Adhesion to fibronectin via α5ß1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.

EGFL7 reduces CNS inflammation in mouse.

Extracellular matrix (ECM) proteins secreted by blood-brain barrier (BBB) endothelial cells (ECs) are implicated in cell trafficking. We discovered that the expression of ECM epidermal growth factor-like protein 7 (EGFL7) is increased in the CNS vasculature of patients with multiple sclerosis (MS), and in mice with experimental autoimmune encephalomyelitis (EAE). Perivascular CD4 T lymphocytes colocalize with ECM-bound EGFL7 in MS lesions. Human and mouse activated T cells upregulate EGFL7 ligand αvß3 integrin and can adhere to EGFL7 through integrin αvß3. EGFL7-knockout (KO) mice show earlier onset of EAE and increased brain and spinal cord parenchymal infiltration of T lymphocytes. Importantly, EC-restricted EGFL7-KO is associated with a similar EAE worsening. Finally, treatment with recombinant EGFL7 improves EAE, reduces MCAM expression, and tightens the BBB in mouse. Our data demonstrate that EGFL7 can limit CNS immune infiltration and may represent a novel therapeutic avenue in MS.

Cutting Edge: Human CD49e- NK Cells Are Tissue Resident in the Liver.

Most knowledge on NK cells is based on studies of what are now known as conventional NK cells in the mouse spleen or human peripheral blood. However, recent studies in mice indicate the presence of tissue-resident NK cells in certain organs, such as the liver, that display different markers and transcription factor dependencies as compared with conventional NK cells. In this study, we provide evidence from cytometry by time-of-flight analysis and humanized mice indicating that human CD49e- NK cells are tissue resident in the liver. Thus, these studies indicate that tissue-resident NK cells are evolutionarily conserved in humans and mice, providing a foundation to explore their role in human disease.

Identification of Stage-Specific Surface Markers in Early B Cell Development Provides Novel Tools for Identification of Progenitor Populations.

Whereas the characterization of B lymphoid progenitors has been facilitated by the identification of lineage- and stage-specific surface markers, the continued identification of differentially expressed proteins increases our capacity to explore normal and malignant B cell development. To identify novel surface markers with stage-specific expression patterns, we explored the reactivity of CD19(+) B cell progenitor cells to Abs targeted to 176 surface proteins. Markers with stage-specific expression were identified using a transgenic reporter gene system subdividing the B cell progenitors into four surface IgM(-) stages. This approach affirmed the utility of known stage-specific markers, as well as identifying additional proteins that selectively marked defined stages of B cell development. Among the stage-specific markers were the cell adhesion proteins CD49E, CD11A, and CD54 that are highly expressed selectively on the most immature progenitors. This work identifies a set of novel stage-specific surface markers that can be used as a complement to the classical staining protocols to explore B lymphocyte development.

α-Integrin expression and function modulates presentation of cell surface calreticulin.

Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the prophagocytic signal for macrophages. Cell adhesion is a physiologically relevant stimulus previously shown to increase calreticulin interaction with α-integrins via the juxtamembrane, cytosolic GFFKR motif. This study assessed whether integrin function can regulate surface calreticulin levels in ICD. We generated calreticulin-null T-lymphoblasts and confirmed the loss of surface calreticulin expression on cells treated with doxorubicin, an ICD inducer. Reconstituted expression with full-length calreticulin targeted to the endoplasmic reticulum (ER) successfully rescued doxorubicin-induced surface calreticulin. Reconstitution with a truncation mutant calreticulin targeted to the cytosol led to constitutively high surface calreticulin that was not further elevated by doxorubicin, suggesting calreticulin released from the stressed ER transits the cytosol before its translocation to the cell surface. When stimulated to engage integrin substrates, doxorubicin-treated wild-type T-lymphoblasts exhibited decreased surface calreticulin compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin was recapitulated for cells in suspension treated with a ß1-integrin-activating antibody, 9EG7. Similarly, cells expressing a truncated α-integrin cytosolic tail, bearing only the juxtamembrane GFFKR calreticulin-binding motif, exhibited low surface calreticulin with doxorubicin treatment under non-adherent conditions. Using partial permeabilization techniques to distinguish between cytosolic and ER staining, we found that ICD inducers promoted the accumulation of cytosolic calreticulin with negligible change in total calreticulin, suggesting that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation. T-lymphoblasts co-treated with an ICD inducer and 9EG7 exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD inducer. This study reveals a previously uncharacterized function of integrins as negative regulators of ICD by suppressing presentation of cell surface calreticulin.

