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Results for “Integrin alpha5”

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PubMed2006

Microfluidic techniques for single-cell protein expression analysis.

BACKGROUND: The analysis of single cells obtained from needle aspirates of tumors is constrained by the need for processing. To this end, we investigated two microfluidic approaches to measure the expression of surface proteins in single cancer cells or in small populations (<50 cells). METHODS: One approach involved indirect fluorescence labeling of cell-surface proteins and channeling of cells in a microfluidic device past a fluorescence detector for signal quantification and analysis. A second approach channeled cells in a microfluidic device over detection zones coated with ligands to surface proteins and measured rates of passage and of retardation based on transient interactions between surface proteins and ligands. RESULTS: The fluorescence device detected expression of integrin alpha5 induced by basic fibroblast growth factor (FGF-2) treatment in MCF-7 cells and that of Her-2/neu in SK-BR-3 cells compared with controls. Experiments measuring passage retardation showed significant differences in passage rates between FGF-2-treated and untreated MCF-7 cells over reaction regions coated with fibronectin and antibody to integrin alpha5beta1 compared with control regions. Blocking peptides reversed the retardation, demonstrating specificity. CONCLUSIONS: Immunofluorescence detection in a microfluidic channel demonstrates the potential for assaying surface protein expression in a few individual cells and will permit the development of future iterations not requiring cell handling. The flow retardation device represents the first application of this technology for assessing cell-surface protein expression in cancer cells and may provide a way for analyzing expression profiles of single cells without preanalytical manipulation.

Antibodies↗
PubMed2001

Focal adhesion kinase is a key mediator of human trophoblast development.

Trophoblast differentiation during the first trimester of pregnancy involves cell proliferation and invasion and extracellular matrix (ECM) remodeling. Reports have indicated that, in a variety of cell types, processes such as proliferation, invasion, and ECM remodeling require the turnover of focal adhesions mediated by a cytoplasmic tyrosine kinase named focal adhesion kinase (FAK). Therefore, in the present study we examined the expression and spatial localization of FAK during early human placental development. Immunocytochemical and immunoblot analysis showed that FAK and a focal adhesion-associated protein named paxillin were highly expressed between the 5th and 8th weeks of gestation, specifically in villous cytotrophoblast and extravillous trophoblast (EVT) cells. Activated FAK, phosphorylated on Tyr-397, colocalized with alpha5 integrin and matrix metalloproteinase-2 (MMP2) expression in EVT cells within a previously characterized intermediate, invasive-restrained region. FAK and paxillin expression dramatically decreased after 10 to 12 weeks of gestation coincident with increasing pO(2) levels. Exposure of human villous explants of 5 to 8 weeks to a 3% O(2) environment resulted in increased trophoblast outgrowth, cell proliferation, and detection of alpha5 integrin and MMP2, as well as increased activation of FAK in EVT cells compared with explants grown in a 20% O(2) environment. To determine whether FAK was a key requisite for trophoblast differentiation, villous explants of 5 weeks gestation were grown in Matrigel in a 3% O(2) environment and incubated with 20-mer antisense FAK oligonucleotides. A dramatic reduction of trophoblast outgrowth was observed in antisense-treated explants compared with missense and control cultures, and, in addition, cell proliferation and MMP2 activity in antisense-treated explants were dramatically reduced. These data suggest that FAK is a key kinase involved in early trophoblast cell differentiation and plays a role in regulating cell proliferation and motility during early placental development.

Antisense Elements (Genetics)↗
PubMed2004

TGF-beta2-induced cell surface tissue transglutaminase increases adhesion and migration of RPE cells on fibronectin through the gelatin-binding domain.

