Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells

B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.     

Induction of protein tyrosine phosphorylation in human natural killer cells by triggering via alpha 4 beta 1 or alpha 5 beta 1 integrins

Recent studies have shown that cell-surface integrins expressed on platelets, fibroblasts, or carcinoma cell lines serve not only as adhesion receptors that connect the extracellular matrix to the cytoskeleton, but also as signal-transducing molecules involved in altering cellular patterns of tyrosine phosphorylation. In this present report we provide evidence that adhesion of freshly purified human natural killer (NK) cells to fibronectin (FN) induces tyrosine phosphorylation of intracellular proteins of approximate molecular mass of 60, 70, and 120 kD. Increases in phosphorylation induced by NK cell binding to immobilized FN were partially blocked by EILDV- (CS-1) or RGD-containing peptides, which compete specifically for a distinct binding site for either alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, within the FN molecule. The presence of either one of the inhibitory peptides alone inhibited tyrosine phosphorylation primarily during short-term (30 minutes) and, to a lesser extent, during long- term (2 to 3 hours) periods of adhesion. These observations indicate that triggering either via alpha 4 beta 1 or alpha 5 beta 1 integrins, which are constitutively expressed on NK cells, induces protein tyrosine phosphorylation. Moreover, FN fragments of 40 or 120 kD, known to contain the binding sites for alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, used as immobilized substrates for NK cell adhesion, were able to initiate tyrosine kinase activity. The induced tyrosine phosphorylation was observed mainly on intracellular proteins of greater than 50 kD molecular weight. We have identified a 70-kD tyrosine phosphoprotein as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions. Thus, interaction of NK cells with immobilized extracellular matrix glycoproteins required for migration and extravasation of these cells involves activation of intracellular protein tyrosine kinases and tyrosine phosphorylation of cytoskeleton- associated protein, paxillin, which may play a role in signaling between beta 1 integrins and the underlying cytoskeleton.     

Distribution of integrins on human peripheral blood mononuclear cells

The receptors for fibronectin (FN-R) and vitronectin (VN-R) belong to a family of integral membrane glycoproteins known to be involved in cell- extracellular matrix and cell-cell interactions named integrins (FN-R = beta 1 integrin and VN-R = beta 3 integrin). Adhesion studies using FN- coated plastic dishes and highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) showed a strong binding of monocytes and T lymphocytes to FN but virtually no binding of B cells to FN. Binding of monocytes and T cells to FN could be partially inhibited by a hexapeptide (GRGDSP) containing the adhesive peptide sequence Arg-Gly- Asp (RGD) as well as by an anti-FN-R antibody. The distribution of beta 1 and beta 3 integrin complexes on PBMCs was characterized by immunoprecipitation of detergent extracts of 125I-labeled cells using polyclonal antibodies against these two receptors. Two surface polypeptides corresponding to the alpha and beta chains of FN-R and VN- R were found on all three cell types. To characterize these receptors further, monoclonal antibodies (MoAbs) against the very late antigens (VLAs) 1, 3, and 5 were used for immunoprecipitation studies. Monocytes and T cells reacted with VLA 5 that was previously identified as the human FN receptor, whereas no labeling with anti-VLA 5 could be shown for B cells. When cell populations were cultured in 10% human serum for 24 hours, an increase in beta 1-integrin+ monocytes and T cells was observed. The number of beta 3-integrin+ cells remained essentially unchanged. The presence of beta 1 and beta 3 integrins on monocytes as well as on T and B lymphocytes may be of significance in the ability of these cells to interact with each other and participate in hematopoiesis and certain immune reactions.     

