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Results for “Integrin alpha2”

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PubMed1998

The thrombospondin receptor CD47 (IAP) modulates and associates with alpha2 beta1 integrin in vascular smooth muscle cells.

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a beta1 integrin, rather than alphav beta3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to alpha2 and beta1 integrin subunits and to IAP. A combination of antialpha2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antialphav beta3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between alpha2 beta1 and IAP was detected by the coimmunoprecipitation of both alpha2 and beta1 integrin subunits with IAP. These data suggest that IAP can associate with alpha2 beta1 integrin and modulate its function.

Animals↗

PubMed2003

Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection.

Circulating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detect and quantify platelet mRNA that excludes contamination with leukocyte RNA is mandatory. Applying laser-assisted microdissection and manipulation (LMM) we could isolate platelets from hemalaun-stained cytospins under permanent visual control and after laser-photolysis of nucleated blood cells. For mRNA quantification, the platelet-specific mRNAs were subsequently measured by real time RT-PCR. High-copy beta3 integrin and low-copy a alpha2 integrin as well as tissue factor (TF) transcripts were analyzed in LMM-harvested platelets. In 91.2% (83/91) beta3 integrin was detectable with a mean threshold cycle (CT) value of 32.5+/-3.2 (< or =50,000 cells). The low-copy a 2 integrin mRNA was positive in 84.4% (38/45) with CT mean value of 36.9+/-1.3, indicating that the relative expression of alpha2 integrin mRNA in platelets was about 130 times lower than beta3. The TF transcript was undetectable in all samples. Comparing platelet mRNA from LMM isolation to that from limiting dilution series resulted in a high accordance for beta3 integrin transcript in both, recovery (91.2% vs. 95.2%) and CT value (32.5 vs. 32.8). These results demonstrate that the combination of LMM and real-time RT-PCR is a valuable tool for precise, platelet-specific mRNA analysis without contamination of other cells.

Blood Platelets↗

PubMed2003

Genetic polymorphisms of platelet adhesive molecules: association with breast cancer risk and clinical presentation.

The main platelet adhesive receptors integrin alpha2beta1, integrin alphaIIbbeta3 and glycoprotein (GP) Ibalpha are also expressed in breast carcinoma cells. They play a key role in tumor cell-induced platelet aggregation and in adhesive interactions necessary for tumoral invasion and metastasis. Several polymorphisms affecting these molecules, two in integrin alpha2 (C807T and G1648A). one in integrin beta3 (T1565C) and one in GP Ibalpha (VNTR), influencing their levels, structure, and possibly their function, have been previously described and associated with cardiovascular diseases. In this study, we investigated the association of these polymorphisms with breast cancer risk or clinical presentation. We studied 101 patients with invasive breast cancer. The main prognostic variables were recorded, and genomic PCR analysis of these polymorphisms was performed. A group of 101 control subjects matched on age and sex was studied and compared with patients. No association was found between VNTR (GP Ibalpha) polymorphism and breast cancer risk or presentation. Genotype and allele frequencies of C807T and G1648A polymorphisms of integrin alpha2 were not statistically different in breast cancer patients and controls, although we found an association between the 1648G/G genotype and higher disease stages (III and IV) (p = 0.02). Breast cancer risk was higher in carriers of beta3 integrin T/T genotype (OR = 2.08, 95% CI = 1.04-4.16, p = 0.04). Furthermore, genotype 1565T/T was also associated with axillary nodal metastasis (p = 0.017) and with tumoral diameter greater than 2cm (p = 0.02). Although confirmatory studies are needed, our results suggest that polymorphic genetic variation of integrins expressed in platelets and epithelial breast cells could modify the risk and the biological aggressiveness of breast carcinomas.

Adult↗

PubMed2004

VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.

Vascular endothelial growth factor-A (VEGF-A) is strongly up-regulated in wounded cutaneous tissue and promotes repair-associated angiogenesis. However, little is known about its role in lymphatic regeneration of the healing skin. We studied wound healing in transgenic mice that overexpress VEGF-A specifically in the epidermis and in wild-type mice in the absence or presence of inhibitors of VEGF-A signaling. Surprisingly, transgenic overexpression of VEGF-A in the skin promoted lymphangiogenesis at the wound healing site, whereas systemic blockade of VEGFR-2 prevented lymphatic vessel formation. Studies in cultured lymphatic endothelial cells revealed that VEGF-A induced expression of the alpha1 and alpha2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. VEGF-A-induced lymphatic endothelial cord formation and haptotactic migration were suppressed by anti-alpha1 and anti-alpha2 integrin blocking antibodies, and systemic blockade of the alpha1 and alpha2 integrins inhibited VEGF-A-driven lymphangiogenesis in vivo. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis.