PURPOSE: Migration and adhesion of dislocated retinal pigment epithelial (RPE) cells to a fibronectin-rich extracellular matrix is an initial step in proliferative vitreoretinopathy (PVR). In the present study, the functional role of cell surface tissue transglutaminase (tTG) in adhesion and migration of RPE cells on fibronectin (Fn) and collagen type I (Col I) after stimulation with TGF-beta2 was investigated. METHODS: Cultured human RPE cells were treated with 1.0 ng/mL TGF-beta2 for 24 hours. Cell surface tTG expression was determined by cell fraction analysis. Attachment on Col I, full-length Fn, and its 45-kDa gelatin-binding and 110-kDa cell-binding fragment was measured with an MTT assay. Migration of RPE cells was measured by a Boyden chamber assay, and cell spreading was determined. Experiments were performed in the presence or absence of anti-tTG antibodies and anti-integrin alpha5 and beta1 antibodies. RESULTS: TGF-beta2 markedly induced expression of cell-surface tTG on RPE cells and increased attachment and migration on Fn and Col I. Blocking cell surface tTG inhibited attachment, migration, and spreading on Fn and its 45-kDa gelatin-binding fragment, whereas no effect was seen on Col I and the 110-kDa cell-binding Fn fragment. In contrast, blocking of integrin alpha5 and beta1 suppressed adhesion and migration on full-length Fn and the 110-kDa Fn fragment. CONCLUSIONS: These data demonstrate that TGF-beta2 increases expression of cell surface tTG, which in turn strengthens adhesion, migration, and spreading of RPE cells on Fn through the 45-kDa gelatin-binding Fn fragment. At the onset of PVR, this mechanism may help RPE cells to attach and migrate on Fn-containing matrices.

Adolescent↗
PubMed2000

The HIV-1 vpr protein induces anoikis-resistance by modulating cell adhesion process and microfilament system assembly.

We have previously shown that CD4+ T Jurkat cells constitutively expressing low levels of the human immunodeficiency virus 1 (HIV-1) vpr protein were less susceptible to undergo apoptosis than control cells.1 In this study we have investigated the role of vpr in affecting mechanisms of importance in the control of apoptosis. Vpr-expressing clones consistently aggregated in clusters with time in culture, whereas mock-transfected cells grew as dispersed cultures. The analysis of adhesion molecules involved in cell-to-cell as well as in cell-substrate interactions showed a higher expression of cadherin and integrins alpha5 and alpha6 in vpr-transfected clones with respect to mock-transfected cells. This up-modulation was specifically blocked by cell exposure to antisense oligonucleotides targeted at the vpr. In addition, F-actin microfilament cytoskeletal organization, known to be involved in cell-cell interaction pathways and in the modulation of cell surface molecule expression, was significantly improved in vpr-expressing clones, in which filament polymerization was increased. We thus envisage that vpr viral protein can maintain cell survival via a specific activity on cytoskeleton-dependent cell adhesion pathways, i.e. by inducing anoikis-resistance. These particular effects of vpr might enhance the homing, spreading and survival of the infected lymphocytes, thus contributing to virus persistence in the course of acute HIV-1 infection.

Actin Cytoskeleton↗
PubMed1998

Production of inducible nitric oxide is required for monocytic differentiation of U937 cells induced by vitamin E-succinate.

Many putative differentiating agents arrest cell growth prior to progression of the cell through differentiation. Vitamin E-succinate is known to be a potent modulator of haematopoietic differentiation as well as an inhibitor of cell growth in vitro and in vivo. In this study, we examined whether vitamin E-succinate could modulate the monocytic differentiation of U937 human monoblasts. Treatment with vitamin E-succinate for 1-4 days inhibited the proliferation of U937 cells. Vitamin E-succinate also induced monocytic differentiation as indicated by the increase in nitro blue tetrazolium reduction activity, and the expression of monocyte specific cell surface antigen, CD11c, and integrins alpha5 and beta1. The monocytic differentiation of U937 cells was also induced when the cells were cultured in fibronectin-coated wells. Monocytic differentiation was enhanced when the cells were treated with both vitamin E-succinate and fibronectin, suggesting that vitamin E-succinate and fibronectin synergistically act on monocytic differentiation of U937 cells. During monocytic differentiation of U937 cells induced by vitamin E-succinate and/or fibronectin, nitric oxide was detected in supernatants. The production of nitric oxide was not detected when monocytic differentiation of U937 cells was induced by phorbol-12 myristate 13-acetate, a well known inducer of macrophage-like cell differentiation. Vitamin E-succinate and/or fibronectin induced monocytic differentiation was blocked by the treatment of nitric oxide synthase inhibitor, N-G-monomethyl-L-arginine. In contrast, treatment of cells with sodium nitroprusside, a chemical nitric oxide donor, stimulated monocytic differentiation of U937 cells at an early time point. Taken together, these results suggest that nitric oxide is an important intermediator at an early stage of vitamin E-succinate- and/or fibronectin-induced monocytic differentiation of U937 cells.

