Cytoplasmic Domain Of E-cadherin Research Articles

Reconstitution of the full transmembrane cadherin-catenin complex

The dynamic regulation of epithelial adherens junctions relies on all components of the E-cadherin-catenin complex. Previously, the complexes have been partially reconstituted and composed only of α-catenin, β-catenin, and the E-cadherin cytoplasmic domain. However, p120-catenin and the full-length E-cadherin including the extracellular, transmembrane, and intra-cellular domains are vital to the understanding of the relationship between extracellular adhesion and intracellular signaling. Here, we reconstitute the complete and full-length cadherin-catenin complex, including full-length E-cadherin, α-catenin, β-catenin, and p120-catenin, into nanodiscs. We are able to observe the cadherin in nanodiscs by cryo-EM. We also reconstitute α-catenin, β-catenin, and p120-catenin with the E-cadherin cytoplasmic tail alone in order to analyze the affinities of their binding interactions. We find that p120-catenin does not associate strongly with α- or β-catenin and binds much more transiently to the cadherin cytoplasmic tail than does β-catenin. Overall, this work creates many new possibilities for biochemical studies understanding transmembrane signaling of cadherins and the role of p120-catenin in adhesion activation.

E-cadherin clone 36 nuclear staining dictates adverse disease outcome in lobular breast cancer patients

Breast cancer is a heterogeneous disease and additional biomarkers for individually predicting patient outcomes are needed. Aberrant membrane E-cadherin immunoexpression has been demonstrated in lobular breast cancer. Also, E-cadherin nuclear staining has been reported, associating with prognosis in various tumors. Here, we explore whether membrane or nuclear staining of E-cadherin has the potential to dictate prognosis of patients with lobular breast cancer. We selected a cohort of 285 consecutively diagnosed lobular breast cancer patients and performed immunohistochemistry for E-cadherin (clones 36, EP700Y, and NCH38) and P-cadherin (clone 56C1) in representative formalin-fixed paraffin-embedded blocks. All patients were female, HER2-negative and surgically treated in a single institution. Survival curves were computed by Kaplan-Meier analysis. Hazard ratios and respective 95% confidence intervals were estimated using Cox regression models. Statistical significance was set at p < 0.05. Nuclear staining for E-cadherin clone 36 was frequent (35%), contrarily to other antibodies tested. Negative correlation was found between nuclear and membrane E-cadherin clone 36 immunostaining (rs = −0.30, p < 0.001), whereas positive correlation was found between membrane immunoexpression of E-cadherin clone 36 and P-cadherin (rs = 0.31, p < 0.001). Patients with any evidence of E-cadherin clone 36 nuclear immunostaining disclosed significantly worse overall survival, disease-specific-survival and disease/progression-free survival (hazard ratio = 2.059, 95% confidence interval 1.313–3.230; hazard ratio = 1.980, 95% confidence interval 1.121–3.495; and hazard ratio = 2.341, 95% confidence interval 1.403–3.905, respectively). Differences in survival were more remarkable when considering nuclear E-cadherin immunoexpression in ≥50% tumor cells. Poorer survival was maintained in multivariable analysis, after adjusting for age, menopausal and PR status, treatment course, vascular invasion, tumor grade and stage. Our results support the use of antibodies against the cytoplasmic domain of E-cadherin, such as clone 36, which may reveal nuclear immunostaining and indicate more aggressive clinical course in patients with lobular breast cancer. We hypothesize that E-cadherin is cleaved and translocated to nucleus functioning as transcription factor.

Epithelial-to-Mesenchymal Transition (EMT) and Signal Transducers and Activators of Transcription (STAT3) Are a Novel Therapeutic Strategy for Hepatocellular Carcinoma (HCC) Prognosis