Alteration of integrins by interleukin-1alpha in human pancreatic cancer cells

INTRODUCTION: Adhesion of tumor cells to extracellular matrix (ECM) proteins plays an important role in tumor invasion and metastasis. AIMS: To investigate the expression of integrins in human pancreatic cancer cell lines and its alteration by interleukin (IL)-1alpha to examine the mechanism of adhesion of metastatic human pancreatic cancer cells to ECM proteins. METHODOLOGY: The expression of integrin subunits and their alteration by IL-1alpha were examined by flow-cytometric analysis and cellular enzyme-linked immunosorbent assay in three metastatic human pancreatic cancer cell lines (AsPC-1, BxPC-3, and SW1990) and two nonmetastatic cancer cell lines (PaCa-2 and PANC-1). In addition, assays of cancer cell adhesion to ECM proteins were performed to investigate if increased integrin expression actually affected the adhesive interaction between cancer cells and the putative integrin ECM ligands. RESULTS: The alpha(6) subunit expressed in metastatic cancer cells was enhanced by IL-1alpha. Metastatic cancer cells also showed preferential adherence to laminin compared with nonmetastatic cancer cells, and this was enhanced by IL-1alpha. CONCLUSION: In pancreatic cancer, the enhancement of alpha(6)beta(1) integrin by IL-1alpha through IL-1 receptor type I, as well as the expression of alpha(6)beta(1) integrin, plays an important role in metastasis formation.     

Laminin-5 stimulates hepatocellular carcinoma growth through a different function of alpha6beta4 and alpha3beta1 integrins

Hepatocellular carcinoma (HCC) growth severely affects prognosis. Ki-67, a known marker of cell proliferation, is a negative prognostic factor in HCC. Growth factors such as the epidermal growth factor (EGF) induce HCC cell proliferation but do not explain the great heterogeneity of HCC growth. Laminin-5 (Ln-5) is an extracellular matrix protein (ECM) present in the tissue microenvironment of HCC. The two main receptors for Ln-5, integrins alpha3beta1 and alpha6beta4, are expressed on the cell surface of HCC cells. The aim of this study is to investigate an alternative mechanism of HCC growth whereby Ln-5 promotes HCC cell proliferation through alpha3beta1 and alpha6beta4. HCC tissues containing Ln-5 display a larger diameter and higher number of positive cells for Ki-67, a well known proliferative index, as determined by double immunofluorescence staining and real-time PCR on microdissected tissues. In vitro, Ln-5, but not collagen I, collagen IV or fibronectin, induces proliferation as much as EGF does, via Erk phosphorylation as a consequence of beta4 integrin phosphorylation. However, the two HCC cell lines do not proliferate in presence of Ln-5 despite beta4 integrin and Erk1/2 activation. After transfection with alpha3 integrin, in the presence of Ln-5 one of these HCC cell lines acquires a proliferative activity whereas one of the proliferative HCC cell lines, knocked-down for alpha3 integrin, loses its proliferative activity. Conclusions: Our study suggests a new mechanism of HCC growth whereby Ln-5 stimulates proliferation via a different function of alpha6beta4 and alpha3beta1.     

Expression of beta 1 integrins in normal human keratinocytes

The majority of cell adhesive events to the extracellular matrix are mediated by cell surface receptors, beta 1 integrins. Keratinocytes express at least six different polypeptides of beta 1 integrin class, namely beta 1, alpha 2, alpha 3, alpha 5, and alpha 6 (alpha 6 is mainly associated with beta 4 polypeptide). These epithelial cells use alpha 2 beta 1 as a collagen receptor and alpha 3 beta 1 as a fibronectin receptor, while alpha 6 beta 4 is the major basement membrane receptor. Expression of alpha 5 beta 1 complex is low. Processing of beta 1 integrins is fast in keratinocytes; half-maximal maturation takes only 3 hours. In addition to their function in cell-matrix interactions, beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) have also a role in maintaining keratinocyte cell-cell interactions. It is possible that resting basal keratinocytes use beta 1 integrins as cell-cell adhesion receptors, and during activation, like in wound healing, these receptors relocalize to mediate events involving cell-matrix interactions.     

Src kinase activation by direct interaction with the integrin beta cytoplasmic domain

Src tyrosine kinases transmit integrin-dependent signals pivotal for cell movement and proliferation. Here, we establish a mechanism for Src activation by integrins. c-Src is shown to bind constitutively and selectively to beta3 integrins through an interaction involving the c-Src SH3 domain and the carboxyl-terminal region of the beta3 cytoplasmic tail. Clustering of beta3 integrins in vivo activates c-Src and induces phosphorylation of Tyr-418 in the c-Src activation loop, a reaction essential for adhesion-dependent phosphorylation of Syk, a c-Src substrate. Unlike c-Src, Hck, Lyn, and c-Yes bind more generally to beta1A, beta2, and beta3 cytoplasmic tails. These results invoke a model whereby Src is primed for activation by direct interaction with an integrin beta tail, and integrin clustering stabilizes activated Src by inducing intermolecular autophosphorylation. The data provide a paradigm for integrin regulation of Src and a molecular basis for the similar functional defects of osteoclasts or platelets from mice lacking beta3 integrins or c-Src.     

Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

The alpha4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins alphavbeta3, alpha3beta1, and alpha6beta1. An antibody against the G domain (residues 919-1207; G(919-1207)) of the alpha4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G(919-1207) to support endothelial cell adhesion. In particular, endothelial cell adhesion to G(919-1207) is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins alphavbeta3 and beta1 and a combination of antibodies against alpha3 and alpha6 integrin subunits inhibit endothelial cell attachment to G(919-1207). Moreover, both alphavbeta3 and alpha3beta1 integrin bind with high affinity to G(919-1207). Together, our studies demonstrate that the G domain of laminin alpha4 chain is a specific, high affinity ligand for the alphavbeta3 and alpha3beta1 integrin heterodimers and that these integrins, together with alpha6beta1, function cooperatively to mediate endothelial cell-alpha4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the alphavbeta3 integrin in angiogenesis.     

Role of angiotensin type-1 and angiotensin type-2 receptors in the expression of vascular integrins in angiotensin II-infused rats

Angiotensin II plays an important role in vascular remodeling through effects that involve, in part, interactions of vascular smooth muscle cells with extracellular matrix via integrins, which belong to a family of transmembrane receptors. We hypothesized that angiotensin (Ang) II regulates expression of vascular integrins and their ligands in experimental hypertension. Rats were infused subcutaneously with Ang II and received angiotensin type-1 (AT1) receptor blocker losartan, the AT1/angiotensin type-2 (AT2) [Sar1-Ile8]-Ang II, or the vasodilator hydralazine for 7 days. Osteopontin and integrin subunit expression were evaluated immunohistochemically. Ang II enhanced vascular alpha8, beta1, beta3 integrins and osteopontin expression, which were significantly reduced by losartan, [Sar1-Ile8]-Ang II, and hydralazine. Although Ang II increased vascular alpha5 subunit expression, this was additionally increased by losartan. Losartan was the only treatment that induced alpha1 subunit expression. These results demonstrate that AT1 and AT2 receptors have countervailing effects on vascular integrin subunit expression that may influence their effects on vascular remodeling and extracellular matrix composition.     

Steroid regulation of human placental integrins: suppression of alpha2 integrin expression in cytotrophoblasts by glucocorticoids.

Maintenance of uterine-placental attachment during human pregnancy may depend at least partly on adhesive interactions between cytotrophoblasts and their extracellular matrix (ECM). Such interactions are often mediated by integrins, signal-transducing heterodimeric transmembrane glycoproteins. We previously showed that glucocorticoid (GC) suppressed the expression of collagen and laminin in human placenta; here we show that GC also modulates the expression by human cytotrophoblasts of the integrin subunits alpha2 and beta1, components of a known receptor for these ECM ligands. Cytotrophoblasts were isolated from human term placentas, cultured up to 4 days in the presence of 0-1000 nM dexamethasone (DEX), and assayed for 1) integrin messenger RNA (mRNA) levels by Northern hybridization, 2) integrin subunit synthesis after [35S]methionine labeling, or 3) cell surface integrin levels after 125I labeling by lactoperoxidase. In four independent experiments, 100 nM DEX reduced mRNA levels for integrin alpha2 to 6+/-1% of the control value. This effect was similar between 1-4 days of treatment and was dose dependent between 1-1000 nM DEX. Cortisol treatment (100 nM) inhibited levels of integrin alpha2 mRNA, but 100 nM testosterone, estradiol, and progesterone were less effective, suggesting that this response was specific to GC. In immunoprecipitation studies, treatment of cytotrophoblasts with 100 nM DEX for 2 days reduced the rates of synthesis of the alpha2 integrin subunit as well as its expression on the cell surface to 1-10% of control levels. DEX effects on the beta1 integrin subunit were less dramatic. DEX reduced beta1 mRNA levels to only 69+/-8% of control levels, a smaller reduction compared with effects on alpha2 integrin mRNA. DEX inhibited beta1 protein synthesis and cell surface expression to 60-70% of control levels. In all experiments, DEX had no effect on total protein synthesis. Thus, our results demonstrate that GC treatment specifically and markedly down-regulates expression of alpha2 integrin subunit by human cytotrophoblasts. This finding is consistent with the concept that uterine-placental adherence across gestation may be regulated by coordinate effects on ECM ligands and cellular adhesion receptors.     