Antigens, CD↗
PubMed2001

Cytoskeletal-induced alterations in the adhesion of HT-1080 fibrosarcoma cells to extracellular matrix.

We have investigated the adhesion of the human fibrosarcoma cell line, HT-1080, transfected with glial fibrillary acidic protein (GFAP) to a variety of extracellular matrix macromolecules (ECM) including collagen type IV, laminin, and fibronectin. The GFAP-transfectants demonstrated altered adhesiveness to extracellular matrix substrates when compared to controls. GFAP-positive, heavy metal-induced fibrosarcoma cells were more adherent to plastic and collagen type IV than were the parental or uninduced cells. In contrast, GFAP-positive fibrosarcoma cells were less adherent to laminin- or fibronectin-coated dishes than controls. Time course adhesion studies over 9 days showed that the heavy metal-induced fibrosarcoma cells progressively became more adherent to collagen type IV and less adherent to laminin- or fibronectin-coated dishes than did uninduced cells. However, with the removal of heavy metal from the medium, the HT-1080 fibrosarcoma cells were restored to their original adhesive potential. By phase microscopy, uninduced and induced HT-1080 cells demonstrated different morphological features and remained viable in an anchorage-dependent fashion on collagen type IV as a substrate. By way of contrast, GFAP-induced HT-1080 cells were not particularly viable in monolayer culture and readily detached from laminin as a substrate. The expression of beta1 integrin in GFAP-positive fibrosarcoma cells was decreased following heavy metal induction by Western blot analyses. In contrast, the expression of alpha2 integrin was increased whereas alpha5 integrin was unchanged in HT-1080 cells following the induction of GFAP. Gelatin zymography showed that 72 kDa collagenase was less expressed in GFAP-induced clones than in controls. Our data suggest that the forced expression of the intermediate filament, GFAP, in HT-1080 cells may modulate cell adhesion to different ECM substrates through alterations in expression of integrins.

Blotting, Western↗
PubMed2003

Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V.

The expressions of integrin alpha5, beta1, and alpha6 were studied in H7721 cells by means of flow cytometric and RT-PCR method after transfected with sense and antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V). The transfected cells were characterized by Northern blot. It was found that the order of expression from high to low was beta1>alpha5>alpha6. Transfection of sense GnT-V up-regulated alpha5 and alpha6, but not beta1 subunit, while antisense GnT-V down-regulated alpha5 and beta1, but not alpha6. The alterations of surface integrin subunits were quite compatible with the changes of their mRNAs. Using enzyme-labeled lectin analysis, it was shown that alpha5 subunit contained only C(2)C(2) biantennary N-glycan, which was not regulated by sense and antisense GnT-V. In contrast, beta1 subunit contained both biantennary and tri-/tetra-antennary N-glycans with GlcNAcbeta1,6Manalpha1,6-branch, and the latter was up- and down-regulated by the sense and antisense GnT-V, respectively. Therefore, the amount of biantennary N-glycans on beta1 subunit, but not the integrin protein, was correlated to the cell adhesion to fibronectin and laminin, which was reduced and elevated in the sense and antisense GnT-V-transfected cells, respectively, as we previously reported.

Cell Line↗
PubMed1999

The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function.

alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.

Amino Acid Sequence↗
PubMed2006

Effect of cyclic strain and plating matrix on cell proliferation and integrin expression by ligament fibroblasts.