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide and is the second most common cancer in Egypt. Poor diagnosis of HCC is correlated with vascular invasion and metastasis. Epithelial-to-mesenchymal transition (EMT) is the main step in the tumor invasion process whereby epithelial cells lose cell polarity with each other and then undergo a dramatic remodeling of the cytoskeleton. Also, EMT plays a pivotal role in metastasis when epithelial cell layers lose cell-cell contacts in tumor progression due to the loss of E-cadherin and increasing the ability of the spread into surrounding tissues. Signal transducers and activators of transcription (STAT) play a different cellular function as signal transducers in the cytoplasm and transcription activators in the nucleus. STAT3 gene plays a crucial role to affect EMT in cancer progression by promoting cell proliferation and survival through its function as a transcription factor of the tumor. The aim of our study is to investigate the gene expression of STAT3 in HCC (HepG-2) cell lines and the expression of cell differentiation and proliferation markers. Western blotting was used to examine the protein expression of STAT3, E-cadherin, and β-catenin signaling. Our results showed that STAT3 expression levels were detected, as well as a significant increase in the expression of proliferation marker as β-catenin and a significant decrease of differentiation marker as E-cadherin protein expression levels in HepG-2 cell lines. Conclusion Our finding provides novel evidence for using a molecular gene therapy as STAT3, which showed an effect on EMT that plays a pivotal role in the prognosis of HCC. The loss of E-cadherin expression is a hallmark of EMT, because β-catenin is associated with the cytoplasmic domain of E-cadherin. We have emphasized the significant role of EMT and STAT3 in HCC progression, which could be a potential application as a novel therapeutic strategy for HCC treatment.

Force Bistability in Adhesion Switch

The lateral junctions of epithelial cells must display mechanical stability to form barriers, but also significant plasticity to allow for reorganization during development and repair. In this issue, Biswas et al. (1) extend the use of fluid supported-lipid bilayers (SLBs) to reconstitute function of the key adhesion molecule that initiates lateral junctions of epithelial cells, E-cadherin, and to further study the requirements for mechanical forces in activating the cytoplasmic complex that links E-cadherin to the cytoskeleton (1).

The N-cadherin cytoplasmic domain confers anchorage-independent growth and the loss of contact inhibition.

Tumor growth is characterized by anchorage independence and the loss of contact inhibition. Previously, we showed that either a red fluorescent protein (DsRed)-tagged N-cadherin or E-cadherin cytoplasmic domain (DNCT or DECT) could function as a dominant negative inhibitor by blocking the cell surface localization of endogenous E-cadherin and inducing cell dissociation. Here, we show that expression of DNCT abrogated contact inhibition of proliferation and conferred anchorage-independent growth. DNCT expression induced the relocation of the tumor suppressor Merlin from the cell surface to intracellular compartments. Although DNCT expression induced redistribution of TAZ from the cytoplasm to the nucleus, YAP/TAZ signaling was not activated. An E-cadherin–α-catenin chimera that functions as a β-catenin–independent cell adhesion molecule restored contact inhibition and anchorage-dependency of growth. Addition of the SV40 large T antigen nuclear localization signal reversed the effects of DNCT expression, indicating that DNCT functioned outside of the nucleus.

E-cadherin cytoplasmic domain inhibits cell surface localization of endogenous cadherins and fusion of C2C12 myoblasts.

ABSTRACTMyoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca2+-dependent cell–cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell–cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell–cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell–cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

Epithelial cadherin is present in bovine oviduct epithelial cells and gametes, and is involved in fertilization-related events

Fertilization is a calcium-dependent process that involves sequential cell–cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein β-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and β-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine–induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. β-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell–cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm–OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.

E-cadherin is under constitutive actomyosin-generated tension that is increased at cell–cell contacts upon externally applied stretch

Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and αE-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell-cell contacts, although tension is further increased at cell-cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell-cell junctions but throughout the cell surface.

γ-Secretase-Dependent Cleavage of E-Cadherin by Staurosporine in Breast Cancer Cells

E-cadherin is a transmembrane protein that serves as a cell adhesion molecule component of the adherens junction. We previously showed that cadmium induced γ-secretase-dependent E-cadherin cleavage via oxidative stress. In this study, we report that staurosporine (STS)-induced apoptosis induces caspase-2 and/or -8-dependent E-cadherin cleavage. STS increased γ-secretase-dependent cleavage of E-cadherin in breast cancer cells through caspase activation. The ability of the γ-secretase inhibitor DAPT and the caspase inhibitor zVAD-FMK to block E-cadherin cleavage provided support for these results. The cleavage of E-cadherin was blocked by caspase-2 and -8 inhibitors. Immunofluorescence analysis confirmed that, along with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulated in the cytosol. In the presence of an inhibitor of γ-secretase or caspase, the cleavage of E-cadherin was partially blocked. Our findings suggest that activation of caspase-2/-8 stimulated the disruption of cadherin-mediated cell-cell contacts in apoptotic cells via γ-secretase activation.