Proteolytic and non-proteolytic activation of keratinocyte-derived latent TGF-ß1 induces fibroblast differentiation in a wound-healing model using rat skin.

Transforming growth factor-ß1 (TGF-ß1) reportedly causes the differentiation of fibroblasts to myofibroblasts during wound healing. We investigated the mechanism underlying the activation of latent TGF-ß1 released by keratinocytes in efforts to identify promising pharmacological approaches for the prevention of hypertrophic scar formation. A three-dimensional collagen gel matrix culture was prepared using rat keratinocytes and dermal fibroblasts. Stratified keratinocytes promoted the TGF receptor-dependent increase in α-smooth muscle actin (α-SMA) immunostaining and mRNA levels in fibroblasts. Latent TGF-ß1 was found to be localized suprabasally and secreted. α-SMA expression was inhibited by an anti-αv-integrin antibody and a matrix metalloproteinase (MMP) inhibitor, GM6001. In a two-dimensional fibroblast culture, α-SMA expression depended on the production of endogenous TGF-ß1 and required αv-integrin or MMP for the response to recombinant latent TGF-ß1. In keratinocyte-conditioned medium, MMP-dependent latent TGF-ß1 secretion was detected. Applying this medium to the fibroblast culture enhanced α-SMA production. This effect was decreased by GM6001, the anti-αv-integrin antibody, or the preabsorption of latent TGF-ß1. These results indicate that keratinocytes secrete latent TGF-ß1, which is liberated to fibroblasts over distance and is activated to produce α-SMA with the aid of a positive-feedback loop. MMP inhibition was effective for targeting both keratinocytes and fibroblasts in this model.

Type I interferon and NF-κB activation elicited by herpes simplex virus gH/gL via αvß3 integrin in epithelial and neuronal cell lines.

αvß3 integrin represents a novel sensing system which detects herpes simplex virus (HSV) and bacterial constituents. In cooperation with Toll-like receptor 2 (TLR2), it elicits an innate response that leads to activation of type I interferon (IFN), NF-κB, and a specific set of cytokines. We report that this defensive branch is functional in cells which represent experimental models of epithelial, including keratinocytic, and neuronal cells. These are the major targets of HSV in vivo. HSV entered the three cell lines via distinct routes. Hence, the defensive response was independent of the route of virus entry. Soluble gH/gL sufficed to elicit type I IFN and NF-κB activation and represents the viral pathogen-associated molecular pattern (PAMP) of this defense system.

Staphylococcal enterotoxin B compromises the immune tolerant status in the airway mucosa.

BACKGROUND: The breakdown of immune tolerance plays a critical role in allergic disorders; the mechanism of breaching immune tolerance remains largely unknown. OBJECTIVE: The present study aimed to investigate the role of Staphylococcal enterotoxin B (SEB) in the interference of the immune tolerance in the nasal mucosa. METHODS: The immune tolerant components, tolerogenic dendritic cells (TolDC) and regulatory T cells (Treg), were assessed in the surgically removed nasal mucosa from patients with allergic rhinitis (AR) or non-AR chronic rhinitis. The contents of SEB and integrin alphavbeta6 (avb6) in the nasal epithelium were assessed using enzyme-linked immunoassay. The ability of avb6 on TolDC induction and the effect of SEB on suppression of avb6 in nasal epithelial cells were observed in cell culture. RESULTS: Compared with that in the non-AR nasal mucosa, the frequencies of TolDCs/Tregs were lower, the contents of SEB were higher and the contents of avb6 were lower in the AR nasal mucosa. Avb6 had the ability to induce the development of TolDCs in vitro; the latter had the ability to induce Treg development. The expression of avb6 was detected in nasal epithelial cells in culture that could be suppressed by SEB. CONCLUSIONS AND CLINICAL RELEVANCE: The components of immune tolerance machinery, TolDCs and Tregs were suppressed in the AR nasal mucosa. The increases in SEB and decreases in avb6 in nasal epithelium are associated with the compromises of immune tolerance in the nasal mucosa. SEB has the ability to suppress the expression of avb6 in nasal epithelial cells.

The role of dystroglycan in PDGF-BB-dependent migration of activated hepatic stellate cells/myofibroblasts.