The role of cell surface integrins in cell migration, proliferation, and attachment to matrix molecules is well known. Integrin-matrix interactions have been implicated in mechanotransduction and load transmission from the outside to the inside of the cell. In this study, the effect of cyclic strain on the cell proliferation, attachment, and expression of integrin subunits beta1, beta3, and alpha5 was determined in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) fibroblasts grown on polystyrene, Type I collagen, laminin, elastin, and fibronectin. ACL fibroblast proliferation was not affected by growth substrate whereas MCL cells reached confluence more rapidly on fibronectin compared with collagen or polystyrene. Exposure to 5% cyclic strain resulted in a significant decrease in ACL and MCL fibroblast proliferation on fibronectin and Type I collagen. MCL cells showed a greater strain-dependent inhibition of cells grown on a fibronectin substrate than those grown on collagen. This matrix-dependent effect of strain on cell proliferation was not seen with ACL cells. Attachment of ACL and MCL fibroblasts was stronger to fibronectin compared with Type I collagen, laminin, and polystyrene. In the absence of applied load, the expression of beta1, beta3, and alpha5 subunits was not substrate dependent and the expression of beta1 and alpha5 integrin subunits was higher in MCL cells than ACL cells on all substrates. In contrast, the expression of beta3 integrin subunit was higher in ACL cells than MCL cells. In response to 5% strain, beta1, and alpha5 expression increased in all fibroblasts with MCL cells having a higher magnitude of expression. beta3 expression showed a 90% increase in response to load when grown on laminin for both MCL and ACL fibroblasts and demonstrated no change in expression on Type I collagen or fibronectin. The duration of applied strain from 2 versus 22 h had no effect on cell proliferation or integrin expression.

Animals↗
PubMed1999

Studies on the phenotype of migrant thymic stem cells.

Stem cells first enter the thymus around the 11th to 12th days of gestation in BALB/c mouse embryos. The phenotype of these stem cells has been difficult to determine because their entry occurs when the thymic primordium is very small and involves too few stem cells to allow studies by flow cytometry. We have been able to microdissect the thymus from embryos during this stage and immunophenotype cells in sections using a sensitive tyramide amplification system. Our results show that migrant stem cells express CD45, c-kit, CD44, CD34 and alpha4 integrin, but other markers such as CD62L, CD25, Thy-1.2, CD3epsilon, alpha5 integrin and RAG-1 expression are detected only after stem cell entry. These results should help to improve the isolation and characterization of migrant thymic stem cells.

Animals↗
PubMed1999

Overlapping and independent functions of fibronectin receptor integrins in early mesodermal development.

Mouse embryos deficient in fibronectin (FN-null) die at E8.5 with mesodermal defects. Eight integrin heterodimers alpha3beta1, alpha4beta1, alpha5beta1, alpha8beta1, alphavbeta1, alphavbeta3, alphavbeta6, and alphaIIbbeta3 can bind to FN. However, embryos deficient in each of these integrins exhibit less severe defects than do FN-null embryos, raising questions as to which integrin(s) are the key FN receptors for these early FN-dependent processes. alpha5beta1 is believed to be the key receptor and alpha5-null embryos display mesodermal defects similar to, although less severe than, those of FN-null. Here we report that the alpha5-null mutation exhibits a more severe phenotype on a 129Sv (129) than on a C57BL/6 (B6) background, as does the FN-null mutation. While alpha5-null/B6 embryos develop normal headfolds, alpha5-null/129 embryos have headfold defects similar to those of FN-null. The differences between FN-null and alpha5-null embryos, however, cannot be attributed to genetic background. FN-null embryos never form somites, whereas in alpha5-null/129 embryos the somites do condense but fail to epithelialize. Second, we examined double mutants carrying all possible pairwise combinations of null mutations in alpha3, alpha4, and alpha5 integrin genes. There was no evidence for any synergy between paired mutations, suggesting that these integrin genes do not have overlapping functions during early embryonic development. Finally, we examined double-mutant embryos deficient in both alpha5 and alphav integrin genes. These double-mutant embryos have an amniotic defect similar to that of FN-null embryos, but die even earlier with a defect in gastrulation. These studies thus revealed a gradation in the severity of defects in the mutations alpha5(-/-); alphav(-/-) > FN(-/-) (129) > FN(-/-) (B6) > alpha5(-/-) (129) > alpha5(-/-) (B6), and in each step in this series there is a certain degree of phenotypic overlap, suggesting that the defects arising from these mutations may result from disruptions of the same embryonic process.

