Alternative molecular mechanisms for force transmission at adherens junctions via ß-catenin-vinculin interaction.

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

Branched actin networks push against each other at adherens junctions to maintain cell-cell adhesion.

Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes.

Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity.

Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and ß- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.

Differential expression of cell adhesion molecules in an ionizing radiation-induced breast cancer model system.

Cell-cell adhesion is mediated by members of the cadherin-catenin system and among them E-cadherin and ß-catenin are important adhesion molecules for epithelial cell function and preservation of tissue integrity. To investigate the importance of cell adhesion molecules in breast carcinogenesis, we developed an in vitro breast cancer model system wherein immortalized human breast epithelial cell line, MCF-10F, was malignantly transformed by exposure to low doses of high linear energy transfer (LET) α particle radiation (150 keV/µm) and subsequent growth in the presence or absence of 17ß-estradiol. This model consisted of human breast epithelial cells in different stages of transformation: i) parental cell line MCF-10F; ii) MCF-l0F continuously grown with estradiol at 10(-8) (Estrogen); iii) a non-malignant cell line (Alpha3); and iv) a malignant and tumorigenic cell line (Alpha5) and the Tumor2 cell line derived from the nude mouse xenograft of the Alpha5 cell line. Expression levels of important cell adhesion molecules such as α-catenin, ß-catenin, γ-catenin, E-cadherin and integrin were found to be higher at the protein level in the Alpha5 and Tumor2 cell lines relative to these levels in the non-tumorigenic MCF-10F, Estrogen and Alpha3 cell lines. In corroboration, cDNA expression analysis revealed elevated levels of genes involved in the cell adhesion function [E-cadherin, integrin ß6 and desmocollin3 (DSc3)] in the Alpha5 and Tumor2 cell lines relative to the levels in the MCF-10F, Estrogen and Alpha3 cell lines. Collectively, our results suggest that cell adhesion molecules are expressed at higher levels in malignantly transformed breast epithelial cells relative to levels in non-malignant cells. However, reduced levels of adhesion molecules observed in the mouse xenograft-derived Tumor 2 cell line compared to the pre-tumorigenic Alpha5 cell line suggests that the altered expression levels of adhesion molecules depend on the tumor tissue microenvironment.

Cell-to-cell contact dependence and junctional protein content are correlated with in vivo maturation of pancreatic beta cells.

In this study, we investigated the cellular distribution of junctional proteins and the dependence on cell-cell contacts of pancreatic beta cells during animal development. Fetus and newborn rat islets, which display a relatively poor insulin secretory response to glucose, present an immature morphology and cytoarchitecture when compared with young and adult islets that are responsive to glucose. At the perinatal stage, beta cells display a low junctional content of neural cell adhesion molecule (N-CAM), α- and ß-catenins, ZO-1, and F-actin, while a differential distribution of N-CAM and Pan-cadherin was seen in beta cells and nonbeta cells only from young and adult islets. In the absence of intercellular contacts, the glucose-stimulated insulin secretion was completely blocked in adult beta cells, but after reaggregation they partially reestablished the secretory response to glucose. By contrast, neonatal beta cells were poorly responsive to sugar, regardless of whether they were arranged as intact islets or as isolated cells. Interestingly, after 10 days of culturing, neonatal beta cells, known to display increased junctional protein content in vitro, became responsive to glucose and concomitantly dependent on cell-cell contacts. Therefore, our data suggest that the developmental acquisition of an adult-like insulin secretory pattern is paralleled by a dependence on direct cell-cell interactions.

p120 catenin/αN-catenin are molecular targets in the neuroprotection and neuronal plasticity mediated by atorvastatin after focal cerebral ischemia.

Atorvastatin (ATV), a 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, exerts beneficial effects on stroke through several pleiotropic mechanisms. However, its role following cerebral ischemia is not completely understood yet. We evaluated the effect of ATV treatment on the synaptic adhesion proteins after a transient middle cerebral artery occlusion (t-MCAO) model in rats. Ischemic male Wistar rats were treated with 10 mg/kg ATV. The first dose was 6 hr after reperfusion, then every 24 hr for 3days. Our findings showed that ATV treatment produced an increase in pAkt ser473 and a decrease in pMAPK 44/42 protein levels 12 and 24 hr postischemia in the cerebral cortex and the hippocampus. However, p120 catenin and αN-catenin became drastically increased throughout the temporal course of postischemia treatment (12-72 hr), mainly in the hippocampus. Neurological recovery was observed at 48 and 72 hr, supported by a significant reduction of infarct volume, neuronal loss, and glial hyperreactivity after 72 hr of postischemia treatment with ATV. ATV treatment also up-regulated the association of p120(ctn) , αN-catenin to PSD-95, accompanied by a reduction of RhoA activation and the recovery of MAP2 immunoreactivity, these being significantly affected by the focal cerebral ischemia. Our findings suggested that p120(ctn) and αN-catenin synaptic adhesion proteins are crucial molecular targets in ATV-mediated neuroprotection and neuronal plasticity after focal cerebral ischemia.

T3 regulates E-cadherin, and ß- and α-catenin expression in the stomach during the metamorphosis of the toad Rhinella arenarum.