Hepatic stellate cells are embedded in the loose connective tissue matrix within the space of Disse. This extracellular matrix contains several basement membrane components including laminin, but its composition changes during liver injury because of the production of extracellular matrix components found in scar tissue. These changes in extracellular matrix composition and in cell-extracellular matrix interactions may play a key role in hepatic stellate cell transdifferentiation. In this communication we used early passages of mouse hepatic stellate cells (activated HSC/myofibroblasts) to study the platelet-derived growth factor BB (PDGF-BB)-dependent expression and regulation of ß-dystroglycan and its role in activated HSC/myofibroblast migration. We used Northern and Western analysis to study dystroglycan expression and confocal microscopy to investigate changes in subcellular distribution of the protein. Activated HSC migration was investigated using an in vitro wound-healing assay. PDGF-BB induced significant changes in dystroglycan regulation and subcellular distribution of the protein. Whereas steady-state levels of dystroglycan mRNA remained constant, PDGF-BB increased dystroglycan transcription but shortened the t(1/2) by 50%. Moreover, PDGF-BB changed dystroglycan and α5-integrin cellular distribution. Cell migration experiments revealed that PDGF-BB-dependent migration of activated HSC/myofibroblasts was completely blocked by neutralizing antibodies to fibronectin, α5-integrin, laminin, and ß-dystroglycan. Overall, these findings suggest that both laminin and fibronectin and their receptors play a key role in PDGF-BB-induced activated HSC migration.

Identification of immunoreactive regions of homology between soluble epidermal growth factor receptor and α5-integrin.

Proteins encoded by the epidermal growth factor receptor (EGFR/HER1/ERBB1) gene are being studied as diagnostic, prognostic, and theragnostic biomarkers for numerous human cancers. The clinical application of these tissue/tumor biomarkers has been limited, in part, by discordant results observed for epidermal growth factor receptor (EGFR) expression using different immunological reagents. Previous studies have used EGFR-directed antibodies that cannot distinguish between full-length and soluble EGFR (sEGFR) expression. We have generated and characterized an anti-sEGFR polyclonal antiserum directed against a 31-mer peptide (residues 604-634) located within the unique 78-amino acid carboxy-terminal sequence of sEGFR. Here, we use this antibody to demonstrate that sEGFR is coexpressed with EGFR in a number of carcinoma-derived cell lines. In addition, we show that a second protein of ~140 kDa (p140) also is detected by this antibody. Rigorous biochemical characterization identifies this second protein to be α5-integrin. We show that a 26-amino acid peptide in the calf domain of α5-integrin (residues 710-735) is 35% identical in sequence with a 31-mer carboxy-terminal sEGFR peptide and exhibits an approximately 5-fold lower affinity for anti-sEGFR than the homologous 31-mer sEGFR peptide does. We conclude that the carboxy terminus of sEGFR and the calf-1 domain of α5-integrin share a region of sequence identity, which results in their mutual immunological reactivity with anti-sEGFR. We also demonstrate that anti-sEGFR promotes three-dimensional tissue cohesion and compaction in vitro, further suggesting a functional link between sEGFR and α5-integrin and a role of the calf-1 domain in cell adhesion. These results have implications for the study of both EGFR and sEGFR as cancer biomarkers and also provide new insight into the mechanisms of interaction between cell surface EGFR isoforms and integrins in complex processes such as cell adhesion and survival signaling.

Pharmacology and placental transfer of a human alphav integrin monoclonal antibody in rabbits.

BACKGROUND: Intetumumab is a human IgG1 anti-alphav-integrin monoclonal antibody that inhibits angiogenesis. Integrin binding and angiogenesis are important in reproduction including fertilization, implantation, and embryofetal development. These studies were designed to determine the pharmacological relevance of the rabbit for the evaluation of potential effects on embryofetal development and to evaluate the placental transfer of intetumumab in rabbits. METHODS: In vitro pharmacology studies evaluated the binding of intetumumab to rabbit cells and the inhibition of vessel sprouting from rabbit aorta. For the evaluation of placental transfer, pregnant rabbits (8/group) were injected intravenously with intetumumab 50 or 100 mg/kg every 2 days from Gestation Day (GD)7 to GD19. Maternal sera, fetal homogenates/sera, and amniotic fluid were collected at necropsy on GD19 or GD28 for evaluation of intetumumab concentrations. Clinical condition of the dams was monitored and fetuses were screened for abnormalities. RESULTS: Intetumumab (5-40 microg/mL) inhibited aortic cell adhesion to vitronectin and vessel sprouting from rabbit aortic rings. Immunohistochemical staining of rabbit tissues demonstrated binding of intetumumab to placenta. Administration of intetumumab to pregnant rabbits was well tolerated by the dams and the fetuses did not show major abnormalities. Fetal exposure to intetumumab relative to maternal exposure was <0.1% on GD19 and 100-130% on GD29. CONCLUSIONS: The rabbit is a pharmacologically relevant species for evaluation of potential developmental effects of intetumumab. Intetumumab crosses the rabbit placenta during the fetal period (GD 19-28).

