Differential expression of laminin chains and their integrin receptors in human gastric mucosa.

The proliferating cells of the gastric mucosa are found among the pit and mucous neck cells. These cells migrate upward to renew the surface epithelium and downward to restitute the glandular cells. As the epithelial basement membranes (BMs) function as substrate for cell adhesion and migration as well as signals for their differentiation, we studied, by indirect immunofluorescence microscopy, the distribution of different laminin chains and their integrin receptors in adult human stomach. The immunoreactivity for laminin alpha 2 chain localized to the BMs of glands and the lower parts of the gastric pits whereas the laminin alpha 3 chain (laminin-5/kalinin) immunoreactivity was strictly confined to BMs underneath the surface epithelium and the upper parts of the pits. Proliferating mucosal epithelial cells, identified by Ki-67 antibodies, were confined to the areas containing both alpha 2 and alpha 3 laminin chains. The alpha 1, beta 1, and gamma 1 laminin chains were found in all BMs of the mucosa whereas the beta 2 chain was prominent in mucosal blood vessels and also detectable in some glands. Among the laminin integrin receptors, the alpha 3 and beta 4 subunits were seen to be expressed in cells along the BMs with the alpha 3 laminin chain. The alpha 6 integrin, on the other hand, was seen in all gastric epithelia. The present results demonstrate that in the adult human stomach laminin alpha 2 and alpha 3 chains show zonal distribution in BM underlying gastric mucosal epithelium whereas other laminin chains show a more general distribution.     

Integrin Dependence of Calu-1 Cell Motility on Endothelial Extracellular Matrix Proteins

We recently showed that the motility of the malignant Calu-1 human epidermoid lung carcinoma cells correlates to their expression levels of α2, α3, α6, and β1 integrin subunits. To determine a causative relationship underlying this correlation, here we measured Calu-1 cell adhesion to and migration on laminin, collagen IV, human umbilical vein endothelial cell monolayers, and endothelial cell extracellular matrix in the presence of function-blocking antibodies against the suspect integrin subunits. Blocking individual α subunits did not affect adhesion to or motility on laminin, but when used in pair-wise combinations, monoclonal antibody treatments significantly decreased tumor cell motility on, without diminishing adhesion to, laminin and the other substrates. Blocking all three α subunits at once or the β1 subunit alone abolished migration on laminin; however, the latter treatment also abolished adhesion, whereas the former treatment did not. By contrast, blocking the β1 subunit significantly reduced motility on collagen IV, endothelial cell monolayers, and endothelial cell extracellular matrix, but always without affecting adhesion. These results suggest a separation of roles and mechanisms of different integrins in adhesion and motility.     

Role of Integrin Receptors for Fibronectin,Collagen and Laminin in the Regulation of Ovarian Carcinoma Functions in Response to a Matrix Microenvironment

Integrins play an important role in cellular matrix interactions requisite for cancer cell adhesion, growth, migration and invasion. In this study, we have investigated the expression of integrin subunits α3, α6, αv and β1 in normal ovaries, benign ovarian tumors and ovarian carcinomas of different pathological grades. The expression of these integrins in ovarian cancer cell lines was also investigated, and their role in sustaining proliferation, adhesion, migration and invasion in cohort with the activation of signaling pathways in response to extracellular matrices (ECM) was evaluated. We demonstrate a differential expression pattern of α3, α6, αv and β1 integrin subunits in ovarian carcinomas compared to normal ovaries and benign ovarian tumors. Ovarian cancer cell lines (Hey, Ovcar3 and Peo.36) demonstrated significantly high expression of α3, α6, αv and β1 integrin subunits. A significant increase in proliferation and adhesion (P<0.05) in response to collagen 1 (Coll) and laminin (LM), ligands for integrin receptor α3β1 and α6β1 was observed in ovarian cancer cell lines. On the other hand, fibronectin (FN), a receptor for αvβ1 integrin, increased proliferation in all ovarian cancer cell lines studied but only enhanced adhesion in Hey cell line (P<0.05). Neutralizing antibodies against α3, α6, αv and β1 integrin subunits inhibited ECM-induced proliferation, but increased adhesion to ECM was inhibited by β1 integrin subunit antibody. No suppression of Coll, LM and FN-induced (Hey cells only) adhesion was observed in the presence of α3 or αv subunit antibodies but LM-induced adhesion was inhibited by blocking α6 subunit functions. LM, FN and Coll enhanced chemotactic migration in Hey cells, but direct invasion across ECM was observed only in the presence of LM and Coll. Blocking antibodies against α3, α6 and β1 integrin subunits inhibited both chemotactic migration and invasion of Hey cells in response to respective ECM. Adhesion of ovarian cancer cells to FN, Coll and LM activated Ras, Erk and Akt pathways. Neutralizing αv and β1 functions did not inhibit FN-induced activation of Ras and Erk pathways but inhibited the Akt pathway. On the other hand, antibodies against α6 and β1 subunits, but not α3 subunit, inhibited LM-induced activation of Ras but did not inhibit the downstream Akt pathway. Neutralizing β1 subunit function however, inhibited LM-induced Erk activation. Coll-induced activation of Ras, Erk and Akt pathways was inhibited by α3 and β1 integrin subunit antibodies. These results indicate that α3β1, αvβ1 and α6β1 integrin mediate proliferation, adhesion, migration and invasion of ovarian cancer cells in response to ECM and targeting these integrins to modulate integrin–ECM interactions in tumor cells may be a promising tool to reduce the dissemination of ovarian carcinoma in vivo.     

