Α5 Integrin Subunit Research Articles

αVβ8 integrin is a Schwann cell receptor for fibrin

The interaction of Schwann cells with molecules in the extracellular environment following peripheral nerve injury is a critical aspect of nerve repair. A principal component of this material is fibrin, which derives from fibrinogen infiltrating into the nerve after the injury. This study was undertaken to identify cell surface receptor(s) that mediate the interaction of Schwann cells with fibrin. We found that adhesion of Schwann cells to fibrin could be effectively inhibited by low concentrations of RGD-containing peptides. Among RGD-sensitive integrins expressed by Schwann cell, αVβ8, but not α5β1, was found to bind to fibrin-Sepharose. In contrast, both of these integrins bound to fibronectin-Sepharose. We also found that αV, but not α5 or β1 integrin subunit, accumulated in focal contacts of Schwann cell plated on fibrin. Taken together, these results strongly suggest that αVβ8 integrin is a Schwann cell receptor for fibrin.

Collagen IV regulates Caco-2 migration and ERK activation via α1β1- and α2β1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.

Integrin αvβ3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin α2β1

The interplay between the collagen-binding integrin, α2β1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type α2β1 (α2β1A) or α2β1 with a Y783/795F mutation in the cytoplasmic tail of the β1 subunit (α2β1Amut) in the β1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the α1 and α2 integrin subunits and do not adhere to collagen even after transfection with β1A. Cells expressing α2β1Amut contracted three-dimensional collagen lattices less efficiently than those expressing α2β1A. PDGF-BB significantly stimulated lattice contraction by GD25-α2β1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130 Cas were phosphorylated when GD25-α2β1A cells, but not GD25-α2β1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130 Cas only in GD25-α2β1A cells. However, when cultured within collagen lattices, FAK and p130 Cas phosphorylation were stimulated in both α2β1A- and α2β1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-α2β1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-α2β1Amut cells were mediated by the αvβ3 integrin. Phosphorylation of p130 Cas, but not FAK, in GD25-α2β1Amut cells seeded in collagen lattices also depended on αvβ3. Our results show that PDGF-BB stimulation of fibroblast–collagen interactions is mediated by the αvβ3 integrin when β1 integrin function is impaired.

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit.

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.

Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar

Objective: The development of the periodontium involves the coordinated expression of numerous extracellular matrix (ECM) macromolecules and their receptors (integrins). The aim of this study was to determine the expression of selected hard and soft tissue matrix molecules and the integrin α5β1 in the periodontal tissues, during cementogenesis in the rat molar. Methods: Using immunohistochemical methods, the distribution of the extracellular matrix proteins, fibronectin, tenascin, and bone sialoprotein (BSP), as well as the integrin subunits α5 and β1 were studied in rats aged 3, 5 and 8 weeks. Results: Fibronectin was widely distributed in the gingival epithelium, gingival connective tissue and in the periodontal ligament. Tenascin expression was less marked compared with fibronectin, but was more distinctly associated with cells and peri-cellular areas of the epithelial-connective tissue interface, the gingiva and within the periodontal ligament. The fibronectin-receptor α5β1 integrins were expressed by epithelial cells, periodontal ligament cells and gingival fibroblasts. A notable finding was the increased staining intensity of fibronectin, tenascin and α5β1 integrin in all 5-week old molar sections in the periodontal ligament matrix and cells, apical to the cemento-enamel junction (CEJ) along the alveolar crest (AC) ridge height. Bone sialoprotein was distinctly associated with the hard tissues of the periodontium as acellular cementum and alveolar bone matrix expressed bone sialoprotein throughout all sections, in all age groups. Conclusions: In conclusion, this study has demonstrated the selective distribution of several hard and soft tissue matrix molecules during periodontogenesis. The results highlight the complex nature of interactions of various proteins and molecules during development. The interactions between these molecules and their specific roles in development and regeneration await further investigation.

Role of multiple β1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3

ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the α6β1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the α9β1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express α6β1 and α9β1 integrins at their surfaces. Antibodies to the α6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human α9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes α4β1 and α9β1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the α9 integrin subunit in mouse eggs. In contrast to our ability to detect α6β1, we were unable to convincingly detect α9β1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its ∼IC 50 for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect α9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the α6 and α7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the α4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.

Partial characterization of cell-type X collagen interactions.