Regulation of postangiogenic neovessel survival by beta1 and beta3 integrins in collagen and fibrin matrices

We used the aortic ring model of angiogenesis to investigate the role of beta(1) and beta(3) integrins in postangiogenic vascular survival in collagen and fibrin matrices. Confocal microscopy studies showed that both beta(1) and beta(3) integrins were expressed in endothelial cells and pericytes of sprouting neovessels. Antibody blocking experiments demonstrated that beta(1) integrins but not beta(3) integrins were required for angiogenic sprouting in collagen. Conversely, in fibrin, blockade of both integrins was needed to inhibit angiogenesis whereas treatment with either antibody alone was ineffective. Antibody-mediated blockade of beta(1) but not beta(3) integrins accelerated vascular regression in collagen. In contrast, both anti-beta(1) and -beta(3) integrin antibodies were required to promote neovessel breakdown in fibrin. These results demonstrate that angiogenic sprouting and postangiogenic neovessel survival in collagen are critically dependent on beta(1) integrins. They also indicate that these processes involve a redundant repertoire of beta(1) and beta(3) integrins when angiogenesis occurs in fibrin. Thus, pharmacologic targeting of integrin receptors aimed at blocking neovessel formation and survival must be tailored to the specific extracellular matrix environment in which angiogenesis takes place.     

Expression of {beta}1 integrin receptors during rat pancreas development--sites and dynamics

The integrin receptors link to extracellular matrix proteins and exert a dynamic role in development by providing the physical basis for cell adhesion and controlling cell growth. In the present study, we examined changes in the expression of beta1 integrins and its associated alpha-subunits to islet cell development in the rat pancreas. A significant increase in protein expression of integrin alpha3, alpha6, and beta1 was observed from fetal to postnatal life. High mRNA levels of these integrin subunits was detected at embryonic d 18 and dropped significantly after birth with relatively low expression throughout postnatal life. Integrins alpha3, alpha5, alpha6, and beta1 were expressed in a cell-specific manner in the pancreas with high integrin immunoreactivity in duct and islet regions during fetal life, and a progressive increase later into postnatal life. The coexpression with islet and putative islet precursor markers during fetal and postnatal development suggest a role for these integrin subunits in differentiation and maturation of islets. Functional studies in vitro showed that anti-beta1 antibody treatment inhibited islet cell adhesion to extracellular matrices and disrupted islet architecture. Blockade of beta1 integrin receptor and knockdown beta1 mRNA resulted in a decrease in the expression of insulin mRNA and increased islet cell death. These results suggest that progression in islet cell development is accompanied by and dependent upon cell adhesion via beta1 integrin and its respective alpha-subunits and suggest that the beta1 family of integrins may play a critical role in islet cell architecture, development, integrity, and function.     

Human endothelial cells express integrin receptors on the luminal aspect of their membrane.

Endothelial cells (EC) form a dynamic interface between blood and the rest of the body. EC surface properties promoting adhesion of reactive plasma proteins and/or circulating cells might be of pivotal importance for the homeostasis of blood and tissues. EC express multiple integrin receptors that promote their attachment to the subendothelial matrix proteins. Among these receptors, alpha v beta 3 is of particular relevance on EC, since it is abundantly expressed and can bind many different matrix and plasma proteins. It is still unknown whether integrin receptors are selectively located to the basal side of EC membrane or may also be exposed on the cell surface in contact with blood. This issue was addressed using different experimental approaches. First, selective surface radioiodination using lactoperoxidase (LPO)-latex beads and immunoprecipitation analysis were performed. We found that cultured EC, similarly to human skin fibroblasts (HSF), expose alpha v beta 3 on both their apical (free) and basal (substratum-attached) surfaces. This held also for other integrins such as alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1. Immunoprecipitation data were verified by morphological techniques. Immunofluorescence and immunogold-staining of EC with alpha v beta 3, as well as with beta 1 subfamily antibodies, showed a diffuse and granular distribution of these integrins on EC surface. alpha v beta 3 and beta 1 integrins were also detected on the apical membrane of EC at higher magnification by scanning electron microscopy (SEM). Finally, data obtained on cultured EC were confirmed in vivo on immunogold-labeled ultrathin cryosections of human vessels by transmission electron microscopy (TEM). Data indicate, that in addition to their role in promoting EC attachment to extracellular matrix proteins, integrin receptors of EC can be exposed to blood-stream and eventually be available for binding of plasma proteins and circulating cells.     

