Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2ß1 Binding in TGFß1-Evoked Renal Tubular Epithelial Cells.

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2ß1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFß1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2ß1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFß1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2ß1 signaling, ahead of establishing Peptide 5 as a potential intervention.

Lysyl oxidase is associated with increased thrombosis and platelet reactivity.

Lysyl oxidase (LOX) is overexpressed in various pathologies associated with thrombosis, such as arterial stenosis and myeloproliferative neoplasms (MPNs). LOX is elevated in the megakaryocytic lineage of mouse models of MPNs and in patients with MPNs. To gain insight into the role of LOX in thrombosis and platelet function without compounding the influences of other pathologies, transgenic mice expressing LOX in wild-type megakaryocytes and platelets (Pf4-Lox(tg/tg)) were generated. Pf4-Lox(tg/tg) mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox(tg/tg) mice, indicating a higher propensity for thrombus formation in vivo. Exploring underlying mechanisms, we found that Pf4-Lox(tg/tg) platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox(tg/tg) platelets to the collagen sequence GFOGER implies that the collagen receptor integrin α2ß1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis.

Cooperation between IL-7 Receptor and Integrin α2ß1 (CD49b) Drives Th17-Mediated Bone Loss.

Th17 cells are critical effectors in inflammation and tissue damage such as bone erosion, but the mechanisms regulating their activation in this process are not fully understood. In this study, we considered the cooperation between cytokine receptors and integrin pathways in Th17-osteoclast function. We found that human Th17 cells coexpress IL-7R and the collagen-binding integrin α2ß1 (CD49b), and IL-7 increases their adhesion to collagen via α2ß1 integrin. In addition, coengagement of the two receptors in human Th17 cells cooperatively enhanced their IL-17 production and their osteoclastogenic function. The functional cooperation between IL-7R and α2ß1 integrin involves activation of the JAK/PI3K/AKT (protein kinase B) and MAPK/ERK pathways. We also showed that IL-7-induced bone loss in vivo is associated with Th17 cell expansion. Moreover, blockade of α2ß1 integrin with a neutralizing mAb inhibited IL-7-induced bone loss and osteoclast numbers by reducing Th17 cell numbers in the bone marrow and reducing the production of IL-17 and the receptor activator of NF-κB ligand. Thus, the cooperation between IL-7R and α2ß1 integrin can represent an important pathogenic pathway in Th17-osteoclast function associated with inflammatory diseases.

Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin α2ß1 and GPVI: The role of Pyk2 and c-Cbl.

Phosphatidylinositol 3-kinaseß (PI3Kß) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kß activation downstream of integrin αIIbß3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kß upon recruitment of the two main platelet collagen receptors, integrin α2ß1 and GPVI. PI3Kß-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2ß1, but occurred normally upon GPVI ligation. Integrin α2ß1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kß activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KßKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kß activity also hampered GPVI-induced tyrosine-phosphorylation and activation of PLCγ2, preventing intracellular Ca2+ increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2+ increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kß inhibitor TGX-221. These results outline important differences in the regulation of PI3Kß by GPVI and integrin α2ß1 and suggest that inhibition of Pyk2 may target PI3Kß activation in a selective context of platelet stimulation.

Implication of α2ß1 integrin in anoikis of MCF-7 human breast carcinoma cells.

Silencing of α2ß1 integrin expression significantly promoted anchorage-dependent apoptosis (anoikis) and drastically reduced clonal activity of MCF-7 human breast carcinoma cells. Depletion of α2ß1 enhanced the production of apoptotic protein p53 and of inhibitor of cyclin-dependent protein kinases, p27, while downregulating antiapoptotic protein Bcl-2 and multifunctional protein cMyc. Blocking the expression of α2ß1 had no effect on activity of protein kinase Akt, but it sharply increased the kinase activity of Erk1/2. Pharmacological inhibition of Erk1/2 had a minor effect on anoikis of control cells, while it reduced anoikis of cells with downregulated α2ß1 to the level of control cells. The data show for the first time that integrin α2ß1 is implicated in the protection of tumor cells from anoikis through a mechanism based on the inhibition of protein kinase Erk.