Type X collagen is a short-chain non-fibrillar collagen that is deposited exclusively at sites of new bone formation. Although this collagen has been implicated in chondrocyte hypertrophy and endochondral ossification, its precise function remains unclear. One possible function could be to regulate the processes of chondrocyte hypertrophy through direct cell-type X collagen interactions. Adhesions of embryonic chick chondrocytes, and cell lines with known expression of collagen-binding integrins (MG63 and HOS), were assayed on chick type X collagen substrates, including the native, heat-denatured and pepsin-digested collagen, and the isolated C-terminal non-collagenous (NC1) domain. Type X collagen supported the greatest level of adhesion for all cell types tested. The involvement of the alpha2beta1 integrin in type X collagen-cell interaction was demonstrated by adhesion studies in the presence of Mg(2+) and Ca(2+) ions and integrin-function-blocking antibodies. Cells expressing alpha2beta1 integrin (chick chondrocytes and MG63 cells) also adhered to heat-denatured type X collagen and the isolated NC1 domain; however, removal of the non-collagenous domains by limited pepsinization of type X collagen resulted in very low levels of adhesion. Both focal contacts and actin stress-fibre formation were apparent in cells plated on type X collagen. The presence of alpha2 and beta1 integrin subunits in isolated chondrocytes and epiphyseal cartilage was also confirmed by immunolocalization. Our results demonstrate, for the first time, that type X collagen is capable of interacting directly with chondrocytes and other cells, primarily via alpha2beta1 integrin. These findings are atypical from the fibrillar collagen-cell interactions via collagen binding integrins in that: (1) the triple-helical conformation is not strictly required for cell adhesion; (2) the NC1 domain is also involved in the adhesion of alpha2beta1-expressing cells. These data form the basis for further studies into the mechanism and biological significance of type X collagen deposition in the growth plate.

Immunohistochemical expression of integrins and extracellular matrix proteins in non-small cell lung cancer: correlation with lymph node metastasis

Purpose: For patients with non-small cell lung cancer (NSCLC), the biggest threat to survival is metastasis. During metastatic cascade, tumor cells interact with extracellular matrix (ECM) through certain adhesion molecules such as integrins. The aim of this study was to analyze the distribution of the main integrins and ECM in a series of patients with NSCLC to assess their distribution and correlate with lymph node (LN) metastasis of NSCLC. Methods: Formalin-fixed and paraffin-embedded tissues of NSCLC with ( n=45) or without ( n=23) regional LN metastasis were obtained form 68 surgically treated patients. The expression of fibronectin, collagen type IV, tenascin and the integrin subunits (α2, α3, α4, α5 and β1) was studied by immunohistochemistry. Chi-square and Fisher's exact tests were used to compare groups and parameters. Results: Extensive (>50% of section area) fibronectin and collagen type IV staining were seen in 22 and 55% of tumors, respectively, with focal areas of immunoreactivity seen in another 75 and 38% of tumors, respectively. Tenascin staining showed just focal areas of immunoreactivity in 21% of tumors. Interstitial collagen matrices were more frequently lost in LN metastasis ( P=0.007). Integrins α2, α5 and β1 expressions were present in 9, 12 and 26% of tumors, respectively. The expression of integrins α5 and β1 was significantly associated with LN metastasis ( P=0.04 and 0.005, respectively). Conclusions: Increased expression of integrins α5 and β1, and lost expression of collagen matrices significantly correlated with LN metastasis of NSCLC. These findings suggested that enhanced expression of integrins and disrupted collagen stroma in NSCLC might promote tumor cell survival and invasiveness.

Differential regulation of cell adhesive functions by integrin alpha subunit cytoplasmic tails in vivo.

Cell adhesion to fibronectin (FN) is crucial for early vertebrate morphogenesis. In Xenopus gastrulae, several distinct integrin-dependent adhesive behaviors can be identified: adhesion of cells to FN, assembly of FN fibrils, and initiation of cell spreading and migration in response to mesoderm inducing signals. We have taken a chimeric integrin approach to investigate the role of the integrin alpha cytoplasmic tail in the specification of these developmentally significant adhesive functions. Cytoplasmic tail-deleted alpha4 constructs and alpha4-ectodomain/alpha-cytoplasmic tail chimeras were generated and expressed in whole embryos. Normal gastrula cells lack integrin alpha4 and, correspondingly, are unable to adhere to the alpha4 ligand, the V-region of FN. The ability of alpha4 constructs to promote adhesive behaviors was established by placing tissue explants or dissociated cells on an FN V-region fusion protein that lacks the RGD (Arg-Gly-Asp)/synergy sites or treating whole embryos with antibodies that block endogenous integrin-FN interactions. We found that each alpha4 cytoplasmic domain deletion mutant and alpha-tail chimera examined could support cell attachment; however, activin induction-dependent cell spreading, mesoderm cell and explant motility, and the ability to assemble FN matrix on the blastocoel roof varied with specific alpha subunit tail sequences. These data suggest that alpha cytoplasmic tail signaling and changes in integrin activation state can regulate a variety of developmentally significant adhesive behaviors in both space and time.