Analysis of E-Cadherin as a Tension Sensor at Epithelial Cell-Cell Junctions

Cells are mechanosensitive systems in which the detection of external mechanical forces by proteins that span the plasma membrane leads to changes in cell organization, differentiation and proliferation. Cell-cell contacts are thought to be sites of mechano-transduction, and a major cell adhesion protein E-cadherin is a candidate protein to transduce forces between cells under tension. To directly test whether force transduction occurs through E-cadherin, we inserted a force sensor module into the E-cadherin cytoplasmic domain. The force sensor (TSMod) is a 32-aa-long flexible peptide capped by mTFP and Venus that together form a FRET pair. FRET efficiency scales inversely with linker extension, and thus reports the tension exerted on the sensor within the host protein. Using FRET intensity ratio imaging in live MDCK cells, we show that the E-cadherin/TSmod construct is recruited to cell-cell contacts like its endogenous counterpart. We observe decreased FRET both at cell-cell contacts and at the plasma membrane distant from intercellular junctions, indicating that the E-cadherin cytoplasmic domain is under tension regardless of its recruitment to intercellular contacts. Significantly, we observe increased FRET upon actin cytoskeleton disruption or depletion of alpha-catenin, a putative linker between actin and E-cadherin, regardless of subcellular location. These results indicate that the E-cadherin complex is under tension through the catenin complex and actin cytoskeleton, and that this is a constitutive state that is independent of cell-cell adhesion.

Abstract 3623: Monoclonal antibody against the 4th extracellular domain of N-cadherin affects FGF2 mediated pathways in prostate cancer

Abstract Introduction: Fibroblast growth factor 2 (FGF2) has been reported to synergize with N-cadherin, a cell surface marker of epithelial mesenchymal transition (EMT), to enhance migration and invasion in breast cancer and other cancers. Recent studies from our group suggested that N-cadherin promotes castration resistance and metastasis in prostate cancer. In addition, our studies showed that antibodies against N-cadherin ectodomains blocked prostate cancer invasion and castration resistant growth. To investigate the mechanism of action of N-cadherin antibodies in prostate cancer cells, we seek to determine the contribution of the different N-cadherin domains in promoting invasion and growth in prostate cancer cells, with particular focus on interaction with FGF2. Methods: Chimeric constructs, in which a N-cadherin domain was swapped with an E-cadherin domain, were generated and ectopically expressed in androgen dependent prostate cancer cell line LNCaP. The following combinations were made: 1/ NE: N-cadherin ectodomains linked to E-cadherin cytoplasmic domain (NE), 2/ EN: the reverse of NE, 3/ NEN: swapping N-cadherin ectodomain 4 for that of E-cadherin. Full length N-cadherin and empty vector sublines served as positive and negative controls. All cell lines were assayed for invasion and growth in androgen depleted media in combination with recombinant FGF2, FGF2 neutralizing antibody and N-cadherin antibodies. Results: Ectopic expression of N-cadherin in N-cadherin negative cells result in a 5-fold increase of FGF2 secreted in the media. In vitro studies have shown that the addition of recombinant FGF2 enhanced invasion by 56%± 0.04 in N-cadherin positive cells while treatment with FGF2 neutralizing antibody resulted in a 65%± 0.03 reduction in invasion. Chimeric cell lines which harbor extracellular domain 4 of N-cadherin, N and NE cell lines, showed &amp;gt;56%± 0.11 enhanced invasion to the addition of recombinant FGF2 while EN, NEN, and control cell lines did not. Monoclonal antibodies developed against regions in the extracellular domain of N-cadherin block the response to recombinant FGF2 with 2A9 (antibody raised against extracellular domain 4) being the least responsive. Conclusions: These studies suggest that the relationship between N-cadherin and FGF2 is involved in prostate cancer progression. Therapeutic targeting of N-cadherin with monoclonal antibodies, alone or in combination with other drugs targeting those potentially involved in downstream pathways may have significant clinical benefit. Future experiments include in vivo models where chimeric cell lines will be implanted in mice as xenografts, and will be monitored for tumor growth, local invasion, and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3623. doi:10.1158/1538-7445.AM2011-3623

KLRG1, Cadherins and their Interaction (35.29)