Expression patterns of integrins on quiescent and invasive smooth muscle cells and impact on cell locomotion

Migration and invasion of human arterial smooth muscle cells (haSMCs) are essential steps during the development of atherosclerosis, restenosis, and transplant vasculopathy. The molecular mechanisms leading to these processes are only incompletely understood. Due to their contact to the surrounding extracellular matrix, integrins have been shown to be essentially involved in cell locomotion. Therefore, the function of integrins during this process was analyzed in an in vitro model which was based on the defined quiescent and invasive phenotypes of human haSMCs induced by cell culture conditions. Flow-cytometric analysis of integrin expression between both phenotypes showed a strong upregulation of alpha 5 beta 1 (13.1x) and a modest upregulation of alpha vs beta 3 (3.4x) and alpha IIb (3.0x) in invasive haSMCs in comparison to quiescent ones. Other integrins analyzed (alpha 2, alpha 3, alpha 4, beta 1) did not show differential regulation. Functional inhibition of alpha 5 beta 1 reduced cell migration (-29%+/-8), invasion (-49%+/-16), collagen contraction (-125%), and attachment to fibronectin. Although, there was a clear discrepancy between alpha 5 beta 1 and alpha vs beta 3 expression levels, inhibition of alpha vs beta 3 (-45%+/-9) reduced haSMC invasion equally. Interestingly, alpha vs beta 3 unlike alpha 5 beta 1 blockade caused a significant stimulation of collagen contraction (+52% vs 154%) with possible implications on vascular remodeling. In conclusion, alpha 5 beta 1 blockade or combined alpha 5 beta 1/alpha v beta 3 blockade by specific antibodies or selective RGD peptides together with local drug delivery strategies could be a promising strategy for the therapy of restenotic lesions or atheromatous plaques.     

Altered glycosylation of integrin adhesion molecules in colorectal cancer cells and decreased adhesion to the extracellular matrix.

The integrin mediated interactions between tumour cells and the surrounding extracellular matrix are thought to play crucial parts in the complex process of invasion and metastasis. It has been previously shown that the expression of integrins is differently diminished in a chain-specific manner in human colorectal cancer. To further characterise the integrins still expressed in colorectal carcinomas, immunoblots with monoclonal antibodies against the beta 1 integrin subunit have been performed. In isolated cell membranes of colorectal cancers a second smaller beta 1 chain (105 kD non-reduced) was found as well as the mature beta 1 chain (116 kD non-reduced) present in normal mucosa of the colon. This smaller beta 1 chain comigrates with the diminished glycosylated precursor form of the beta 1 chain. The role of N-glycosylation for the function and expression of integrins in vitro was therefore investigated, with deoxymannojirimycin (DMJ) and deoxynojirimycin (DNJ) as specific inhibitors of N-glycan processing. Pretreatment of human colon adenocarcinoma derived HT-29 cells with DMJ resulted in an expression of the 105 kD beta 1 precursor chain and of smaller forms of the alpha 1, alpha 3, alpha 6, and alpha v integrin subunits in a time and dose dependent manner. HT-29 cells treated with DMJ adhered poorly to laminin (8% of untreated controls), collagen type IV (40%), and fibronectin (35%). Pretreatment of the cells with DNJ did not alter the molecular weight of the integrin chains expressed and reduced HT-29 adhesion to laminin and fibronectin only to 68% and 49% respectively. Adhesion to collagen type IV was increased to 124% by DNJ. These results show that N-glycan processing is essential for the function and expression of integrins in human colorectal cancer cells. An altered glycosylation of these adhesion receptors may contribute to a more invasive or metastatic phenotype in colorectal cancer.     