Animals↗
PubMed1996

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Animals↗
PubMed1999

Characterization of integrin expression in islets isolated from hamster, canine, porcine, and human pancreas.

The reasons for the failure of clinical islet transplantation remain obscure. Islet isolation, however, exposes the islet to variety of cellular stresses, including disruption of the cell-matrix relationship, an event associated with apoptosis. The cell-matrix relationship is characterized by an interaction between cell surface integrin receptors and matrix molecules of the surrounding basement membrane (BM). The purpose of this study was to characterize integrin expression and the distribution of the peri-insular BM in human, porcine, canine, and hamster pancreas, and after routine islet isolation. Whereas islets in the porcine pancreas do not have a demonstrable BM, islets in the human, canine, and hamster pancreas have an almost continuous BM with very little direct exocrine to endocrine cell-cell contact. After islet isolation, the BM was destroyed, only to be reestablished during the period of culture. In the pancreas of all four species, integrin alpha3 was expressed only on islet cells, and integrin alpha5 was present on islet cells as well as on acinar, centroacinar, and duct cells. Integrin alphaV was detected only in human and canine pancreas. Integrin beta1 was demonstrated only in the human pancreas. In isolated islets, integrin alpha3, alpha5, and alphaV expression decreased during the culture period and the intensity of the staining was observed to be coincident with the distribution of the BM. In summary, this is the first report of integrin expression in hamster, canine, porcine, and human islets. After islet isolation, the altered islet cell-matrix relationship is reflected both in the decrease in integrin expression and in the destruction of the peri-insular BM. These profound changes will need to be considered as the process of islet isolation for transplantation is refined. (J Histochem Cytochem 47:499-506, 1999)

Adult↗
PubMed1999

Expression and function of cell adhesion molecules on fetal liver, cord blood and bone marrow hematopoietic progenitors: implications for anatomical localization and developmental stage specific regulation of hematopoiesis.

The mechanism of localization, migration, and regulation of hematopoiesis at different stages of ontogeny is not well understood, but may relate to the specific adhesive interactions between hematopoietic stem cells and their microenvironment at different ontogenic stages. We studied the expression of cell adhesion molecules (CAM) on fetal liver (FL), umbilical cord blood (UCB) and adult bone marrow (ABM) CD34+ cells, and the adhesion of committed progenitors (CFC) from all three sources to ABM stromal layers and purified extracellular matrix proteins. Compared to ABM CFC, significantly more UCB CFC and fewer FL CFC adhered to ABM stroma. Adhesion of FL CFC to fibronectin (FN), the 75 kD RGD containing FN fragment and the 33-66 kD COOH-terminal heparin binding FN fragment was also significantly less than that of ABM CFC. Like ABM CFC, the adhesion of FL CFC was mediated through alpha4beta1 and alpha5beta1 integrins. Of note, more FL CD34+ cells expressed alpha5 integrins and the number of alpha4, alpha5 and beta1 integins per cell (mean channel frequency) was similar or higher for FL CD34+ cells than ABM CD34+ cells. Further, treatment of FL CFC with a beta1 integrin activating antibody (8A2), increased adhesion of FL CFC to FN to the same level as that of 8A2 treated ABM CFC. This suggests that the alpha4beta1 and alpha5beta1 integrins on FL CD34+ cells may be present in a low avidity/affinity state. We also show that unlike ABM, FL CD34+ cells expressed alpha2 and that approximately 20% FL CFC adhered to collagen IV. Further, alpha2beta1 integrin on FL CFC was functional since their engagement, either by adhesion to collagen IV or through blocking alpha2 antibodies, transmitted proliferation inhibitory signals. In contrast to alpha4b and alpha5beta1 integrin dependent adhesion, alpha2beta1 dependent adhesion of FL CFC to collagen IV was not enhanced after treatment with 8A2. The reason for this is not clear but suggests that alpha2 integrins on FL CFC are maximally activated. This novel adhesive interaction with collagen IV, reminiscent of that described for CML progenitors, may have a role in the extramedullary localization of FL hematopoiesis or its developmental stage-specific regulation by its microenvironment. Studies to evaluate these possibilities are underway.