Abstract The killer cell lectin-like receptor G1 (KLRG1) is a unique inhibitory receptor expressed on a phenotypically mature subset of resting NK cells as well as effector memory CD8+ T cells. KLRG1 is a C-type lectin inhibitory receptor that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. KLRG1 ligands have been recently identified and are members of the broadly expressed cadherin family, N-, R-, and E-cadherin. In this study, we attempt to elucidate the consequences of cadherin engagement by KLRG1 as well as KLRG1 engagement by cadherin. We found that E-cadherin engagement by KLRG1 influences the dynamics of cell adhesion. Along this line, we showed that E-cadherin and KLRG1 interaction regulates the signaling properties of E-cadherin. These data are supported by the fact that recombinant extracellular domain of N-cadherin blocks KLRG1 tetramer binding to cadherin expressing cells. In addition, we show that recombinant extracellular domain of N-cadherin functions to regulate KLRG1 signaling. We also demonstrated ex vivo that KLRG1 engagement by E-cadherin induces a significant inhibition of TCR signaling. Notably, upon analysis of E-cadherin mutants, we discovered that E-cadherin's cytoplasmic domain regulates its cell surface expression. Taken together, our data suggest that immune receptor interaction with cadherin may have an impact not only on immune cells such as NK cells but also on cells expressing cadherin such as epithelial cells. Supported by NIH grant AI 58181.

Dendritic Cells Break Bonds to Tolerize

Typically, dendritic cells (DCs) induce peripheral tolerance under steady state but immunity during inflammation or infection. In this issue of Immunity, Jiang et al. (2007) identify disruption of E-cadherin interactions as a unique maturation pathway by which DCs are capable of mediating tolerance.

Disruption of E-Cadherin-Mediated Adhesion Induces a Functionally Distinct Pathway of Dendritic Cell Maturation

The maturation of dendritic cells (DCs) after exposure to microbial products or inflammatory mediators plays a critical role in initiating the immune response. We found that maturation can also occur under steady-state conditions, triggered by alterations in E-cadherin-mediated DC-DC adhesion. Selective disruption of these interactions induced the typical features of DC maturation including the upregulation of costimulatory molecules, MHC class II, and chemokine receptors. These events were triggered at least in part by activation of the beta-catenin pathway. However, unlike maturation induced by microbial products, E-cadherin-stimulated DCs failed to release immunostimulatory cytokines, exhibiting an entirely different transcriptional profile. As a result, E-cadherin-stimulated DCs elicited an entirely different T cell response in vivo, generating T cells with a regulatory as opposed to an effector phenotype. These DCs induced tolerance in vivo and may thus contribute to the elusive steady-state "tolerogenic DCs."

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.

Phosphorylation of β-Catenin by AKT Promotes β-Catenin Transcriptional Activity

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation is poorly understood. We have demonstrated that AKT, which is activated downstream from epidermal growth factor receptor signaling, phosphorylates beta-catenin at Ser552 in vitro and in vivo. AKT-mediated phosphorylation of beta-catenin causes its disassociation from cell-cell contacts and accumulation in both the cytosol and the nucleus and enhances its interaction with 14-3-3zeta via a binding motif containing Ser552. Phosphorylation of beta-catenin by AKT increases its transcriptional activity and promotes tumor cell invasion, indicating that AKT-dependent regulation of beta-catenin plays a critical role in tumor invasion and development.

Increased Internalization of p120-uncoupled E-cadherin and a Requirement for a Dileucine Motif in the Cytoplasmic Domain for Endocytosis of the Protein

E-cadherin is a member of the cadherin family of Ca2+-dependent cell-cell adhesion molecules. E-cadherin associates with beta-catenin at the membrane-distal region of its cytosolic domain and with p120 at the membrane-proximal region of its cytoplasmic domain. It has been shown that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. Further, p120 knockdown by small interference RNA resulted in dose-dependent elimination of cell surface E-cadherin. Consistent with these observations, we found that selective uncoupling of p120 from E-cadherin by introduction of amino acid substitutions in the p120-binding site increased the level of E-cadherin endocytosis. The increased endocytosis was clathrin-dependent, because it was blocked by expression of a dominant-negative form of dynamin or by hypertonic shock. A dileucine motif in the juxtamembrane cytoplasmic domain is required for E-cadherin endocytosis, because substitution of these residues to alanine resulted in impaired internalization of the protein. The alanine substitutions in the p120-uncoupled construct reduced endocytosis of the protein, indicating that this motif was dominant to p120 binding in the control of E-cadherin endocytosis. Therefore, these results are consistent with the idea that p120 regulates E-cadherin endocytosis by masking the dileucine motif and preventing interactions with adaptor proteins required for internalization.