Glucocorticoids increase alpha5 integrin expression and adhesion of synovial fibroblasts but inhibit ERK signaling, migration, and cartilage invasion.

OBJECTIVE: In rheumatoid arthritis (RA), integrins mediate cell adhesion, migration, and invasion, and their expression is regulated by cytokines and growth factors. The aim of this study was to investigate whether hormones such as cortisol or other steroids can influence integrin expression and function in the synovial cells of patients with RA. METHODS: We performed immunofluorescence and fluorescence-activated cell sorting analyses to quantify surface integrin levels. Adhesion and migration assays were performed to study the function of synovial fibroblasts (SFs). ERK activation was measured by cellular activation of a signaling enzyme-linked immunosorbent assay. Invasion of SFs into cartilage was determined in the SCID mouse coimplantation model of RA in vivo. RESULTS: In RA, expression of integrin subunits alpha5, alphav, and beta1 was higher at the site of invasion compared with the sublining zone. Testosterone and 17beta-estradiol had no influence on integrin levels, but cortisol up-regulated expression of the alpha5 subunit in a time-dependent and dose-dependent manner. In addition, cortisol increased the adhesion of SFs to fibronectin and inhibited ERK signaling upon integrin activation or upon stimulation with tumor necrosis factor. Small interfering RNA or a neutralizing antibody to alpha5 integrin increased SF migration, indicating that up-regulated alpha5 integrin is responsible for an immobile phenotype. In addition, in the SCID mouse model, SF invasion into cartilage was attenuated by glucocorticoid treatment in vivo. CONCLUSION: Glucocorticoids increase integrin expression and the adhesion of cells to fibronectin, inhibit ERK signaling, and down-regulate the invasiveness of SFs in vivo. This study demonstrates that an important antiinflammatory aspect of glucocorticoids is regulating the expression and function of alpha5 integrin.

Proteinase-activated receptor-2 mediates the expression of integrin alpha5 and beta1 in Helicobacter pylori-infected gastric epithelial AGS cells.

BACKGROUND/AIM: Proteinase-activated receptor-2 (PAR2), which is activated by trypsin, is known to be associated with expression of adhesion molecule integrins. We previously demonstrated that Helicobacter pylori induced the expression of integrin alpha(5) and beta(1) in human gastric epithelial cells. The present study aims to investigate whether H. pylori in a Korean isolate (HP99) induces the expression of PAR2, which mediates the expression of integrin alpha(5) and beta(1) and thus cell adhesion to fibronectin in gastric epithelial AGS cells. METHODS AND RESULTS: mRNA expressions of PAR2, trypsinogen 1 and 2, and integrin alpha(5) and beta(1) were assessed by RT-PCR analysis while protein levels of PAR2, trypsin as well as integrin alpha(5) and beta(1) were determined by Western blot analysis. The activity of trypsin in the medium was determined by fluorometric analysis. Cell adhesion assay was performed colorimetrically. H. pylori induced the expressions of PAR2 and integrin alpha(5) and beta(1) of the cells. H. pylori increased mRNA expression of trypsinogen 1 and 2 as well as the level and activity of trypsin in the medium. H. pylori induced cell adhesion to fibronectin. H. pylori-induced expression of integrin alpha(5) and beta(1) and adhesion of the cells to fibronectin were inhibited in the cells transfected with PAR2 antisense oligonucleotide or treated with a soybean trypsin inhibitor, anti-integrin alpha(5) antibody or beta(1) antibody. CONCLUSION: H. pylori induces the expression of integrin alpha(5) and beta(1) and adhesion of the cells to fibronectin through PAR2 which is induced and may be activated by trypsin in H. pylori-infected gastric epithelial cells. PAR2 may have an important role in gastric cell adhesion and possibly carcinogenesis associated with H. pylori.

Cooperation between integrin alpha5 and tetraspan TM4SF5 regulates VEGF-mediated angiogenic activity.