Modulation of integrin function in hematopoietic progenitor cells by CD43 engagement: possible involvement of protein tyrosine kinase and phospholipase C-gamma

Attachment of cells to extracellular matrix components is critical for the regulation of hematopoiesis. CD43 is a mucin-like transmembrane sialoglycoprotein expressed on the surface of almost all hematopoietic cells. A highly extended structure of extracellular mucin with negative charge may function as a repulsive barrier to hematopoietic cells. However, some investigators have shown that CD43 has proadhesive properties, and engagement of CD43 has been reported to upregulate integrin-mediated cell adhesion in T cells. We found that cross-linking of CD43 with monoclonal antibodies (MoAbs) enhanced integrin alpha4beta1 (very late antigen [VLA]-4) and alpha5 beta1 (VLA-5)-dependent adhesion of human cord blood CD34(+) cells to fibronectin. CD34(+) CD38(hi), but not CD34(+)CD38(-/low) cells responded significantly to the stimulus, suggesting that committed, but not stem and more immature progenitors are sensitive to CD43-mediated activation of integrin. To elucidate the molecular mechanism leading to integrin activation, we used the growth factor-dependent cell line MO7e. Cross-linking of CD43 induced tyrosine phosphorylation of several intracellular molecules including the protein tyrosine kinase Syk, the proto-oncogene product Cbl, and phospholipase C (PLC)-gamma2 in MO7e cells. Moreover, protein tyrosine kinase inhibitor herbimycin A and PLC inhibitor U73122 both blocked CD43-induced enhancement of adhesion to fibronectin. These results indicate that signals mediated through CD43 may increase integrin affinity to fibronectin via a pathway dependent on protein tyrosine kinase and PLC-gamma activation in hematopoietic progenitors.     

Integrin alpha6beta1 plays a significant role in the attachment of hepatoma cells to laminin.

BACKGROUND/AIMS: Tumor invasion and metastasis consist of a series of complex events. During this process, the ability of tumor cells to adhere to laminin, a major component of basement membranes, is required at various steps. The expression of laminin-binding integrins and the extent of tumor metastasis and progression appear to be related. In hepatocellular carcinoma, increased expression of laminin-binding integrins is observed. However, little is known concerning the possible functional interactions between laminin-binding integrins and laminin. Therefore, we investigated the participation of laminin-binding integrins in the attachment of hepatoma cells to laminin. METHODS: Human hepatoma cell lines (KIM-1, KYN-1, 2) were used. We investigated the expression of integrin alpha1, alpha2, alpha3, alpha6, beta1, and beta4 subunits on hepatoma cells by immunocytochemical and flow cytometric analysis. Participation of these integrin subunits in the attachment of hepatoma cells to laminin was evaluated by an inhibition of cell adhesion assay. RESULTS: Integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunits were expressed at the marginal areas of hepatoma cells, while the integrin beta4 subunit was scarcely detected. Laminin promoted the attachment of hepatoma cells in a dose-dependent manner. Although anti-integrin alpha1, alpha2, beta3 and beta4 subunit antibodies did not inhibit cell attachment to laminin, anti-integrin alpha6 and beta1 subunit antibodies inhibited the attachment by 50% or more. CONCLUSIONS: These findings indicate that integrin alpha6beta1 is very important in the attachment of hepatoma cells to laminin, suggesting the participation of this integrin in metastasis and invasion of hepatoma cells.     

The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression.