Animals↗

PubMed2004

Crystal structure of EMS16 in complex with the integrin alpha2-I domain.

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin alpha2beta1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin alpha2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Angstrom resolution reveals that the collagen-binding site of the alpha2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the alpha2-I domain. The bound alpha2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the alpha2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the alpha2-I domain, suggesting that EMS16 may have the potential to bind specifically to the alpha2-I domain in a metal ion-independent fashion.

Amino Acid Sequence↗

PubMed2005

[Effects of dermal template on the biological behaviors of fibroblasts during wound healing].

OBJECTIVE: To study the effects of dermal template on the biological behaviors of fibroblasts during wound healing. METHODS: A total of 120 rats were made full-thickness wound modes on the dorsum and divided into 4 groups, in group 1, the wounds were allowed to heal by contraction(ConT); in group 2, the wounds covered with full-thickness skin grafts (FTSG); in group 3, the wounds were with split thickness skin grafts (STSG); and in group 4, the wounds were covered by dermal regeneration template with overlying thin split-thickness autograft (ADMT). The specimens were obtained at one week, two weeks, four weeks, six weeks, and twelve weeks respectively. The expressions of alpha smooth muscle actin(alpha-SMA, characteristic of MFB), fibronectin (FN), integrin alpha2 beta1 and transforming growth factor beta1 (TGF-beta1) were examined by immunohistochemical analysis. RESULTS: Positive expression of alpha-SMA, FN, integrin alpha2 beta1 and TGF-beta1 in ADMT groups was significantly lower than that in STSG group and ConT group, but higher than that in FTSG group (P < 0.05). CONCLUSION: Dermal regeneration template can inhibit the transformation of FB to MFB and restrain the expression of FN, integrin alpha2 beta1, and TGF-beta1 in fibroblasts which might reduce the possibility of hypertrophic scarring, and improve wound healing.

Actins↗

PubMed2003

Invasion of interstitial matrix by a novel cell line from primary peritoneal carcinosarcoma, and by established ovarian carcinoma cell lines: role of cell-matrix adhesion molecules, proteinases, and E-cadherin expression.

OBJECTIVE: Primary peritoneal carcinosarcomas are similar to ovarian carcinomas in that they can metastasize by intraperitoneal dissemination; therefore, invasion of the submesothelial interstitial (stromal) matrix is an integral part of the pathology. Our objective was to study cell-matrix interactions that may influence invasive behavior of a novel, primary peritoneal carcinosarcoma cell line (PC880), and to assess how these cell-matrix interactions are different from frequently studied cultured ovarian carcinoma cells NIH:OVCAR-3, SKOV-3, and ES-2. We also wanted to determine how the expression of the cell-cell adhesion molecule E-cadherin is related to invasive behavior. METHODS: The PC880 cell line was established from ascites fluid of a patient diagnosed with primary peritoneal carcinosarcoma. Adhesion assays were done in titer plates coated with individual matrix components. Cell migration in monolayer cultures was assessed by the scratch wound assay method. Invasion assays were done using a three-dimensional type I collagen gel. Cytokeratin, vimentin, and E-cadherin were detected by Western blotting. E-cadherin mRNA was detected by RT-PCR. RESULTS: PC880 cells adhered well to fibronectin, laminin, and vitronectin in an integrin-dependent manner. The cells also adhered to type I collagen and invaded a three-dimensional type I collagen matrix. The invasiveness of the PC880 cells was moderated by pretreatment of the collagen matrix with heparin or chondroitin sulfate (82 and 63% of control invasiveness, respectively), indicating a role of cell surface proteoglycans in promoting invasive phenotype. Treatment of PC880 cells with sodium chlorate also decreased invasiveness (80% of control), further confirming the role of cell surface proteoglycans. Treatment of PC880 cells with function-blocking antibody to alpha2 integrin decreased invasiveness (57% of control), indicating the role of integrins in promoting the invasive phenotype. The protease inhibitors GM6001, E-64, and AEBSF decreased invasiveness (35, 57, and 37% of control, respectively) of PC880 cells. The ES-2 cells also adhered to type I collagen, and invaded the three-dimensional type I collagen matrix; however, inhibitors such as heparin, chondroitin sulfate, function-blocking antibody to alpha2 integrin, E-64, and AEBSF were less effective in moderating the invasiveness. Inhibition of invasiveness with sodium chlorate was the same as in PC880 cell, while GM6001 did not inhibit invasiveness at all. The NIH:OVCAR-3 and SK-OV-3 cells were previously found to adhere to type I collagen, but these cells did not invade the three-dimensional type I collagen matrix. In a monolayer culture PC880 and ES-2 cells had significantly higher motility than NIH:OVCAR-3 and SK-OV-3 cells. Only these noninvasive cell lines expressed E-cadherin protein or mRNA. CONCLUSIONS: PC880 is the first cell line established from primary peritoneal carcinosarcoma, and the cytoskeletal composition indicated that these cells represent the sarcomatous elements of the tumor. PC880 cells, similar to ES-2 cells, adhered to type I collagen, and invaded a three-dimensional collagen matrix. The invasion of the interstitial matrix by both the peritoneal carcinosarcoma and the ovarian carcinoma cell line was mediated by cell surface proteoglycans, alpha2 integrin, and proteases. The invasive cell behavior of PC880 and ES-2 cells correlated with a high degree of motility, and with the lack of expression of the cell-cell adhesion molecule E-cadherin.