The metamorphosis of Rhinella arenarum was induced precociously for 5 days, then blocked for 3 months to evaluate the role of thyroid hormones as modulators of morphoregulatory molecules such as E-cadherin, and ß- and α-catenin during epithelium remodeling. We then performed an immunohistochemical and morphometric study of these molecules in the larval stomach. We show that 3,5,3'-triiodothyronine exerts a positive regulatory effect on E-cadherin and ß- and α-catenin expression in stomach epithelium. This suggests continuous synthesis of E-cadherin, and ß- and α-catenin; synthesis essentially is thyroid hormone-independent during premetamorphosis and early prometamorphosis, but it becomes thyroid hormone-dependent during metamorphic climax.

Expression of cell adhesion molecules in the normal and T3 blocked development of the tadpole's kidney of Bufo arenarum (Amphibian, Anuran, Bufonidae).

Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, beta-and alpha-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of beta-and alpha-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of beta-and alpha-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, beta-catenin and alpha-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.

Expression of cell adhesion molecules in the normal and T3 blocked development of the tadpole's kidney of Bufo arenarum (Amphibian, Anuran, Bufonidae) / Expressão das moléculas de adesão celular no desenvolvimento normal e com a inibição do hormônio tireoidea do rim nas larvas do Bufo arenarum (Amphibia, Anura, Bufonidae)

Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, β-and α-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of β-and α-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of β-and α-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, β-catenin and α-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.

Moléculas de adesão celular atuam como tradutores do ambiente extracelular para o citoesqueleto e o núcleo e, conseqüentemente, induzindo mudanças no padrão da expressão das proteínas estruturais. Neste estudo, observamos os efeitos da inibição do hormônio tireóidea (TH) e detenção da metamorfose na expressão da E-caderina, β- e α- catenina no desenvolvimento do rim do Bufo arenarum. As moléculas de adesão celular durante o desenvolvimento têm uma expressão temporal e espacial seletiva, sugerindo um papel específico na nefrogênese. Com o propósito de estudar os mecanismos de controle da expressão das moléculas de adesão durante o desenvolvimento renal, bloqueou-se a metamorfose do B. arenarum com uma substancia goitrogênica que bloqueia a síntese de TH. A expressão da E-caderina nos tubos proximais é independente do controle da tireóide. Entretanto, o bloqueio da síntese de TH provoca uma sobre elevação da E-caderina nos dutos coletores, nos tubos distais e nos glomérulos. A expressão da β- e α-catenina nos dutos coletores, nos tubos distais, nos glomérulos e no mesênquima mesonéfrico é independente da TH. O bloqueio da TH causa uma sobre-regulação da β- e α-catenina nos tubos proximais. Em contraste com a E-caderina, a expressão da caderina desmossomal demogloína 1 (Dsg-1) é ausente no controle durante a metamorfose da fase larval dos rins e se expressa em algumas células intersticiais nas larvas tratadas com KClO4. De acordo com este trabalho, a expressão Dsg-1 é subregulada pela TH. Demonstramos que a expressão da E-caderina, Dsg-1, β-catenina e α-catenina são afetadas de forma diferencial pelos níveis de TH, sugerindo um dependência hormonal destas proteínas na metamorfose renal do B. arenarum.

[Immunohistochemical expression of the E-cadherin-catenin complex in gastric cancer]. / Expresión inmunohistoquímica del complejo E-caderina-catenina en cáncer gástrico. Relación con variables clínico-morfológicas y sobrevida de pacientes.

BACKGROUND: The E-cadherin/catenin complex plays an essential role in the control of epithelial differentiation. Abnormal expression in tumors correlates with histological grade, advanced stage and poor prognosis. AIM: To evaluate the expression pattern of E-cadherin/catenin complex in gastric carcinoma and analyze their association with tumor clinicopathological features and patient survival. MATERIAL AND METHODS: Inmunohistochemical staining of E-cadherin, alpha and ss-catenin was performed from paraffin specimens of 65 gastric carcinomas. RESULTS: Abnormal expression of E-cadherin, alpha and ss-catenin was demonstrated in 82%, 85% and 88% of gastric carcinomas, respectively. There was a significant correlation between abnormal expression and Lauren pathological classification and depth of infiltration, but not with tumor stage, positive lymph node metastases and survival. CONCLUSION: Abnormal expression of E-cadherin, alpha and ss-catenin occurs frequently in gastric carcinoma and correlates with histological grade.

Expresión inmunohistoquímica del complejo E-caderina-catenina en cáncer gástrico: Relación con variables clínico-morfológicas y sobrevida de pacientes / Immunohistochemical expression of the E-cadherin-catenin complex in gastric cancer

Background: The E-cadherin/catenin complex plays an essential role in the control of epithelial differentiation. Abnormal expression in tumors correlates with histological grade, advanced stage and poor prognosis. Aim: To evaluate the expression pattern of E-cadherin/catenin complex in gastric carcinoma and analyze their association with tumor clinicopathological features and patient survival. Material and Methods: Inmunohistochemical staining of E-cadherin, alpha and ß-catenin was performed from paraffin specimens of 65 gastric carcinomas. Results: Abnormal expression of E-cadherin, alpha and ß-catenin was demonstrated in 82 percent, 85 percent and 88 percent of gastric carcinomas, respectively. There was a significant correlation between abnormal expression and Lauren pathological classification and depth of infiltration, but not with tumor stage, positive lymph node metastases and survival. Conclusion: Abnormal expression of E-cadherin, alpha and ß-catenin occurs frequently in gastric carcinoma and correlates with histological grade.