Collagen type IV,laminin, α-smooth muscle actin (αSMA), α1 and α6 integrins expression in the liver with metastases from malignant gastrointestinal tumours

Basement membrane proteins and integrins can profoundly affect the biological behaviour of metastasic tumour cells. Using light and ultrastructural immunohistochemistry, we showed the presence of alterations in the occurrence of collagen type IV and laminin, and the expression of α1 and α6 integrin chains in the livers of patients with metastases from gastric, colorectal and pancreatic cancers. The myofibroblast-like cells in the metastatic stroma were studied. Parallel expressions of α-SMA, collagen type IV and α1 integrin chain, and appearance of laminin and α6 integrin chain immunoreactivity in the extratumoral liver tissue were markedly increased in sinusoids associated with metastases. Furthermore, ultrastructural immunohistochemistry detected tumour cells adhered to amorphous laminin deposits in the metastases. Laminin occurrence in liver sinusoids was visible as fine amorphous deposits in the space of Disse. The similarity between α-SMA-positive stromal cells in metastatic stroma and hepatic stellate cells (HSCs) was established by the presence of lipid droplets in their cytoplasm. The immune deposits of α1 and α6 integrin chains were observed on the hepatocyte microvilli and on the membrane of sinusoidal endothelial cells. These findings suggest that metastatic cells produce stimuli that induce HSCs activation and sinusoidal changes. In addition, the enhanced parallel expression of αSMA, collagen type IV, laminin and of α1 and α6 integrin chains in sinusoids associated with metastases, might potentiate the further dissemination of tumour cells in new liver areas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alteration in the expression of endothelial cell integrin receptors α5β1 and α2β1 and α6β1 after in vitro infection with a clinical isolate of human cytomegalovirus

Summary. Human cytomegalovirus infection of human umbilical vein endothelial cells reduces the ability of these cells to bind to fibronectin, collagen type IV and laminin. This suppression requires active virus, since UV-inactivated virus did not alter the binding ability of these cells to adhere to fibronectin, collagen type IV, and laminin. In an attempt to elucidate the molecular mechanism of this altered interaction, the surface expression of α5β1, α2β1, α3β1, and α6β1 integrins on cytomegalovirus-infected endothelial cells was examined using attachment inhibition assay and flow cytometric analysis. The results presented here show that infection with human cytomegalovirus selectively alters the expression of integrin on human endothelial cells, with the ability to induce downregulation of α5β1 and α2β1 (p=0.001 and p=0.03, respectively), while significantly upregulating α6β1 (p=0.03), and marginally upregulating α3β1 (p=0.05). Received April 15, 1996 Accepted July 20, 1996     

Alpha 6 beta 4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype.