Tetraspan TM4SF5 is highly expressed in a diverse number of tumor types. Here we explore the mechanistic roles of TM4SF5 in angiogenesis. We found that TM4SF5 overexpression correlates with vascular endothelial growth factor (VEGF) expression in SNU449 hepatocytes and with vessel formation in clinical hepatocarcinoma samples. Conditioned media from TM4SF5-expressing cells enhanced viability and tube formation of primary human umbilical vein endothelial cells, and outgrowth of endothelial cells from aorta ring segments, which was abolished by treatment with an anti-VEGF antibody. TM4SF5 retained integrin alpha(5) on the cell surface for VEGF induction, and preincubation with anti-integrin alpha(5) antibody abolished TM4SF5-mediated VEGF expression and secretion. TM4SF5-mediated effects required integrin alpha(5), c-Src, and signal transducer and activator of transcription 3 (STAT3). In addition, tumors from nude mice injected with TM4SF5-expressing cells and from clinical human hepatocarcinoma tissues showed enhanced integrin alpha(5) expression, vessel formation, and signaling activity, which were inhibited by administration of anti-integrin alpha(5) or -VEGF antibody. This study suggests that TM4SF5 facilitates angiogenesis of neighboring endothelial cells through VEGF induction, mediated by cooperation between TM4SF5 and integrin alpha(5) of epithelial cells.

Antibody-mediated blockade of integrin alpha v beta 6 inhibits tumor progression in vivo by a transforming growth factor-beta-regulated mechanism.

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.

Alphav integrin-targeted immunoconjugates regress established human tumors in xenograft models.

PURPOSE: Targeted delivery of cytotoxic agents to solid tumors through cell surface antigens can potentially reduce systemic toxicity and increase the efficacy of the targeted compounds. The purpose of this study was to show the feasibility of treating solid tumors by targeting alpha(v) integrins with antibody-maytansinoid conjugates and to test the relative in vivo activities of several linker-maytansinoid chemistries. EXPERIMENTAL DESIGN: CNTO 364, CNTO 365, and CNTO 366 are targeted cytotoxic agents created by conjugating the CNTO 95 anti-alpha(v) integrin antibody with three distinct maytansinoid-linker structures. These structures were designed to have varying degrees of chemical substitution surrounding the disulfide bond linking the cytotoxic agent to the antibody. A model conjugate was shown to be specifically cytotoxic in vitro and highly active against established human tumor xenografts in immunocompromised rats. The in vivo antitumor activities of CNTO 364, CNTO 365, and CNTO 366 were compared in rat xenograft models. RESULTS: CNTO 365, with a linker chemistry of expected intermediate stability, was shown to be substantially more active than the other two conjugates with lesser or greater substitution around the disulfide linkage. CONCLUSION: CNTO 95-maytansinoid immunoconjugates are potent antitumor agents against alpha(v) integrin-expressing human carcinomas. These studies show for the first time the feasibility of targeting alpha(v) integrins on solid tumors with tumor-activated prodrugs. The DM4 linker-maytansinoid configuration of CNTO 365 was substantially more active in the models tested here when compared with alternative configurations with greater or lesser chemical substitution surrounding the linker.

Influence of arginine-glycine-aspartic acid (RGD), integrins (alphaV and alpha5) and osteopontin on bovine sperm-egg binding, and fertilization in vitro.

Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, has been identified in cow oviduct epithelium and fluid. To investigate the potential role OPN in fertilization, we evaluated the ability of RGD peptide (arginine-glycine-aspartic), RGE peptide (arginine-glycine-glutamic acid), integrins alphaV and alpha5 antibodies and OPN antibody to influence bovine in vitro sperm-egg binding and fertilization. Treatment of sperm or oocytes with the RGD peptide prior fertilization significantly decreased in vitro sperm-egg binding and fertilization compared to the non-treated controls or those treated with RGE peptide. Binding and fertilization were also significantly decreased when in vitro matured bovine oocytes or sperm were pre-incubated with integrins alphaV and alpha5 antibodies at concentration ranging from 5 to 20 microg/mL. Addition of a rabbit polyclonal IgG antibody against purified bovine milk OPN with sperm or/and oocytes decreased (P<0.05) fertilization compared to the in vitro-fertilized control. These data provided evidence that integrin ligands existed on bovine oocytes and spermatozoa that contained RGD recognition sequences, and that antibody to OPN, a protein that contains that RGD sequence, was capable of reducing sperm-egg binding and fertilization in vitro.