Anchorage-dependent cells that are prevented from attaching to an extracellular matrix substrate stop proliferating and may undergo apoptosis. Cell adhesion to a substrate is mediated by the integrin family of cell surface receptors, which are known to elicit intracellular signals upon cell adhesion. We show here that Chinese hamster ovary cells expressing the alpha 5 beta 1 integrin, which is a fibronectin receptor, do not undergo apoptosis upon serum withdrawal when the cells are plated on fibronectin. However, the alpha v beta 1 integrin, which is also a fibronectin receptor and binds fibronectin on the same RGD motif as alpha 5 beta 1, did not prevent apoptosis on fibronectin of the same cells. The cytoplasmic domain of the integrin alpha 5 subunit was required for the alpha 5 beta 1-mediated cell survival on fibronectin. The fibronectin-mediated survival effect appeared to be independent of the level of tyrosine phosphorylation of the focal adhesion kinase, which is induced by integrin-mediated cell attachment. The expression of the Bcl-2 protein, which counteracts apoptosis, was elevated in cells attaching to fibronectin through alpha 5 beta 1; cells attaching through alpha v beta 1 survived only if exogenous Bcl-2 was provided. Thus, alpha 5 beta 1, but not the closely related alpha v beta 1 integrin, appears to suppress apoptotic cell death through the Bcl-2 pathway.     

Immunolocalization of integrin receptors in normal lymphoid tissues

The integrin superfamily of cell adhesion receptors consists of heterodimeric glycoproteins composed of unique alpha and beta subunits. These receptors mediate cell-substrate and cell-cell adhesive properties for a variety of cell types. This investigation has focused on the histologic distribution of the beta 1 subfamily of integrins within lymphoid tissues including tonsil, lymph node, spleen, thymus, and appendix. The dendritic cells of both follicular center and thymic origin express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6, as well as the beta 1 integrin subunits. Most lymphoid cells in normal tissues do not express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits, or the alpha v beta 3 integrin. The beta 1 subunit is expressed by all lymphocytes but with variable intensity. Increased levels of the alpha 5 and beta 1 subunits are observed in the follicular light zone, suggesting a role for these integrins in B-cell activation. Although the alpha 4 subunit is expressed by all lymphoid cells, an increased expression of alpha 4 and decreased expression of beta 1 by the mantle zone B-cell compartment is noted in comparison with the decreased expression of alpha 4 and increased expression of beta 1 by follicular center B-cells. These studies suggest that alpha 4 may be paired with a beta subunit other than beta 1 on the mantle zone lymphoid population. Thus, integrin expression by cells of lymphoid tissues varies with location and function and differs significantly from integrin expression observed on circulating and cultured peripheral blood lymphocytes.     

Phenotyping of chondrocytes from human osteoarthritic cartilage: chondrocyte expression of beta integrins and correlation with anatomic injury

Chondrocyte-ECM (extracellular matrix) interactions are believed to play a pivotal role in the development and metabolic homeostasis of articular cartilage. Cell surface adhesion molecules have been reported to modulate chondrocyte binding to ECM (collagen, fibronectin, laminin) and they also act as transducers of critical signals in many biological processes such as growth, differentiation, migration and matrix synthesis. Recently, it has been shown that normal human articular chondrocytes strongly express beta1 integrins, which are constituted by a common chain (beta1) and a variable alphachain, but the behaviour of these molecules in human osteoarthritic cartilage has not been extensively investigated. We studied the expression of beta integrins (beta1-5, alpha1-6, av chains), LFA-1, ICAM-1 and CD44, on freshly isolated chondrocytes obtained from 10 osteoarthritic patients undergoing surgical knee replacement. Chondrocytes were isolated by enzymatic digestion from three zones of each articular cartilage with a differing degree of macroscopic and microscopic damage. Integrin expression and cell cycle analysis were carried out by flowcytometry. Chondrocytes from costal cartilages of 5 human foetuses were also studied. Chondrocytes from osteoarthritic cartilage expressed high levels of beta1 integrin and, at different percentages, all the alphachains. The alphachain most frequently expressed was alpha1, foilowed by alpha3, alpha5, alpha2, alphav. Integrin expression decreased from the least to the most damaged zone of articular cartilage and cell cycle analysis showed that proliferating chondrocytes (S phase) were prevalent on the latter zone. beta2, beta3, beta2, beta5, CD44, LFA-1/ICAM-1 complex were very low expressed. Fetal chondrocytes strongly expressed beta1 and beta5 chains. These data provide evidence to show that integrin expression on human chondrocytes changes in osteoarthritis and suggest that perturbations of chondrocyte-ECM signalling occur in the development of the disease. The different pattern of expression of beta1 and beta5 chains on adult and fetal chondrocytes leads to speculate that integrins play a key role in control of cartilage morphogenesis and differentiation.