A novel role for integrin-linked kinase in epithelial sheet morphogenesis

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.     

Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes

Extracellular Ca(2+) (Ca(2+)(o)) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca(2+) signaling during Ca(2+)(o)-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca(2+)(o)-induced increase in intracellular free calcium (Ca(2+)(i)) and expression of terminal differentiation genes, while promoting apoptosis. Ca(2+)(o) also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca(2+)(o) concentration ([Ca(2+)](o)) from 0.03 to 2 mm rapidly induced the co-localization of alpha-, beta-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca(2+)(o)-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca(2+)(o)-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca(2+)(o)-induced rise in Ca(2+)(i). Inhibition of CaR expression blocked the Ca(2+)(o)-induced tyrosine phosphorylation of beta-, gamma-, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca(2+)(o)-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling.     

Role of nectin in formation of E-cadherin-based adherens junctions in keratinocytes: analysis with the N-cadherin dominant negative mutant

E-cadherin is a Ca(2+)-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin-based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin-based AJs in keratinocytes.     

Socs3 induction by PPARγ restrains cancer-promoting inflammation

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.     

Rat seminiferous epithelium contains a unique junction (Ectoplasmic specialization) with signaling properties both of cell/cell and cell/matrix junctions

The seminiferous epithelium contains unique actin related cell-cell junctions, termed ectoplasmic specializations (ESs). Turnover of these junctions is fundamental to sperm release and to movement of spermatocytes from basal to adluminal compartments of the epithelium during spermatogenesis. In this study we report several novel observations related to the spatial and temporal distribution of integrin-related signaling molecules at ESs. We confirm the presence of beta(1)-integrin at these sites and further demonstrate co-localization of integrin linked kinase (ILK). beta(1)-Integrin and ILK were shown by immunoprecipitation to associate in whole cell lysates of seminiferous epithelium. This observation provides the first evidence for a direct beta(1)-integrin/ILK interaction in noncultured epithelium. Pan-cadherin and beta-catenin antibodies did not react at ESs. Rather, antibodies reacted with desmosome-like junctions that are present both at basal junctional complexes between Sertoli cells and at sites of attachment to spermatogenic cells. Focal adhesion kinase (FAK), a known integrin-associated molecule, did not codistribute with beta(1)-integrins and did not associate with these adhesion molecules in immunoprecipitation studies. Although FAK was expressed in the epithelium, it appeared to be limited to the cytoplasm of early spermatogenic cells. Significantly, polyclonal antibodies against phosphotyrosine-containing residues reacted strongly at ESs, with highest levels detected during sperm release and turnover of basal junction complexes. Our observations indicate that ESs share cell signaling features both of cell-cell junctions and of cell-extracellular matrix junctions.     

The mechanism of contribution of integrin linked kinase (ILK) to epithelial-mesenchymal transition (EMT)

Integrin linked kinase (ILK) is ubiquitously expressed serine/threonine protein kinase, a binding partner of β1 and β3 integrin subunit as a cytoplasmic effector of integrin receptors that functionally links them to the actin cytoskeleton.We postulate that ILK is important enzyme involved in epithelial-mesenchymal transition (EMT) a critical event in the process of cancer progression. Commonly used EMT molecular markers include among others increased expression of N-cadherin and vimentin, nuclear localization of β-catenin, and the decrease of E-cadherin synthesis. In this study we were able to show that N-cadherin expression in melanoma cells is dependent on ILK signaling and the translocation of β-catenin to the nucleus. Silencing of ILK expression by siRNA significantly inhibited the stabilization and subsequent nuclear translocation of β-catenin and the expression of N-cadherin, a crucial molecule in the EMT, which facilitates association with fibroblast and endothelial cells during invasion of various cancers. The results allow to cautiously speculate on the important role of ILK in the cross-talk between integrins and cadherins accompanying EMT in melanoma.     

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.     

Roles of the intercellular adhesion molecule nectin in intracellular signaling

The nectin family comprises four Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules. Each nectin homophilically and heterophilically trans-interacts and causes intercellular adhesion, which organizes a variety of intercellular junctions in cooperation with, or independently of, cadherin. Nectin furthermore induces activation of Cdc42 and Rac small G proteins through c-Src, which eventually regulates formation of the cadherin-based adherens junctions through reorganization of the actin cytoskeleton, gene expression through activation of a mitogen-activated protein kinase cascade, and cell polarization through cell polarity proteins. We describe here the roles of nectin in intracellular signaling.     

An ELMO2-RhoG-ILK network modulates microtubule dynamics

ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin–dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca²⁺-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes.     