Integrin alpha5 is involved in fibronectin-induced human extravillous trophoblast invasion.

To identify the molecules involved in human extravillous trophoblast (EVT) invasion, we raised murine mAbs that react with EVTs and obtained one mAb (CHL3) that inhibited invasion of a human choriocarcinoma-derived cell line, BeWo cells. The N-terminal 22 aminoacid sequence of the CHL3 antigen (150kDa) purified from placental tissue completely matched that of integrin alpha5, which is known to interact with fibronectin. Double immunohistochemical staining and flow cytometry confirmed the reactivity of CHL3 with integrin alpha5 and its expression on the surface of BeWo cells and human EVTs isolated from villous explant cultures. CHL3 mAb inhibited the attachment of human EVTs and BeWo cells to fibronectin-coated dishes, but not to Matrigel dishes. In the Matrigel invasion assay supplemented with or without fibronectin, the invasion of isolated EVTs and BeWo cells was attenuated by treatment with CHL3 without affecting cell proliferation. During invasion assays, the production of matrix metalloproteases 2 and 9 was not changed by CHL3. These findings suggest that interaction with fibronectin through integrin alpha5 plays an important role in human extravillous trophoblast invasion.

[Regulatory effect of integrin alpha5 and beta1 on proliferation inhibition of K562 cells induced by interferon alpha-2b].

BACKGROUND & OBJECTIVE: Integrin beta1 can inhibit the proliferation of chronic myelocytic leukemia (CML) ph+ cells. The dysfunction of integrin beta1 might accelerate the growth of CML ph+ cells. This study was to explore the effects of integrin alpha5 and beta1 on the proliferation inhibition of K562 cells induced by IFNalpha-2b. METHODS: The expression indexes of integrin alpha5 and beta1 on K562 cells, the binding capability of K562 cells to fibronectin (FN), and K562 cell-FN binding blocking induced by integrin alpha5 and beta1 antibodies were evaluated by flow cytometry (FCM). The viability of K562 cells, treated with IFNalpha-2b (10,000 u/ml), was observed by MTT assay. The mRNA level of focal adhesion kinase (FAK) in K562 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) 48 h after treatment of interferon alpha-2b (IFNalpha-2b). RESULTS: The positive rates of integrin alpha5 and beta1 were significantly higher on K562 cells than on bone marrow mononuclear cells from healthy donors [(97.59+/-1.04)% vs. (64.05+/-2.38)%, (99.24+/-0.52)% vs. (72.40+/-3.56)%, P<0.05). IFNalpha-2b could not change the expression of integrin alpha5 and beta1 on K562 cells, but improved the binding capability of K562 cells to FN, which could be blocked by anti-alpha5 and/or anti-beta1 antibodies. IFNalpha-2b enhanced the expression of FAK gene, and inhibited the proliferation of K562 cells. The anti-alpha5 and anti-beta1 antibodies improved the inhibitory effect of IFNalpha-2b on the proliferation of K562 cells, and blocked IFNalpha-2b-induced increase of FAK gene expression. CONCLUSION: IFNalpha-2b could inhibit the proliferation of K562 cells through restoring the function of integrin alpha5 and beta1, enhancing binding capability of integrin alpha5 and beta1 to FN, and up-regulating FAK gene expression.

Gap junction-microtubule associations in rat alveolar epithelial cells.

Connexin 43 (Cx43) is a predominant gap junction (GJ) protein expressed by alveolar epithelial cells (AEC) in primary cell culture. Cx43 trafficking, assembly, and turnover are regulated by multiple mechanisms, including those mediated by integrins, by extracellular matrix, and by the cytoskeleton. Immunocytochemical double labeling demonstrates association of microtubules with internalization of Cx43-positive GJ plaques. Antibodies against the alpha 5-integrin subunit block cell-matrix interactions without effect on tubulin expression, whereas inhibition of MAP kinase kinase by PD-98059 reduces tubulin expression, based on both Western blot and immunostaining. To examine direct association of microtubules (MT) with GJ plaques, we treated day 3 AEC for 0.5-24 h with colchicine, an inhibitor of tubulin polymerization. After 60 min, MTs were disassembled, whereas Western blot analysis showed no change in tubulin expression. In parallel, colchicine initiated redistribution of immunopositive Cx43 from the membrane to the cytosol. These observations support the premise that direct association of the cytoskeleton with gap junctions plays a significant role in regulation of Cx43 expression and distribution through integrin-mediated signal transduction pathways.