We studied the expression and distribution of different laminin chains, the alpha 6 beta 4 integrin and type VII collagen, i.e., components of the epithelial adhesion complex, in gastric carcinomas and in suggested preneoplastic stages of this malignancy. Intestinal-type gastric carcinomas showed strong reactivity for laminin alpha 1, alpha 3, beta 1, and beta 3 chains, the components of laminin-1 and -5, at the interface between malignant cells and tumor stroma. The reactivities were continuous throughout the carcinomas, even in structures invading through the smooth muscle layers of the gastric wall. The expression of different laminin chains was accompanied by strong polarized reactivity for the alpha 6 beta 4 integrin, which is a receptor for both laminin-1 and laminin-5. Collagen type VII was only occasionally present at sites showing reactivity for laminin-5 and was totally absent from the cell islands invading through the gastric wall. Intestinalized gastric epithelium showed a similar expression pattern of laminins and the alpha 6 beta 4 integrin as the gastric carcinomas. Our results suggest that gastric carcinomas use the alpha 6 beta 4 integrin and newly deposited laminin-1 and -5, accompanied by the disappearance of type VII collagen, as their mechanism of adhesion during the invasion through surrounding tissues. Unlike in previous studies, the reactivity for the laminin-5 protein was not restricted to the invading cells but surrounded the malignant glandular structures throughout the tumor. Our results also show that both intestinal-type gastric carcinoma, and intestinal metaplasia mimic the gastric surface epithelium in the expression pattern of laminins and the beta 4 integrin subunit. This supports previous studies proposing a pathogenetic sequence from intestinal metaplasia to gastric carcinoma.     

The Differential Effect of Endothelial Cell Factors on <Emphasis Type="Italic">In Vitro</Emphasis> Motility of Malignant and Non-malignant Cells

Motility of cancer cells plays a critical role in tumor metastasis, and as such is a target for intervention. The motility of malignant Calu-1 human lung epithelial carcinoma cells is upregulated when placed on a human umbilical vein endothelial cell monolayer, while that of non-malignant L132 human lung epithelial cells is not. To dissect the factor(s) causing such differential behaviors, the motile responses of both cell lines to endothelial cell factors—secreted to the media, on the endothelial cell surface, and secreted to the extracellular matrix—and to individual extracellular matrix proteins were compared. Cell motility was quantified by tracking the cell movement on a surface with time-lapse video microscopy, which was analyzed with the persistent random walk model of motility. None of the factors tested had a remarkable effect on L132 cell motility, but the Calu-1 cell motility was significantly upregulated by endothelial cell extracellular matrix and by laminin, fibronectin, collagen I and collagen VI individually. Flow cytometry analysis revealed significantly higher expression levels of integrin subunits β1, α2, α3, and α6, which are known receptors for these extracellular matrix proteins, on the Calu-1 than L132 cells, implicating a role of these integrins in the observed motile behaviors of these cell lines.     

Use of RNA interference to inhibit integrin (α6β4)-mediated invasion and migration of breast carcinoma cells

The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the α6β4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the α6β4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced α6β4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of β4 expression in these cells augmented the formation of α6β1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the α6β4 integrin in invasion and migration that has been demonstrated previously by expression of the β4 subunit in β4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the α6β4 integrin may be a useful approach to prevent carcinoma cell progression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells

Early metastasis is the primary cause of death in melanoma patients. The adhesion receptor integrin αvβ3 contributes to tumor cell functions that are potentially involved in melanoma growth and metastasis. We tested whether integrin αvβ3 supports metastasis of human melanoma cells when injected into the bloodstream of immune deficient mice. Comparing variants of the same melanoma cell type that expressed either αvβ3, αIIbβ3 or no β3 integrin, we found that only αvβ3 strongly supported metastasis. Inhibition of tumor cell αvβ3 function reduced melanoma metastasis significantly and prolonged animal survival. To understand mechanisms that allow αvβ3, but not αIIbβ3 to support melanoma metastasis, we analyzed proteolytic and migratory activities of the melanoma cell variants. Melanoma cells expressing αvβ3, but not those expressing αIIbβ3 or no β3 integrin, produced the active form of metalloproteinase MMP-2 and expressed elevated mRNA levels of MT1-MMP and TIMP-2. This indicates an association between αvβ3 expression and protease processing. Furthermore, αvβ3 expression was required for efficient melanoma cell migration toward the matrix proteins fibronectin and vitronectin. The results suggest that expression of integrin αvβ3 promotes the metastatic phenotype in human melanoma by supporting specific adhesive, invasive and migratory properties of the tumor cells and that the related integrin αIIbβ3 cannot substitute for αvβ3 in this respect. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of integrin alpha(v)beta3 in the early phase of liver metastasis: PET and IVM analyses