Animals↗

PubMed1997

Anti-alpha3 integrin antibody induces the activated form of matrix metalloprotease-2 (MMP-2) with concomitant stimulation of invasion through matrigel by human rhabdomyosarcoma cells.

Proteolytic enzymes, such as matrix metalloproteases (MMPs), play a pivotal role in cancer invasion and metastasis. Invasive human rhabdomyosarcoma cells (RD) secreted proMMP-2 (72-kDa progelatinase). We found that anti-alpha3 and -alpha2 integrin antibodies induced the activated form of MMP-2 and enhanced proMMP-2 secretion by RD cells. The effect of anti-alpha2 integrin antibody was less prominent than that seen with anti-alpha3 integrin antibody. Moreover, we have found that anti-alpha3 and -alpha2 integrin antibodies enhanced RD-cell invasion through matrigel (reconstituted basement membrane) by 2.6- and 2.0-fold respectively this process was abrogated by neutralizing antibody to MMP-2. These data suggest that signaling events induced by anti-alpha3 integrin antibody may be involved in RD-cell invasion as a result of modulation of matrix-metalloprotease expression.

Antibodies, Anti-Idiotypic↗

PubMed2001

Contraction-dependent apoptosis of normal dermal fibroblasts.

The mechanisms underlying the contraction-dependent apoptosis of primary fibroblasts are of prime importance in understanding anchorage-dependent survival/apoptosis of dermal fibroblasts. As integrins are essential extracellular matrix receptors in fibroblasts, their role in anchorage-dependent apoptosis/survival of fibroblasts was analyzed. Primary human fibroblasts displayed a marked reduction of apoptosis in mechanically relaxed collagen matrices in the presence of adhesion-blocking antibodies against alpha1beta1 or alpha2beta1. Anti-alphavbeta3 antibodies had a considerably weaker effect. In additional experiments RD cells, which lack alpha2 integrin, displayed no apoptosis in mechanically relaxed collagen matrices. Their susceptibility to apoptosis was restored after transfection with functional alpha2 integrin, and it could be blocked again by adhesion-blocking antibodies against alpha2beta1 integrin. Therefore we conclude that apoptosis of human primary fibroblasts in contractile collagen matrices is - at least in part - inhibited by adhesion-blocking anti-integrin antibodies, suggesting that the mode of apoptosis in this case is different from anoikis. Further, apoptosis in a mechanically relaxed collagen matrix could be abrogated by depolymerization of F-actin using cytochalasin D and also by disturbing actin-myosin interaction using 2,3-butanedione monoxime, indicating a possible dependence of apoptosis on mechanical forces and/or cell shape.

Antibodies↗

PubMed1999

Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2.

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the alpha1beta1 and alpha2beta1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen. These cells require exogenous BMP-2, as well as ascorbic acid and beta-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat alpha1 and/or alpha2 integrin subunit, or anti-type I collagen (Col-I), antibodies were added to human recombinant (hr)BMP-2-treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-collagen-I or both anti-integrin-alpha1 and -alpha2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5-1.8% control) of these cells. In all cases, adding anti-alpha1 or anti-alpha2 antibodies separately produced partial effects, while their combined effect approached that of anti-collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by alpha1 and alpha2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.

Alkaline Phosphatase↗

PubMed2001

Impaired platelet function among platelet donors.

