The scaffold protein Ajuba suppresses CdGAP activity in epithelia to maintain stable cell-cell contacts.

Levels of active Rac1 at epithelial junctions are partially modulated via interaction with Ajuba, an actin binding and scaffolding protein. Here we demonstrate that Ajuba interacts with the Cdc42 GTPase activating protein CdGAP, a GAP for Rac1 and Cdc42, at cell-cell contacts. CdGAP recruitment to junctions does not require Ajuba; rather Ajuba seems to control CdGAP residence at sites of cell-cell adhesion. CdGAP expression potently perturbs junctions and Ajuba binding inhibits CdGAP activity. Ajuba interacts with Rac1 and CdGAP via distinct domains and can potentially bring them in close proximity at junctions to facilitate activity regulation. Functionally, CdGAP-Ajuba interaction maintains junctional integrity in homeostasis and diseases: (i) gain-of-function CdGAP mutants found in Adams-Oliver Syndrome patients strongly destabilize cell-cell contacts and (ii) CdGAP mRNA levels are inversely correlated with E-cadherin protein expression in different cancers. We present conceptual insights on how Ajuba can integrate CdGAP binding and inactivation with the spatio-temporal regulation of Rac1 activity at junctions. Ajuba provides a novel mechanism due to its ability to bind to CdGAP and Rac1 via distinct domains and influence the activation status of both proteins. This functional interplay may contribute towards conserving the epithelial tissue architecture at steady-state and in different pathologies.

Defining functional interactions during biogenesis of epithelial junctions.

In spite of extensive recent progress, a comprehensive understanding of how actin cytoskeleton remodelling supports stable junctions remains to be established. Here we design a platform that integrates actin functions with optimized phenotypic clustering and identify new cytoskeletal proteins, their functional hierarchy and pathways that modulate E-cadherin adhesion. Depletion of EEF1A, an actin bundling protein, increases E-cadherin levels at junctions without a corresponding reinforcement of cell-cell contacts. This unexpected result reflects a more dynamic and mobile junctional actin in EEF1A-depleted cells. A partner for EEF1A in cadherin contact maintenance is the formin DIAPH2, which interacts with EEF1A. In contrast, depletion of either the endocytic regulator TRIP10 or the Rho GTPase activator VAV2 reduces E-cadherin levels at junctions. TRIP10 binds to and requires VAV2 function for its junctional localization. Overall, we present new conceptual insights on junction stabilization, which integrate known and novel pathways with impact for epithelial morphogenesis, homeostasis and diseases.

Activation of the small GTPase Rac is sufficient to disrupt cadherin-dependent cell-cell adhesion in normal human keratinocytes.

To achieve strong adhesion to their neighbors and sustain stress and tension, epithelial cells develop many different specialized adhesive structures. Breakdown of these structures occurs during tumor progression, with the development of a fibroblastic morphology characteristic of metastatic cells. During Ras transformation, Rac-signaling pathways participate in the disruption of cadherin-dependent adhesion. We show that sustained Rac activation per se is sufficient to disassemble cadherin-mediated contacts in keratinocytes, in a concentration- and time-dependent manner. Cadherin receptors are removed from junctions before integrin receptors, suggesting that pathways activated by Rac can specifically interfere with cadherin function. We mapped an important region for disruption of junctions to the putative second effector domain of the Rac protein. Interestingly, although this region overlaps the domain necessary to induce lamellipodia, we demonstrate that the disassembly of cadherin complexes is a new Rac activity, distinct from Rac-dependent lamellipodia formation. Because Rac activity is also necessary for migration, Rac is a good candidate to coordinately regulate cell-cell and cell-substratum adhesion during tumorigenesis.

H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes.

The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85alpha and p110alpha subunits of PI3K with beta-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between beta-catenin and p85alpha. Overexpression of either V12Ras or constitutively active p110alpha induces metabolic stabilization of beta-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of beta-catenin by a mechanism involving its interaction with PI3K.

Caspase activation in the terminal differentiation of human epidermal keratinocytes.

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.

Regulation of cadherin function by Rho and Rac: modulation by junction maturation and cellular context.

Cadherins are cell-cell adhesion receptors whose adhesive function requires their association with the actin cytoskeleton via proteins called catenins. The small guanosine triphosphatases (GTPases), Rho and Rac, are intracellular proteins that regulate the formation of distinct actin structures in different cell types. In keratinocytes and in other epithelial cells, Rho and Rac activities are required for E-cadherin function. Here we show that the regulation of cadherin adhesiveness by the small GTPases is influenced by the maturation status of the junction and the cellular context. E-cadherin localization was disrupted in mature keratinocyte junctions after inhibition of Rho and Rac. However, an incubation of 2 h was required after GTPase inhibition, when compared with newly established E-cadherin contacts (30 min). Regarding other cadherin receptors, P-cadherin was effectively removed from mature keratinocytes junctions by blocking Rho or Rac. In contrast, VE-cadherin localization at endothelial junctions was independent of Rho/Rac activity. We demontrate that the insensitivity of VE-cadherin to inhibition of Rho and Rac was not due to the maturation status of endothelial junction, but rather the cellular background: when transfected into CHO cells, the localization of VE-cadherin was perturbed by inhibition of Rho proteins. Our results suggest that the same stimuli may have different activity in regulating the paracellular activity in endothelial and epithelial cells. In addition, we uncovered possible roles for the small GTPases during the establishment of E-cadherin-dependent contacts. In keratinocytes, Rac activation by itself cannot promote accumulation of actin at the cell periphery in the absence of cadherin-dependent contacts. Moreover, neither Rho nor Rac activation was sufficient to redistribute cadherin molecules to cell borders, indicating that redistribution results mostly from the homophilic binding of the receptors. Our results point out the complexity of the regulation of cadherin-mediated adhesion by the small GTPases, Rho and Rac.