Cadherin/Catenin Complexes in Murine Epidermal Keratinocytes: E-Cadherin Complexes Containing either β-Catenin or Plakoglobin Contribute to Stable Cell-Cell Contacts

The cadherin/catenin complexes expressed by a murine epidermal keratinocyte cell line PDV, expressing E- and P-cadherin, have been analysed using a combination of biochemical and confocal microscopy analysis. Two types of E-cadherin complexes, containing β-catenin or plakoglobin and α-catenin, were detected in PDV cells as in other cell types, while β-cadherin was mainly detected in complexes containing β-catenin and α-catenin in PDV and other murine epidermal keratinocytes. Bio tin-labelling studies have shown that both types of E-cadherin complexes are present at the surface of confluent cells. Furthermore, confocal microscopy analysis indicated that E-cadherin/ plakoglobin complexes are located in stable cell-cell contacts at the middle lateral membranes and associated with α-catenin and the actin cytoskeleton, with a similar distribution to that of the E-cadherin/β-catenin complexes. In addition, E-cadherin/ plakoglobin complexes not associated with α-catenin or the actin cytoskeleton were detected in lower planes of the lateral contacting membranes as well as E-cadherin non-associated with catenins in the more basal planes. These studies support that in murine epidermal keratinocytes both β-catenin- and plakoglobin-containing E-cadherin complexes contribute to the maintenance of stable cell-cell contacts and suggest a differential role of the plakoglobin containing complexes in different epithelial cell types.     

Co-localization of Racl and E-Cadherin in Human Epidermal Keratinocytes

The Racl small GTP-binding protein is known to be involved in reorganization of the actin cytoskeleton and in regulation of intracellular signal transduction. The assembly and maintenance of cadherin-based cell-cell junctions in epidermal keratinocytes is thought to be dependent on activity of Racl. In this study we have generated green fluorescent protein (GFP)-tagged wild type, dominant negative and constitutively active Racl expression vectors and analyzed distribution of Racl following microinjection of human SCC12F epidermal keratinocytes. Wild type, dominant negative and constitutively active GFP-Racl proteins distribute to sites of cell-cell adhesion and co-localize with E-cadherin and the catenins. Disruption of cadherin-based junctions by reduction in extracellular calcium concentrations, or by use of antibodies to E-cadherin, results in redistribution of Racl away from sites of cell-cell interaction but the co-localization with E-cadherin is maintained. In addition, expression of constitutively active GFP-Racl results in formation of membrane ruffles on the apical surface of cells and intracellular vesicles. Interestingly, co-localization of Racl with E-cadherin is maintained in these structures. In contrast to previously published work we find that expression of dominant negative Racl neither disrupts cell-cell adhesion nor prevents assembly of new cadherin-based adhesion structures.     

E-cadherin controls adherens junctions in the epidermis and the renewal of hair follicles

E-cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E-cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E-cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and beta-catenin levels were not strongly altered in the mutant skin. We conclude that E-cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E-cadherin.     

The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca(2+) treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120(CTN), being recruited to cell-cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell-cell contact and E-cadherin redistribution, even in low Ca(2+), which does not normally support stable cell-cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca(2+). Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell-cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca(2+) and by inhibiting Src activity in low (0.03 mM) Ca(2+) in vitro.     

Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.     

Hyaluronan-CD44 interaction with Rac1-dependent protein kinase N-gamma promotes phospholipase Cgamma1 activation, Ca(2+) signaling, and cortactin-cytoskeleton function leading to keratinocyte adhesion and differentiation

In this study we have investigated hyaluronan (HA)-CD44 interaction with protein kinase N-gamma (PKNgamma), a small GTPase (Rac1)-activated serine/threonine kinase in human keratinocytes. By using a variety of biochemical and molecular biological techniques, we have determined that CD44 and PKNgamma kinase (molecular mass approximately 120 kDa) are physically linked in vivo. The binding of HA to keratinocytes promotes PKNgamma kinase recruitment into a complex with CD44 and subsequently stimulates Rac1-mediated PKNgamma kinase activity. The Rac1-activated PKNgamma in turn increases threonine (but not serine) phosphorylation of phospholipase C (PLC) gamma1 and up-regulates PLCgamma1 activity leading to the onset of intracellular Ca(2+) mobilization. HA/CD44-activated Rac1-PKNgamma also phosphorylates the cytoskeletal protein, cortactin, at serine/threonine residues. The phosphorylation of cortactin by Rac1-PKNgamma attenuates its ability to cross-link filamentous actin in vitro. Further analyses indicate that the N-terminal antiparallel coiled-coil (ACC) domains of PKNgamma interact directly with Rac1 in a GTP-dependent manner. The binding of HA to CD44 induces PKNgamma association with endogenous Rac1 and its activity in keratinocytes. Transfection of keratinocytes with PKNgamma-ACCcDNA reduces HA-mediated recruitment of endogenous Rac1 to PKNgamma and blocks PKNgamma activity. These findings suggest that the PKNgamma-ACC fragment acts as a potent competitive inhibitor of endogenous Rac1 binding to PKNgamma in vivo. Most important, the PKNgamma-ACC fragment functions as a strong dominant-negative mutant that effectively inhibits HA/CD44-mediated PKNgamma phosphorylation of PLCgamma1 and cortactin as well as keratinocyte signaling (e.g. Ca(2+) mobilization and cortactin-actin binding) and cellular functioning (e.g. cell-cell adhesion and differentiation). Taken together, these findings strongly suggest that hyaluronan-CD44 interaction with Rac1-PKNgamma plays a pivotal role in PLCgamma1-regulated Ca(2+) signaling and cortactin-cytoskeleton function required for keratinocyte cell-cell adhesion and differentiation.     