BACKGROUND: Platelet transfusions are effective for the prevention and treatment of bleeding in patients with disorders of platelet number and/or function. In recent years plateletpheresis concentrates have outnumbered pooled platelet concentrates, albeit with significant differences between nations. Thus, the platelet quality of individual donors has become increasingly important. The aim of this study was to gain an estimate for the prevalence of impaired platelet function among platelet donors. STUDY DESIGN AND METHODS: We determined the inter-donor variability in platelet plug formation with a PFA-100 analyzer, the prevalence of impaired thromboxane formation, and effects of the density in alpha2 integrin polymorphism and density. RESULTS: (i) Collagen-epinephrine induced closure time (CEPI-CT) showed a great inter-subject variability in platelet donors and was higher than in healthy controls (p = 0.008). One-fifth of donors had abnormal CEPI-CT values and 11% exceeded >300 s (max measurable value). (ii) Decreased serum thromboxane B2 levels were found in 9% of all donors, compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. CEPI-CT correlated inversely with TxB2-levels in donors and controls. (iii) The density of the alpha2-integrin correlated negatively with CEPI-CT and CADP-CT values in controls, but was not responsible for the observed impaired platelet function in donors. (iv) Finally, the ABO blood group system modulates closure times. CONCLUSION: In sum, a large number of platelet donors present with prolonged closure times. Decreased thromboxane formation and frequent platelet donation partly account for this observation.

Adenosine Diphosphate↗

PubMed1997

Differential effects of EGF and amphiregulin on adhesion molecule expression and migration of colon carcinoma cells.

Epidermal growth factor (EGF) is a potent morphogen affecting cell shape and motility through regulation of adhesive interactions. We have characterized the morphological effects of EGF on GP2d and GP5d colon carcinoma cell lines and have compared the ability of the heparin-binding EGF receptor ligand amphiregulin (AR) to elicit the same effects. EGF induced a marked epithelial-mesenchymal transition in both cell lines. This effect was evident at 7 pM EGF and was associated with a reduction in cellular adherens junctions and diminished cell-cell contact; it was also associated with an increase in expression of alpha2-integrin as well as enhanced adhesion to the substratum and cell spreading. These changes in adhesion molecule expression were accompanied by enhanced migration on collagen. Blockade of cell growth with mitomycin C did not prevent the EGF-induced morphological change, showing that the mitogenic and morphogenic responses of the GP cells were separable. The phosphatidyl inositol (PI) 3-kinase inhibitor wortmannin inhibited basal proliferation but had no effect on the EGF-induced morphological change, further suggesting that the PI 3-kinase pathway was not involved in the morphogenic response of these cells. Amphiregulin stimulated proliferation of both cell lines, but could only elicit a modest morphological change if used at considerably higher doses or if growth was blocked with mitomycin C. In cells treated with 55 nM AR, alpha2-integrin expression was slightly increased; however, unlike the EGF case, adherens junctions remained intact. These differences in the ability of EGF and amphiregulin to affect cellular adhesion and migration may be significant factors influencing normal and tumor cell behavior.

Adenocarcinoma↗

PubMed2006

Laminin alpha1 chain LG4 module promotes cell attachment through syndecans and cell spreading through integrin alpha2beta1.

The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.

Alanine↗

PubMed1998

Rapid adhesion and spread of non-adherent colon cancer Colo201 cells induced by the protein kinase inhibitors, K252a and KT5720 and suppression of the adhesion by the immunosuppressants FK506 and cyclosporin A.

We examined alterations in cell morphology and expression of adhesion molecules in response to a general protein kinase inhibitor K252a treatment of non-adherent colon adenocarcinoma Colo201 cells. K252a induced rapid cell adhesion and spreading with concomitant formation of actin stress fibers. A protein kinase A inhibitor KT5720 also induced cell adhesion, but the rate of spread was slower than that seen with K252a. These adhesions were mediated by integrin molecules since cell adhesion required Mg2+, Mn2+ or Ca2+, and was inhibited by monoclonal antibodies for integrins alpha2 and beta1. Indirect immunofluorescence microscopic observations revealed that integrin alpha2 and beta1 molecules in K252a-treated cells were concentrated at sites of focal adhesion, but expressions of integrin molecules were not modulated. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin increased during K252a- or KT5720-induced cell adhesion. Immunosuppressants FK506 and cyclosporin A suppressed the K252a-induced cell adhesion and abolished tyrosine phosphorylation of cellular proteins including FAK and paxillin. Furthermore, W7 and calmidazolium, inhibitors of calmodulin, also inhibited the cell adhesion. Based on findings that FK506 and cyclosporin A are inhibitors of the calcium calmodulin-dependent protein phosphatase, calcineurin, this phosphatase may regulate integrin-dependent cell adhesion and spread of Colo201 cells. This Colo201 cell model provides a pertinent system for studying molecules involved in signal transduction pathways and can shed light on mechanisms of metastasis and invasion of colon carcinoma cells.