Small GTPases and regulation of cadherin dependent cell-cell adhesion.

Cadherins belong to a superfamily of cell-cell adhesion receptors that bind to the same type of molecules (homotypic interaction) in a calcium dependent manner. Different members of the family are found in a wide variety of cell types and cadherin adhesive function plays a role in cell fate, segregation, and differentiation, which ensures the higher order of organisation found in many tissues. This review will focus on the role that cadherin adhesiveness plays in the differentiation of epithelial cells, and how cadherin function can be regulated by proteins of the small GTPase family. In the text, readers are referred to recent reviews and other chapters covering important topics that are not discussed here because of space limitation.

Calcium-induced intercellular adhesion of keratinocytes does not involve accumulation of beta 1 integrins at cell-cell contacts and does not involve changes in the levels or phosphorylation of catenins.

On initiation of terminal differentiation human epidermal keratinocytes detach from the underlying basement membrane as a result of inactivation and subsequent loss of integrins from the cell surface. Assembly of keratinocytes into multilayered sheets requires functional E- and P-cadherin and when stratification is inhibited in low calcium medium differentiating keratinocytes continue to express functional integrins. Using immunofluorescence microscopy, we found that on addition of calcium ions to keratinocyte monolayers there was colocalisation of the beta 1 integrins and E-cadherin along the lateral membranes except for a zone close to the substratum which exclusively contained integrins. Quantitative immunoelectron microscopy showed that on induction of stable cell-cell contacts the density of beta 1 integrins was the same on the apical and lateral membranes, suggesting that the accumulation of integrins on the lateral membranes observed by immunofluorescence microscopy is due to the increased area of contact between adjacent cells and not to an increase in receptor density. There were no changes in the levels of catenins and their degree of phosphorylation after induction of cell-cell contacts. These observations provide new sights into the mechanism of calcium-dependent intercellular adhesion of keratinocytes.

The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts.

Cadherins are calcium-dependent cell-cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell-cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell-cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell- cell contact formation and the remodelling of actin filaments in epithelial cells.

Calcium-induced changes in distribution and solubility of cadherins, integrins and their associated cytoplasmic proteins in human keratinocytes.

Studies with cultured human epidermal keratinocytes have shown that stratification, the movement of differentiating cells out of the basal layer, involves changes in cell-extracellular matrix and cell-cell adhesiveness mediated by receptors of the integrin and cadherin families, respectively. Keratinocytes normally lose their integrins when they initiate terminal differentiation. However, when stratification is inhibited by a low concentration of calcium ions in the medium (0.1 mM) or by addition of antibodies to P- and E-cadherin in standard medium (1.8 mM calcium ions), differentiating, involucrin-positive, cells continue to express functional integrins. In order to investigate the mechanism by which cadherins may regulate integrin expression, we have examined the distribution and detergent solubility of the receptors and associated cytoplasmic proteins in keratinocytes grown as a monolayer in low calcium medium or transferred to standard medium to induce stratification. Within 1 hour of raising the concentration of calcium ions, integrins, cadherins, alpha-catenin, beta-catenin, plakoglobin, vinculin and alpha-actinin appeared to accumulate at cell-cell borders, whereas the focal contact proteins, paxillin and talin, did not. The change in distribution was correlated with decreased solubility in 0.5% Triton X-100 of some of the proteins examined, but the integrins, alpha-actinin, paxillin and talin remained completely soluble. Addition of cytochalasin D inhibited both the redistribution of proteins and subsequent stratification of involucrin-positive cells. Cycloheximide treatment allowed protein redistribution and stratification, but involucrin-positive cells continued to express integrins. These results suggest that stratification requires the interactions of cadherins and integrins with the actin cytoskeleton and that the selective loss of integrins from differentiating cells requires de novo protein synthesis.

Molecular aspects of Schistosoma mansoni female maturation.

Incubation of total protein extracts of Schistosoma mansoni with 3H 17-beta-estradiol and 20-hydroxyecdysone, revealed steroid binding proteins in both, male and female worms. The interaction of nuclear proteins with restriction fragments of the gender and stage-specific gene F-10 was investigated using the "Band-Shift" technique. Distinct male and female nuclear proteins bound to the fragments of this gene. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the estrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females, a band profile similar to that obtained with male worm protein extracts. When Tamoxifen was injected into schistosome infected mice, the eggs produced by females presented an abnormal morphology, compatible with non-viable eggs. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.