To clarify the function of integrin αvβ3 in the early stage of liver metastasis, we investigated the interactions of metastatic cells with their target organ under the actual blood flow by using positron emission tomography (PET). The cells used were CHO-K1 cells and their transfectants bearing human integrin αvβ3 cDNA (αvβ3-CHO-K1 cells). The liver accumulation of αvβ3-CHO-K1 cells was significantly higher than that of CHO-K1 cells after injection via the portal vein, whereas no significant difference was observed in the lung accumulation after tail vein injection, suggesting a specific interaction of αvβ3-CHO-K1 cells with the hepatic sinusoids. Furthermore, to clarify the precise location of each cell in the liver, i.e., to determine whether individual cells were intravascularly localized or had extravasated, we performed intravital fluorescence microscopy (IVM) on the liver by using stable transfectants bearing the green fluorescent protein (GFP) gene, namely, GFP-CHO-K1 and GFP-αvβ3-CHO-K1 cells. Both types of cells remained in the hepatic blood vessels 1 h after injection via the portal vein. On the other hand, expression of integrin αvβ3 promoted the cells to reach the extravascular region after 24 h. These results suggest the possibility that the specific accumulation of αvβ3-CHO-K1 cells in the liver is followed by migration of the cells into the extravascular region. Interestingly, the adhesion of the two types of cells to hepatic sinusoidal endothelial cells in vitro did not correspond to in vivo accumulation of these cells. Therefore, integrin αvβ3 may function to promote extravasation of integrin αvβ3-expressing tumor cells in liver through a process possibly mediated by vitronectin produced by this organ. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional blocking of specific integrins inhibit colonic cancer migration

For more effective oncological management of disseminated colorectal cancer, therapies must be devised that target the different individual stages of metastasis development. Recent work showed that integrin subunits α2, α6 and β4 are involved in the colorectal cancer cell extravasation process. By means of Immunocytochemistry and Western blotting, it was shown that all three integrins are expressed not only in human colorectal cancer cells (HT29) but also in rat colonic cancer cells (DHDK12). Using in vivo models and intravital video microscopy techniques, it was shown that functional blocking of these integrin subunits by specific antibodies produced a significant reduction in cancer cell extravasation and migration. In conclusion, integrin subunits α2, α6 and β4 are expressed in unrelated colorectal cancer cell strains and appear to play a key role in cancer cell migration.     

Organisation of basement membrane components in the human adult and fetal pituitary gland and in pituitary adenomas

 Cell–matrix interactions undoubtedly have a role in the development and maintenance of the complex nonrandom structure of the human pituitary gland. We have extended previous studies by documenting the patterns of immunoreactivity for type IV collagen, laminin and fibronectin in the fetal gland, comparing these with the adult patterns. In both we have examined the differences between the anterior lobe and intermediate zone in an attempt to elucidate the apparent differences in functional response between corticotrophs in the two areas. We have also examined expression of these proteins in a series of pituitary adenomas. Finally, we have immunolocalised β₄ integrin, a component of the α₆β₄ laminin receptor, in the adult gland and in adenomas. In the anterior lobe of the adult gland, type IV collagen and laminin were present in both epithelial and vascular basement membrane. Fibronectin was related to the basement membrane but showed a less continuous distribution. β₄ Integrin was expressed on the basal aspects of pituitary cells, in association with laminin, suggesting that this did identify the α₆β₄ laminin receptor. In addition, immunoreactivity was present on the lateral margins of some pituitary cells, which might indicate a role in cell–cell adhesion. None of the proteins showed specific association with any particular cell type, suggesting that these specific interactions do not regulate differentiation. This pattern of expression had developed in the fetal gland by the second trimester, with expression relating to vessels preceding that in epithelial basement membrane. Type IV collagen, laminin and fibronectin were also expressed in epithelial and vascular basement membrane in the intermediate zone of the adult gland, and around Rathke’s cleft in the fetal gland. However, the organisation differed, with larger groups of cells enclosed within a single basement membrane. Possible vascular connections demonstrated between the posterior lobe and the intermediate zone would permit access of posterior lobe hormones to this zone. Our data confirmed disruption of expression in pituitary adenomas, type IV collagen, laminin and β₄ integrin having a mainly perivascular distribution, with more variable immunoreactivity for fibronectin. Received: 17 February 1997 / Accepted: 17 May 1997     