α2ß1 Integrin.

The α2ß1 integrin, also known as VLA-2, GPIa-IIa, CD49b, was first identified as an extracellular matrix receptor for collagens and/or laminins [55, 56]. It is now recognized that the α2ß1 integrin serves as a receptor for many matrix and nonmatrix molecules [35, 79, 128]. Extensive analyses have clearly elucidated the α2 I domain structural motifs required for ligand binding, and also defined distinct conformations that lead to inactive, partially active or highly active ligand binding [3, 37, 66, 123, 136, 137, 140]. The mechanisms by which the α2ß1 integrin plays a critical role in platelet function and homeostasis have been carefully defined via in vitro and in vivo experiments [76, 104, 117, 125]. Genetic and epidemiologic studies have confirmed human physiology and disease states mediated by this receptor in immunity, cancer, and development [6, 20, 21, 32, 43, 90]. The role of the α2ß1 integrin in these multiple complex biologic processes will be discussed in the chapter.

α2ß1 integrin regulates Th17 cell activity and its neutralization decreases the severity of collagen-induced arthritis.

Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2ß1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2ß1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2ß1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2ß1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2ß1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor-related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2ß1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.

Up-regulation of tension-related proteins in keloids: knockdown of Hsp27, α2ß1-integrin, and PAI-2 shows convincing reduction of extracellular matrix production.

BACKGROUND: Keloid disease is a fibroproliferative disorder, with an ill-defined treatment that is characterized by excessive extracellular matrix deposition. Mechanical tension promotes deposition of extracellular matrix and overexpression of tension-related proteins, which is associated with keloid disease. The aim of this study was to investigate the effect of tension-related proteins on extracellular matrix steady-state synthesis in primary keloid fibroblasts. METHODS: Keloid fibroblasts (n = 10) and normal skin (n = 4) fibroblast cultures were established from passages 0 to 3. A panel of 21 tension-related genes from microarray data were assessed at mRNA (quantitative reverse-transcriptase polymerase chain reaction) and protein (in-cell Western blotting) levels. Three genes were significantly altered in keloid tissue and fibroblasts, and their functional role was assessed using siRNA knockdown. RESULTS: Hsp27, α2ß1-integrin, and PAI-2 were significantly up-regulated (p < 0.05)in keloid tissue and fibroblasts compared with normal skin. Hsp27, α2ß1-integrin, and PAI-2 expression was inhibited by RNA interference. Both the mRNA and protein levels of Hsp27, α2ß1-integrin, and PAI-2 significantly decreased (p < 0.05) in keloid fibroblasts at 48 hours after transfection. After down-regulation of Hsp27, α2ß1-integrin, and PAI-2, the expression of intracellular extracellular matrix was significantly reduced (p < 0.05). Water-soluble tetrazolium salt-1 assay showed that transfection of Hsp27, α2ß1-integrin, and PAI-2 did not influence the viability/metabolic activity of keloid fibroblasts. CONCLUSIONS: This study demonstrates overexpression of key tension-related proteins in keloid tissue and keloid fibroblasts. Knockdown of Hsp27, PAI-2, and α2ß1-integrin by RNA interference attenuates the expression of mRNA and protein levels and certain other extracellular matrix molecules.

Integrin alpha2beta 1 (α2ß1) promotes prostate cancer skeletal metastasis.