Adult↗
PubMed2002

Matrix metalloproteinase-2 is involved in A549 cell migration on laminin-10/11.

We have reported that laminin-10/11 strongly promotes migration of A549 human lung carcinoma cells by activating the alpha3beta1 integrin-dependent signaling pathway. To elucidate the mechanism involved, we investigated whether matrix metalloproteinases (MMPs) are involved in cell migration on laminin-10/11. Here, we demonstrate that laminin-10/11, but not fibronectin which does not greatly promote A549 cell movement, stimulated MMP-2 secretion approximately 3-fold. The cell migration-promoting activity of laminin-10/11 was down-regulated by an MMP inhibitor. In addition, cell motility was significantly increased when cells adhered to a mixture of fibronectin and laminin-10/11 with a concomitant decrease of focal contacts, compared with those adhering to fibronectin alone. The enhanced cell migration was partially suppressed by the MMP inhibitor. Furthermore, an anti-alpha3 integrin, but not an anti-alpha5 integrin, antibody induced the activated form of MMP-2. These data suggest that MMP-2 may play an important role in A549 cell migration on laminin-10/11 through an alpha3beta1 integrin-dependent pathway.

Antibodies↗
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMed2003

Integrin alpha4beta1 regulates migration across basement membranes by lung fibroblasts: a role for phosphatase and tensin homologue deleted on chromosome 10.

Idiopathic pulmonary fibrosis is a disease that is characterized by fibroblast accumulation and activation in the distal airspaces of the lung. We hypothesized that fibrotic lung fibroblasts migrate/invade across basement membranes by integrin-mediated mechanisms as a means of entering alveoli. We demonstrate that in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis, fibronectin signaling is both necessary and sufficient for basement membrane migration/invasion across basement membranes. This effect is mediated through the alpha5beta1 integrin because blockade of fibronectin-alpha5 integrin ligation attenuated this response. In contrast, ligation of alpha4beta1 integrin inhibits basement membrane invasion by normal lung fibroblasts but not by fibrotic lung fibroblasts. This phenotypic difference is not related to surface expression of the alpha4beta1 integrin, as demonstrated by flow cytometry. In normal lung fibroblasts but not in fibrotic lung fibroblasts, we show that ligation of alpha4beta1 integrin induces a significant increase in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. Fibrotic lung fibroblasts express constitutively less PTEN mRNA and protein as well as phosphatase activity in comparison to normal lung fibroblasts. Together, these data suggest that a loss of alpha4beta1 signaling via PTEN confers a migratory/invasive phenotype to fibrotic lung fibroblasts. Furthermore, this study implicates a loss of PTEN function in the pathophysiology of idiopathic pulmonary fibrosis.

Analysis of Variance↗
PubMed1999

Differential expression of integrin subunits in canine knee ligament fibroblasts.

A method for measuring the expression of integrin subunits on the cell surface of knee ligament fibroblasts was developed with use of flow cytometry and immunofluorescence. The ligament cells exhibited uniform size and density, as shown by forward and side-scatter properties, and showed minimal nonspecific binding of isotype control antibodies compared with unstained cells. All cells expressed the alpha5 integrin subunit; lateral collateral ligament cells stained with antibody to alpha5 showed a mean fluorescence intensity 2-fold higher than that of medial collateral ligament cells, 1.5-fold higher than that of posterior cruciate ligament cells, and 3-fold higher than that of anterior cruciate ligament cells, indicating a greater expression of the alpha5 subunit by lateral collateral ligament cells than by medial collateral, posterior cruciate, and anterior cruciate ligament cells. All cells expressed the beta1 integrin subunit; the expression by posterior cruciate ligament cells was 3-fold higher than that by medial collateral ligament or lateral collateral ligament cells and 5-fold higher than that by anterior cruciate ligament cells. All cells expressed the beta3 integrin subunit; the expression by posterior cruciate ligament cells was 1.5, 3, and 4.5-fold greater than that by lateral collateral, anterior cruciate, and medial collateral ligament cells, respectively. Our data suggest there is a differential expression of integrin subunits in knee ligament fibroblasts, and this in part may explain differences in their attachment and adherence to extracellular matrix molecules.

Animals↗