Angiogenesis and the growth potential of craniopharyngiomas

The present study was performed to evaluate the role of neovascularization on the behavior of craniopharyngiomas as well as the contribution of endothelial cell proliferation and migration in the remodeling and expansion of the vascular network associated with angiogenesis. Fourteen primary tumors were studied, all of the adamantinomatous type. CD34 immunostaining, an endothelial cell marker, localized vessels within the connective tissue stroma. MIB-1 immunopositivity was apparent in the nuclei of neoplastic cells, few endothelial cells, and stromal elements. MIB-1 counts were higher in epithelial than connective tissue cells. A positive correlation was found between the number of MIB-1 immunopositive cells and microvessel density (MVD). Immunohistochemistry demonstrated that integrin α_vβ3 expression was restricted to tumor vasculature; the tumor cells were immunonegative. Only 2.5% of vessels detected with CD34 were immunopositive for integrin α_vβ3. At present, no therapeutic implications can be drawn from our observations. More studies are needed to assess whether integrin α_vβ3 antagonists or drugs that arrest the cell cycle of endothelial cells can inhibit angiogenesis in craniopharyngiomas.     

Interaction with the stroma and endothelium

Prostatic carcinoma cells have a propensity to metastasize to bone, and we propose that this phenomenon may be promoted by the adhesion of metastatic cells to bone matrix. Bone matrix is produced by osteoblasts, and we have developed an in vitro model of bone matrix by isolating the substratum deposited by human osteoblast-like U20S cells. The collagenous nature of this matrix was demonstrated by the incorporation of [³H]proline and its subsequent release by purified collagenase. Both U20S matrix and purified type I collagen stimulated the adhesion of human PC-3 prostatic carcinoma cells. Human laminin supported adhesion to a much lesser extent, and PC-3 cells did not adhere to fibronectin. Adhesion of PC-3 cells to U20S matrix closely resembled adhesion to purified type I collagen with respect to (a) inhibition by a collagen-derived peptide and by antibodies raised against α2 or β1 integrin collagen receptor subunits; (b) lack of inhibition by RGD (Arg-Gly-Asp) peptides; (c) stimulation by Mn²⁺ and Mg²⁺ ions but not by Ca²⁺ ion; and (d) stimulation by the phorbol ester PMA (phorbol 12-myristate 13-acetate). This adhesion was also stimulated (2.3-fold) by transforming growth factor β (TGF-β), which is a major bone-derived growth factor. We conclude that human osteoblast-like matrix is an adhesive substrate for PC-3 prostate carcinoma cells. This adhesion appears to be mediated by the interaction of α2β1 integrins on PC-3 cells with matrix-derived collagen. The stimulation of this adhesion by TGF-β suggests that the co-expression of TGF-β and type I collagen in bone may synergistically facilitate the adhesion of metastatic cells to bone matrix proteins and thereby increase their localization in the skeleton.     

Phorbol dibutyrate-induced megakaryocytic differentiation increases susceptibility of K562 cells to SA11 rotavirus infection

Summary.  The role of integrins previously implicated as rotavirus receptors in determining cellular susceptibility to SA11 rotavirus was studied, using phorbol dibutyrate (PDB) treatment of K562 cells to induce megakaryocytic differentiation. Expression of α2β1 integrin was detected after 2 days in PDB, and peaked after PDB treatment for 4–7 days. SA11 titres were increased by 1.8- to 10.8-fold over untreated cells after PDB treatment for 2–7 days, and correlated with levels of α2β1 integrin expression in PDB-treated K562 cells. Accepted April 3, 2001 Received October 30, 2000     

Integrin distribution in gastric carcinoma: Association of β3 and β5 integrins with tumor invasiveness