Men who die of prostate cancer (PCa) do so because of systemic metastases, the most frequent of which are within the skeleton. Recent data suggest that the colonization of the skeleton is mediated in part by collagen type I, the most abundant protein within the bone. We have shown that enhanced collagen I binding through increased expression of integrin α2ß1 stimulated in vitro invasion and promoted the growth of PCa cells within the bone. Accordingly, we sought to determine whether α2ß1 integrin is a potential mediator of skeletal metastasis. To examine whether α2ß1 integrin mediates PCa metastasis, α2 integrin was over-expressed in low-tumorigenic LNCaP PCa cells or selectively knocked-down in highly metastatic LNCaPcol PCa cells. We document that the over-expression of α2 cDNA stimulated whereas α2 shRNA inhibited the ability of transduced cells to bind to or migrate towards collagen in vitro. Correspondingly, α2 integrin knock-down reduced the tumor burden of intra-osseous tumors compared to control-transduced cells. To investigate the clinical significance of α2ß1 expression in PCa, α2ß1 protein was measured in prostatic tissues and in soft tissue and bone metastases. The data demonstrate that α2ß1 protein was elevated in PCa skeletal metastases compared to either PCa primary lesions or soft tissue metastases suggesting that α2ß1 contributes to the selective metastasis to the bone. Taken together, these data support that α2ß1 integrin is needed for the efficient metastasis of PCa cells to the skeleton.

Low α(2)ß(1) integrin function enhances the proliferation of fibroblasts from patients with idiopathic pulmonary fibrosis by activation of the ß-catenin pathway.

Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of α(2)ß(1) integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3ß and high levels of active ß-catenin in the nucleus. Knockdown of ß-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of α(2)ß(1) integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3ß and active ß-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low α(2)ß(1) integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the ß-catenin pathway.

The α2ß1 integrin is a metastasis suppressor in mouse models and human cancer.

Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that ß1 integrins may stimulate metastasis of a number of cancers, expression of the ß1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α2ß1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α2ß1 integrin in normal breast epithelium and loss of α2ß1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α2 integrin, but not genes encoding α1, α3, or ß1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α2ß1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival.

Distinct spatio-temporal Ca2+ signaling elicited by integrin alpha2beta1 and glycoprotein VI under flow.

We studied how integrin alpha2beta1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca(2+) concentration ([Ca(2+)](i)) of FLUO 3-AM-labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca(2+)](i) oscillations. Rapid alpha-like peaks were unaffected by the membrane-impermeable Ca(2+) chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting gamma-like peaks were always preceded by at least one alpha-like peak and abolished by intracellular or extracellular Ca(2+) chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca(2+) responses. Human or mouse platelets lacking GPVI function exhibited alpha-like but not gamma-like Ca(2+) peaks, whereas those lacking alpha2beta1 showed markedly reduced to absent alpha-like and no gamma-like Ca(2+) peaks. Specific alpha2beta1 ligation induced alpha-like but not gamma-like peaks. Thus, alpha2beta1 may generate Ca(2+) signals that are reinforced by GPVI and required for subsequent longer-lasting Ca(2+) oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of alpha2beta1 in response to collagen stimulation.

Effect of VP12 and viperistatin on inhibition of collagen-receptor-dependent melanoma metastasis.

Viperistatin and VP12 isolated from Vipera paleastinae venom showed a potent inhibitory activity against collagen receptors, alpha1beta1 and alpha2beta1 integrins, respectively. Structurally, viperistatin belongs to the disintegrin family of proteins, whereas VP12 is composed of two subunits VP12A and VP12B displaying amino acid sequence homology with heterodimeric C-lectin type proteins. Viperistatin and VP12 used separately and simultaneously inhibited pro-metastatic activities of melanoma cells lines. The level of inhibition of MV3 and HS.939T human cell lines in cell adhesion and migration assays by both compounds was correlated with expression of alpha1beta1 and alpha2beta1 integrins on the cell surface. MV3 cells express collagen receptors to much higher extent than HS.939T and required the application of higher concentrations of inhibitors to block their adhesion to collagen types I and IV. A melanoma cell transmigration assay through a dHMVEC layer revealed that alpha1beta1 integrin plays a significant role in invasion of HS.939T cells, while alpha2beta1 integrin appears to be more important for MV3 cells. In an animal model of hematogenous metastasis of the mouse B16F10 cell line, the inhibitory effect of viperistatin and VP12 was only partial. These data suggest that collagen receptors may be an interesting target for development of new anti-metastatic therapies.

alphavbeta5/beta6 integrin suppression leads to a stimulation of alpha2beta1 dependent cell migration resistant to PI3K/Akt inhibition.