Role for E-cadherin as an inhibitory receptor on epidermal gammadelta T cells

E-cadherin is a homophilic adhesion molecule that maintains homotypic intercellular adhesion between epithelial cells such as epidermal keratinocytes. E-cadherin is also expressed on resident murine epidermal γδ T cells, known as dendritic epidermal T cells (DETCs), but they express another receptor for E-cadherin, α(E)(CD103)β(7) integrin, as well. In this study, we analyzed functional differences between E-cadherin-mediated homophilic binding and heterophilic binding of α(E)β(7) integrin to E-cadherin in heterotypic intercellular adhesion of DETCs to keratinocytes. E-cadherin, but not α(E)β(7) integrin, was downregulated on activation of DETCs in vivo and in vitro. Short-term (1-h) adhesion of DETCs to keratinocytes in vitro was primarily mediated by α(E)β(7) integrin, and blocking of the binding of α(E)β(7) integrin to E-cadherin inhibited the lysis of keratinocytes by DETCs. Stable binding of E-cadherin on DETCs to plate-bound recombinant E-cadherin was observed only after 24-h culture in vitro. Cytokine production and degranulation by DETCs in response to suboptimal TCR cross-linking and mitogen stimulation were augmented by coligation of α(E)β(7) integrin. In contrast, engagement of E-cadherin on DETCs with immobilized anti-E-cadherin Ab, plate-bound recombinant E-cadherin, and E-cadherin on keratinocytes inhibited DETC activation. Therefore, E-cadherin acts as an inhibitory receptor on DETCs, whereas α(E)β(7) integrin acts as a costimulatory receptor. Differential expression of E-cadherin and α(E)β(7) integrin on resting and activated DETCs, as well as their opposite functions in DETC activation, suggests that E-cadherin and α(E)β(7) integrin on DETCs regulate their activation threshold through binding to E-cadherin on keratinocytes.     

IQGAP1 and calmodulin modulate E-cadherin function

Ca(2+)-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin. Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion. On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.     

E-cadherin at the cell periphery is a determinant of keratinocyte differentiation in vitro

The origin of the signal for keratinocyte differentiation is still unknown. Here, we show that Ca(2+)- and density-induced translocation of E-cadherin, but not P-cadherin, is accompanied by induction of differentiation-specific proteins in cultured keratinocytes. Antibodies that artificially cluster cell-surface E-cadherin in low extracellular Ca(2+) also induce differentiation-specific proteins, implicating E-cadherin as a determinant of keratinocyte differentiation in vitro.     

Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin

The association of the cytoskeleton with the cadherin--catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the E-cadherin-catenin-actin complex to the cell periphery. This was accompanied by actin polymerization. Also, it was found that microtubule disruption-induced cell-cell adhesion was significantly reduced in more advanced differentiated keratinocytes. For example, when NHEK cells cultured under high calcium (1.2 mM) for 8 d and then in low calcium for 1 d were treated with nocodazole, there was no induction of cell-cell adhesion. Also long-term treatment of a phorbol ester for 48 h inhibited nocodazole-induced cell-cell adhesion of NHEK. Furthermore, this nocodazole-induced cell-cell adhesion could be observed in squamous cancer cell lines (A431 and SCC-5, -9, and -25) under low calcium condition, but not in the keratinocyte cell lines derived from normal epidermis (HaCaT, RHEK). On the other hand, HaCaT cells continuously cultivated in low calcium media regained a less differentiated phenotype such as decreased expression of cytokeratin 10, and increased K5; these changes were accompanied with inducibility of cell-cell adhesion by nocodazole. Together, our results suggest that microtubule disruption can induce the cell-cell adhesion via activation of endogenous E-cadherin in non- or early differentiating keratinocytes. However, this is no longer possible in advanced terminally differentiating keratinocytes, possibly due to irreversible changes effected by cell envelope barrier formation.     

Microtubule dynamics and Rac-1 signaling independently regulate barrier function in lung epithelial cells

Cadherin-mediated cell-cell adhesion controls the morphology and function of epithelial cells and is a critical component of the pathology of chronic inflammatory disorders. Dynamic interactions between cadherins and the actin cytoskeleton are required for stable cell-cell contact. Besides actin, microtubules also target intercellular, cadherin-based junctions and contribute to their formation and stability. Here, we studied the role of microtubules in conjunction with Rho-like GTPases in the regulation of lung epithelial barrier function using real-time monitoring of transepithelial electrical resistance. Unexpectedly, we found that disruption of microtubules promotes epithelial cell-cell adhesion. This increase in epithelial barrier function is accompanied by the accumulation of beta-catenin at cell-cell junctions, as detected by immunofluorescence. Moreover, we found that the increase in cell-cell contact, induced by microtubule depolymerization, requires signaling through a RhoA/Rho kinase pathway. The Rac-1 GTPase counteracts this pathway, because inhibition of Rac-1 signaling rapidly promotes epithelial barrier function, in a microtubule- and RhoA-independent fashion. Together, our data suggest that microtubule-RhoA-mediated signaling and Rac-1 control lung epithelial integrity through counteracting independent pathways.