Adenocarcinoma↗

PubMed2004

Adhesion of gastric carcinoma cells to peritoneum mediated by alpha3beta1 integrin (VLA-3).

The interaction between gastric carcinoma cells and the peritoneal lining is a key step in peritoneal dissemination. In this study, we examined the roles of the beta1 family of integrin receptors in the adhesion of such cells to the peritoneum. The adhesion of several gastric carcinoma cell lines to peritonea excised from mice was inhibited most by an anti-alpha3 integrin antibody and to a lesser extent by an anti-alpha2 integrin antibody. In the peritoneal implantation of NUGC-4 human gastric carcinoma cells in athymic mice, treatment of the cells with anti-alpha2 or anti-alpha3 integrin antibody reduced the number of disseminated nodules; suppression by the anti-alpha3 integrin antibody was stronger than that by the anti-alpha2 integrin antibody. The cDNAs to human alpha2 and alpha3 integrins were introduced into K562 leukemic cells, which were positive for the integrin beta1 subunit but negative for the alpha2 or alpha3 subunit. The alpha3 integrin-transfected cells adhered to excised peritoneum and to a monolayer of peritoneal mesothelial cells more firmly than did the alpha2 integrin-transfected cells or the mock transfectant. Reverse transcription-PCR was used to analyze the expression of laminin-5 and laminin-10/11, which have been reported to serve as high-affinity ligands for alpha3beta1 integrin. mRNA for these laminin isoforms was found in mesothelial cells from the diaphragm and parietal peritoneum. These results strongly suggest that alpha3beta1 integrin plays an essential role in mediating the initial attachment of cancer cells to the peritoneum, leading to the formation of peritoneal metastasis.

Antibodies↗

PubMed1999

A peptide inhibiting the collagen binding function of integrin alpha2I domain.

Integrin alpha2 subunit forms in the complex with the beta1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin alpha2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet alpha2beta1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant ralpha2I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrin-like domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit ralpha2I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant alpha2I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids, arginine-lysine-lysine (RKK), is essential for ralpha2I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to alpha2I domain in the absence of Mg2+, suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed alpha2beta1 integrin, and the attachment was inhibited by anti-integrin antibodies.

Amino Acid Sequence↗

PubMed2001

Significance of integrin alpha2/beta1 in peritoneal dissemination of a human gastric cancer xenograft model.

In the present study, the role of integrin alpha2/beta1 in peritoneal dissemination of gastric cancer was investigated using an in vivo xenograft model for the highly metastatic MKN-45-P gastric cancer cells. Metastatic ability of MKN-45-P cells was significantly associated with the simultaneous expression of integrin alpha2 and alpha3 subunits. In an in vitro adhesion assay, neutralizing antibody for integrin alpha2 or beta1 subunit inhibited the adhesion of MKN-45-P cells to collagen type I and type IV. Moreover, the injection of anti-beta1 monoclonal antibody reduced the number of cancer cells on the peritoneum in nude mice that had been inoculated with MKN-45-P cells. These results suggest that integrin alpha2/beta1 represents a candidate target molecule available for the prevention of gastric cancer peritoneal dissemination.

Adenocarcinoma↗

PubMed1999

Identification of a heparin binding site and the biological activities of the laminin alpha1 chain carboxy-terminal globular domain.

The carboxy-terminal globular domain (G-domain) of the laminin alpha1 chain has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. In this study, we defined the potential sequences originating from the G-domain of laminin alpha1 chain which possess these functional activities. A series of peptides were synthesized from the G-domain, termed LG peptides (LG-1 to LG-6) and were tested for their various biological activities. In the direct [3H] heparin binding assays, LG-6 (residues 2,335-2,348: KDFLSIELVRGRVK) mediated high levels of [3H]heparin binding, and this peptide also directly promoted cell adhesion and spreading, including B16F10, M2, HT1080, and PC12 cells. The peptide LG-6 also promoted the neurite outgrowth of PC12 cells, mouse granule cells, and chick telencephalic cells. An anti-peptide LG-6 antibody inhibited laminin-1 and peptide LG-6-mediated cell adhesion and neurite outgrowth. Furthermore, an anti-integrin alpha2 antibody also inhibited the cell adhesion activity. These results suggest that peptide LG-6 plays a functional role as a heparin binding site in the G-domain of the laminin alpha1 chain, and this sequence was thus concluded to play a crucial role in regulating cell adhesion and spreading and neurite out-growth which is related to integrin alpha2.

Amino Acid Sequence↗