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alphavbeta5/beta6 and alpha2beta1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of alphavbeta5/beta6 function on alpha2beta1, we decreased the expression of alphav integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha2beta1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alphavbeta5/beta6-dependent limitation of alpha2beta1 function could be overridden by TS2/16, an activating anti-beta1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha2beta1-mediated cell migration observed in the absence of alphav integrins or following activation of alpha2beta1 integrins by the TS2/16. These results suggest that integrins alphavbeta5/beta6 repress alpha2beta1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha2beta1-mediated migration.

The integrin alpha2beta1 agonist, aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production.

BACKGROUND AND PURPOSE: During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin alpha2beta1 agonist. EXPERIMENTAL APPROACH: Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques. KEY RESULTS: Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin alpha2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-beta. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by alpha2beta1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-beta mAb, or a phosphoinositide-3 kinase inhibitor, LY294002. Aggretin stimulated the phosphorylation of PDGFR-beta, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, U73122 and PD98059. CONCLUSIONS AND IMPLICATIONS: Aggretin induces VSMC proliferation and migration mainly through binding to integrin alpha2beta1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.

Autocrine semaphorin3A stimulates alpha2 beta1 integrin expression/function in breast tumor cells.

The axon repulsion factor semaphorin3A (SEMA3A) and its receptor neuropilin-1 (NP-1) are expressed in breast tumor cells, and function as suppressors of tumor cell migration. Based on the knowledge that both SEMA3A and the alpha2beta1 integrin suppress breast tumor cell migration, we studied the impact of SEMA3A signaling on alpha2beta1 integrin expression/function. The incubation of breast tumor cells with SEMA3A increased alpha2 and beta1 integrin levels, and stimulated tumor cell adhesion to the alpha2beta1-binding matrix protein collagen I. Conversely, reducing SEMA3A expression in breast tumor cells decreased alpha2beta1 levels and collagen adhesion. The ability of SEMA3A to increase tumor cell adhesion to collagen was dependent on both the SEMA3A receptor NP-1 and the glycogen synthase kinase-3. The incubation of breast tumor cells with SEMA3A disrupted the actin cytoskeleton, and reduced both tumor cell migratory and invasive behavior. Importantly, using an alpha2beta1-neutralizing antibody, we demonstrated that SEMA3A suppression of tumor cell migration is dependent on alpha2beta1. Our studies indicate that expression of the alpha2beta1 integrin, a suppressor of metastatic breast tumor growth, is stimulated in breast tumor cells by an autocrine SEMA3A pathway.

Acinar epithelial cell laminin-receptors in labial salivary glands in Sjögren's syndrome.

OBJECTIVE: To analyze the epithelial cell-basement membrane attachment, in particular in the secretory end pieces (responsible for secretion of saliva) and in Sjögren's syndrome (SS) characterized by acinar cell failure. METHOD: Immunohistochemistry with laminin receptor chain-specific monoclonal antibodies to integrin (Int) subunits, Lutheran blood group antigen and alpha-dystroglycan. RESULTS: Only acinar cells contained Int alpha1 and alpha2 subunits. This staining was interrupted but strong in controls, but very weak in SS. Both acinar and ductal cells contained Int alpha3, alpha6, b1 and b4 and Lutheran blood group antigen and ductal cells also contained alpha-dystroglycan. These staining patterns were similar in SS and controls. CONCLUSIONS: Binding of the acinar and ductal cells to the basement membrane laminins seems to be mediated by Int alpha3b1, alpha6b1 and alpha6b4 integrin-receptors and Lutheran blood group antigen and alpha-dystroglycan non-integrin receptors. This structure-supporting system is intact in SS, compatible with the maintenance of the tubuloalveolar architecture of the SS glands. The irregular staining pattern of the acinus-specific Int alpha1b1 and alpha2b1 was compatible with a regulated signaling role, which was apparently impaired in SS. Indeed, their laminin counterparts (Lm -1/111 and -2/211) are also aberrant in SS revealing this as the central cell-matrix defect in the syndrome.