Immunolocalization of a variety of integrins using monoclonal antibodies against β3, β5, α3β1 and α6, and polyclonal antibodies against vitronectin receptor (αvβ3) and l were investigated on PLP-fixed paraffin sections of 19 cases of advanced gastric carcinomas. The β5 integrin, which pairs only with the av subunit, was positive in seven cases (37%), and was associated closely with scirrhous invasion (P < 0.05). β5 was positive chiefly in the cytoplasm of carcinoma cells and infrequently in cell membranes. β3, which is another subunit pairing with av, was positive in six cases (32%), and tended to be associated with scirrhous invasion (P < 0.1). β3 was also located chiefly in the cytoplasm. Five of the seven β5-positive cases showed coexpression of β3. Polyclonal antibodies to αvβ3 also showed a significant difference among the amounts of stroma (P < 0.05). Anti-β1 antibodies showed clear positivity in many cases (89%). Of the β1 integrins, α3β1 was positive in a few cases (26%) without any preferential pattern, and laminin receptor subunit α6 stained on cell membranes of neoplastic epithelia in many cases of carcinoma (89%) except for mucinous carcinoma. These distinctive patterns of integrin positivity indicate a close association of β5 and β3 expression with scirrhous invasion in gastric carcinoma.     

Interactions between the serotonergic and histaminergic systems in the central cardiovascular regulation in haemorrhage-shocked rats: involvement of 5-HT1A receptors

Aim and objective:  The aim of the work was to characterise the nAChRs on human PBMC. Method:  PBMC were isolated from human blood buffy coats provided by the blood transfusion service and were used for radioligand binding studies with [³H]-nicotine. RT-PCR experiments were used to determine nAChR subunit expression while immunoblotting experiments were used to confirm that nAChR subunits identified by RT-PCR were translated into protein. Results:  Binding studies suggested the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes. Competition studies showed that only (-)- nicotine, epibatidine and α-bungarotoxin, displaced radiolabelled nicotine from cells. RT-PCR studies demonstrated mRNA for α4, α5, α7, β1 and β2 nAChRs subunits in PBMC. Expression of mRNA for the a5 subunit of nAChR was observed in all lymphocyte samples tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between samples. Western blot analysis showed that protein for α4, α5, and α7 and β2 nAChR subunits was expressed in most, but not all of the PBMC samples tested but some of the bands obtained were faint. Conclusion:  The results obtained suggest that human PBMC contain nAChRs containing α4β2, α4β2α5, and/or α7 subunits. Received 6 August 2008; returned for revision 3 September 2008; received from final revision 3 October 2008; accepted by M. Parnham 6 October 2008     

Adhesion of HT-29 colon carcinoma cells to endothelial cells requires sequential events involving E-selectin and integrin β4

HT-29 colon carcinoma cells attach to TNFα-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to E-selectin. This interaction activates, in the cancer cells, the MAPK SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of E-selectin in activating integrins to modulate adhesion and regulate integrin-mediated events. Blocking the integrins from HT-29 cells (α2, α3, α6, αvβ5, β1 and β4) with specific antibodies revealed a role for β4 integrin in their adhesion to TNFα-treated HUVEC. The β4 integrin-dependent adhesion was maximal after 30 min, whereas the-E-selectin-dependent adhesion was maximal after 15 min. Integrin β4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of E-selectin. Moreover, a recombinant E-selectin/Fc chimera did not induce the phosphorylation of β4. The phosphorylation of β4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of β4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of β4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of β4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of E-selectin to its receptor on carcinoma cells and then the binding of β4 to its own receptor on endothelial cells; 2) that the phosphorylation of integrin β4 contributes to enhance the motile potential of cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration. Abbreviations: ERK – extracellular signal-regulated kinase; GFP – green fluorescent protein; ICAM – intercellular adhesion molecules: JNK – c-Jun NH₂-terminal kinase; MAPK – mitogen-activated protein kinase; MAPKAP K2, MAP kinase-activated protein kinase 2; PI3K – phosphatidyl inositol 3-kinase; SAPK – stress-activated protein kinase; TNFα– tumor necrosis factor-α. This revised version was published online in July 2006 with corrections to the Cover Date.