Evidence that the V832M E-Cadherin Germ-Line Missense Mutation does not Influence the Affinity of α -Catenin for the Cadherin/Catenin Complex

Mutations in E-cadherin are associated with a number of diseases, and have been shown to contribute to disease progression. In particular, 50% of hereditary diffuse gastric cancer cases have inactivating mutations in the E-cadherin gene. An interesting mutation near the beta-catenin-binding site on the cytoplasmic domain of E-cadherin (V832M) was recently reported that produces full-length protein, but exhibits decreased binding of alpha -catenin to the cadherin/catenin complex. The study was done by transfecting mutant E-cadherin into Chinese hamster ovary fibroblast cells. Here we show that the previously reported characteristics of this mutation do not apply to human epithelial cells expressing this mutant protein and suggest that the mechanism whereby the V832M mutation in human E-cadherin promotes gastric cancer is not yet understood.

PAR1b Promotes Cell–Cell Adhesion and Inhibits Dishevelled-mediated Transformation of Madin-Darby Canine Kidney Cells

Mammalian Par1 is a family of serine/threonine kinases comprised of four homologous isoforms that have been associated with tumor suppression and differentiation of epithelial and neuronal cells, yet little is known about their cellular functions. In polarizing kidney epithelial (Madin-Darby canine kidney [MDCK]) cells, the Par1 isoform Par1b/MARK2/EMK1 promotes the E-cadherin-dependent compaction, columnarization, and cytoskeletal organization characteristic of differentiated columnar epithelia. Here, we identify two functions of Par1b that likely contribute to its role as a tumor suppressor in epithelial cells. 1) The kinase promotes cell-cell adhesion and resistance of E-cadherin to extraction by nonionic detergents, a measure for the association of the E-cadherin cytoplasmic domain with the actin cytoskeleton, which is critical for E-cadherin function. 2) Par1b attenuates the effect of Dishevelled (Dvl) expression, an inducer of wnt signaling that causes transformation of epithelial cells. Although Dvl is a known Par1 substrate in vitro, we determined, after mapping the PAR1b-phosphorylation sites in Dvl, that PAR1b did not antagonize Dvl signaling by phosphorylating the wnt-signaling molecule. Instead, our data suggest that both proteins function antagonistically to regulate the assembly of functional E-cadherin-dependent adhesion complexes.

Extracellular Cleavage of E-Cadherin by Leukocyte Elastase During Acute Experimental Pancreatitis in Rats

Cadherins play an important role in cell-cell contact formation at adherens junctions. During the course of acute pancreatitis, adherens junctions are known to dissociate-a requirement for the interstitial accumulation of fluid and inflammatory cells-but the underlying mechanism is unknown. Acute pancreatitis was induced in rats by supramaximal cerulein infusion. The pancreas and lungs were either homogenized for protein analysis or fixed for morphology. Protein sequencing was used to identify proteolytic cleavage sites and freshly prepared acini for ex vivo studies with recombinant proteases. Results were confirmed in vivo by treating experimental pancreatitis animals with specific protease inhibitors. A 15-kilodalton smaller variant of E-cadherin was detected in the pancreas within 60 minutes of pancreatitis, was found to be the product of E-cadherin cleavage at amino acid 394 in the extracellular domain that controls cell-contact formation, and was consistent with E-cadherin cleavage by leukocyte elastase. Employing cell culture and ex vivo acini leukocyte elastase was confirmed to cleave E-cadherin at the identified position, followed by dissociation of cell contacts and the internalization of cleaved E-cadherin to the cytosol. Inhibition of leukocyte elastase in vivo prevented E-cadherin cleavage during pancreatitis and reduced leukocyte transmigration into the pancreas. These data provide evidence that polymorphonuclear leukocyte elastase is involved in, and required for, the dissociation of cell-cell contacts at adherens junctions, the extracellular cleavage of E-cadherin, and, ultimately, the transmigration of leukocytes into the epithelial tissue during the initial phase of experimental pancreatitis.