F-10 nuclear binding proteins of Schistosoma mansoni: structural and functional features.

By incubating total protein extracts of Schistosoma mansoni with 3H-17-beta-estradiol and 20-hydroxyecdysone, steroid binding proteins were detected in both male and female worms. The interaction of nuclear proteins with a restriction fragment of the gender and stage-specific gene F-10 was investigated using the 'band-shift' technique. Male and female nuclear proteins bound in a distinct way to the fragment of this gene containing putative regulatory consensus motifs. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the oestrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females a profile similar to that obtained with male worm protein extracts. This effect of Tamoxifen could not be correlated to inhibition of protein biosynthesis. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.

Modulation of Muc-1 mucin expression in the mouse uterus during the estrus cycle, early pregnancy and placentation.

The Muc-1 protein is an integral membrane protein that is expressed apically by simple secretory epithelia in many different organs. We present in this paper a study on Muc-1 protein expression in the mouse uterus during early pregnancy, placentation and the estrus cycle. Muc-1 immunopositive reaction is found in the decidua by day 8 of pregnancy onwards. The observed pattern was unusual, since Muc-1 protein was present in spherical cytoplasmic granules in granular metrial gland cells. Both the intracellular pattern of expression and the lymphoid origin of these cells were striking results. Muc-1 is thought to be an epithelial differentiation marker, and this is the first report of its expression by non-epithelial cells. Our results on Muc-1 expression in the uterus of cycling mice showed that higher levels of Muc-1 mRNA and protein correlate with higher levels of plasma estrogen in the estrus and proestrus phases. However, in ovariectomized mice without hormone replacement, the endometrium expressed high levels of this protein. These levels could not be substantially changed by estrogen, although progesterone reduced the levels of Muc-1 protein associated with the epithelium. These data together with the normal expression in the cycling mice suggest that progesterone might repress Muc-1 expression during the metestrus and diestrus phases. In cycling mice, when plasma progesterone is at its nadir and the estrogen level is elevated in estrus and proestrus phases, Muc-1 concentration would increase to its basal level, not because of estrogen stimulation, but due to lack of progesterone repression.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial and temporal expression of an epithelial mucin, Muc-1, during mouse development.

The Muc-1 mucin is found as a transmembrane protein in the apical surface of glandular epithelia. To provide insight into possible functions, we have assessed the timing of expression and the distribution of the Muc-1 protein during mouse embryogenesis using three different techniques: RT-PCR, northern blots and immunohistochemistry. Our results indicate that Muc-1 expression correlates with epithelial differentiation in stomach, pancreas, lung, trachea, kidney and salivary glands. Once started, Muc-1 synthesis continually increases with time, mainly due to epithelial area growth. Our data suggest that expression of the Muc-1 gene is under spatial and temporal control during organogenesis. Although Muc-1 is present in different organs, its expression is not induced systemically, but according to the particular onset of epithelial polarization and branching morphogenesis of each individual organ. It is of particular interest that Muc-1 protein can be detected lining the apical surfaces of the developing lumens when the epithelium of these organs is still undergoing folding and branching, and glandular activity has not yet started. We speculate that Muc-1 may participate in epithelial sheet differentiation/lumen formation during early development of the organs known to express it. This speculation is based on: (1) the detection of Muc-1 expression early during organogenesis, (2) the defined apical localization in different epithelia, (3) the decrease in cell-cell interactions when Muc-1 protein is highly expressed and (4) the possible interaction of its cytoplasmic tail with the actin cytoskeleton. However, it remains to be established using in vitro systems, whether the temporal and local expression of the Muc-1 gene coincident with the morphogenetic events described here is relevant for the process.

DNA binding proteins of Schistosoma mansoni recognizing a hexanucleotide motif occurring in genes regulated by steroids.

1. Schistosome proteins interacting with a gene (F-10), which is only expressed in adult females, were investigated. 2. These were also tested using a synthetic oligonucleotide of 20 bp bearing a defined sequence derived from the F-10 gene and containing a hexanucleotide motif, TGTCCT, occurring in genes responsive to steroids. 3. Schistosome proteins (male and female) bound to the F-10 DNA, but only the male proteins bound to the synthetic oligonucleotide with high affinity. 4. The other preparations each produced different binding patterns, although this seemed to lack specificity. 5. These results indicated that the F-10 gene binds different proteins along its structure and suggested that proteins present in the male schistosomes may regulate its expression.

Protein characterization of sexually mature and immature forms of Schistosoma mansoni.

1. Protein composition of different stages of Schistosoma mansoni was compared using specific antisera, 2D polyacrylamide gel electrophoresis and 14-C-leucine incorporation into proteins. 2. Major qualitative differences were detected when an anti-membrane antiserum was used. 3. 2D gel electrophoresis showed that the protein composition varied when mature and immature females were compared, whereas no differences were noted when mature and immature male worms were compared. 4. Experiments measuring protein synthesis by the different schistosome stages confirmed that upon maturation, only the female schistosomes displayed qualitative differences. 5. The protein pattern of the male schistosomes did not vary significantly as a function of development.