[Effect of integrin alpha2beta1 on invasion and migration of neuroblastoma cells].

OBJECTIVE: To study the effect of integrin alpha2beta1 on invasion and migration of SK-N-SH neuroblastoma cells. METHODS: Neuroblastoma SK-N-SH cell line was cultured in the modified eagle's medium. The effects of monoclonal antibodies to integrin alpha2 and integrin beta1 on migration and invasion were measured by inclined test and polycarbonate filters incorporated in modified Transwell chambers respectively. The migration and invasion cells were stained with Gimsa staining and counted under a 200 multiplied microscope. The blocking rate of migration and invasion of cells was calculated. RESULTS: The number of migrated SK-N-SH cells in the anti-alpha2 and anti-beta1 treatment groups (50.9+/-10.5 and 54.3+/-9.0 respectively) was significantly less than that in the control group without monoclonal antibody treatment (98.1+/-7.4) (P<0.01), with a blocking rate of cell migration of 48.1% and 44.5% respectively. The invasion to matrigel of SK-N-SH cells exposed monoclonal antibodies to integrin alpha2 and integrin beta1 was significantly blocked compared with the control SK-N-SH cells, with the number of invasion cells in the anti-alpha2 and anti-beta1 treatment groups of 25.3 +/- 4.4 and 18.8 +/- 3.9 respectively vs 41.5 +/- 4.8 in the control group (P<0.01). The blocking rate of cell invasion in the anti-alpha2 and anti-beta1 treatment groups was 39.0% and 54.7% respectively. CONCLUSIONS: Integrin alpha2beta1 may promote migration and invasion of neuroblastoma cells.

Real-time monitoring approach: assessment of effects of antibodies on the adhesion of NCI-H460 cancer cells to the extracellular matrix.

This paper presents an in situ impedance chip system, which allows studying the effects of drugs on the behaviors of living cell in real-time. A new label-free measurement approach is introduced, which enables to assess the extent of the adhesion of a cell population to the extracellular matrix (ECM)-coated microelectrode array, by adding together all the impedance changes from individually controlled microelectrodes. A high sensitivity was demonstrated and allowed us to monitor cell attachment with the resolution of a "few cells". The dose and pre-incubation time effects of antibodies against beta1-integrin and its subunit alpha2beta1-integrin on the adhesion behavior of NCI-H460 lung cancer cells to collagen type I was extensively studied. Results show that both anti-beta1-integrin and anti-alpha2beta1-integrin inhibit NCI-H460 cells attachment to collagen I. This indicates that beta1-integrin is present on the surface of NCI-H460 cancer cells, and that its subunit alpha2beta1 significantly affects NCI-H460 cells attachment on collagen I.

Ameloblastoma and adenomatoid odontogenic tumor: the role of alpha2beta1, alpha3beta1, and alpha5beta1 integrins in local invasiveness and architectural characteristics.

Ameloblastoma is an odontogenic neoplasm characterized by local invasiveness and a tendency toward recurrence, whereas adenomatoid odontogenic tumor (AOT) is an indolent neoplasm. The objective of the present study was to immunohistochemically analyze the role of alpha2beta1, alpha3beta1, and alpha5beta1 integrins in the cellular events and cell-matrix interactions that occur in these tumors and their consequent repercussions on the architectural arrangement and biologic behavior of these lesions. Paraffin-embedded specimens from 30 ameloblastomas (20 solid and 10 unicystic tumors) and 12 AOTs were submitted to immunohistochemistry using the catalyzed signal amplification system. A difference in the pattern of integrin expression was observed between the various histologic types of ameloblastoma. No significant difference in labeling intensity was observed between unicystic and solid ameloblastomas, but comparison between ameloblastomas and AOT showed a significantly stronger expression of alpha5beta1 integrin in the former (P < .05). Our findings suggest an important role of the integrins studied in the architectural characteristics of ameloblastomas and AOTs and a possible participation of alpha5beta1 integrin in the mechanism of local invasion of ameloblastomas.