diminished lamellipodial protrusion and spreading on fibronectin. The recruitment of the Arp2/3 complex to vinculin may be one mechanism through which actin polymerization and membrane protrusion are coupled to integrin-mediated adhesion.