The function of Xenopus germ cell nuclear factor (xGCNF) in morphogenetic movements during neurulation

The germ cell nuclear factor (GCNF, NR6A1) is a nuclear orphan receptor first described in the mouse testis and subsequently identified as an essential transcription factor in vertebrate embryogenesis. Here, we analyze the phenotype of Xenopus embryos after depletion of embryonic GCNF (xEmGCNF) protein using a specific morpholino antisense oligonucleotide. Morphological defects after xEmGCNF knockdown became obvious from neurulation onward. Among the abnormalities observed, defective formation of the neural tube and a short and curved main body axis were the most remarkable traits. Histological analysis, lineage tracing of injected blastomeres, and Keller sandwich explants revealed that xEmGCNF function is required for different patterns of cell intercalation during neurulation and consequently for the sequence of morphogenetic movements leading to formation of the neural tube. Further characterization of the phenotype at the molecular level showed an abnormal distribution of the extracellular matrix protein fibronectin and a reduction in the expression level of the integrin subunits α5 and α6, the limiting components of the laminin and fibronectin receptors, respectively. We propose integrin-mediated cell–matrix interaction as a process that requires xEmGCNF function and provides, in concert with cadherins-mediated cell–cell interactions, a molecular basis for morphogenetic cell movements during neurulation.

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of α1, α5, α6, and β4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on α1, α5, α6, and β4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the α2, α3, αv, and β1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway.

PTH-related protein modulates PC-3 prostate cancer cell adhesion and integrin subunit profile

Parathyroid hormone-related protein (PTHrP), which has been localized in prostate cancer tissue and cell lines, plays a role in the development of bone metastases, a frequent complication in prostate cancer patients. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. The present experiments examined the ability of PTHrP to influence adhesion of the human prostate cancer cell line PC-3 to several ECM proteins found in normal tissues. Clonal PC-3 cells induced to overexpress PTHrP by stable transfection with PTHrP complementary DNA showed significantly higher adhesion to collagen type 1, fibronectin, and laminin than control (empty vector-transfected) cells. PTHrP-overexpressing cells also exhibited higher expression of the α1, α5, α6, and β4 integrin subunits. These results suggest that PTHrP may play a role in prostate tumor invasion and metastasis by influencing cell adhesion to the ECM via upregulation of specific integrin subunits.

The α 2β 1 and α 3β 1 integrins do not mediate attachment of endometrial cells to peritoneal mesothelium

Objective: To evaluate the possible role of mesothelial α 2β 1 and α 3β 1 integrins in the attachment of endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs). Design: In vitro study. Setting: University medical center. Patient(s): Women of reproductive age (n = 26). Main Outcome Measure(s): Mesothelial cells were grown on collagen IV. Endometrial stromal cells and EECs were plated on mesothelial cells for 1 hour. Before plating, mesothelial cells or endometrial cells were incubated with antibodies to α2, α3, and β1 integrin subunits. The effect of these antibodies on ESC and EEC binding to collagen IV and collagen I was also examined. The expression of collagen I, collagen IV, fibronectin, and laminin by cultured ESCs and EECs was examined. Result(s): The anti-integrin antibodies had no effect on endometrial binding to mesothelium. The β1 integrin antibody decreased binding of ESCs and EECs to the collagen matrices. In culture, ESCs and EECs expressed collagen I, collagen IV, fibronectin, and laminin to varying degrees. Conclusion(s): The initial adhesion of ESCs and EECs to mesothelium is not mediated by β1 integrins. In contrast, ESC and EEC attachment to collagen IV and collagen I, which are present in the submesothelial extracellular matrix, is mediated by β1 integrins.

Proximal Tubular Epithelial Cell Integrins Respond to High Glucose by Altered Cell-Matrix Interactions and Differentially Regulate Matrixin Expression

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mm glucose underwent a reduction of the expression of α3, β1, αvβ3, and α5 integrin subunits, with a concomitant increase of the α2 subunit, compared with cells grown in 5 mm glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mm glucose used mainly αvβ3 and α5β1 integrins to adhere to colIV, whereas in 25 mm glucose they additionally used α2β1. In the case of laminin, a β1-mediated adhesion was observed when HK-2 cells were in 5 mm glucose, whereas in 25 mm glucose, α2β1 and αvβ3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of α3β1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of αvβ3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.

Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells.

Primary and secondary prevention trials suggest that statins possess favorable effects independent of cholesterol reduction. We investigated whether statin therapy may also accelerate reendothelialization after carotid balloon injury. Simvastatin treatment in 34 male Sprague-Dawley rats accelerated reendothelialization of the balloon-injured arterial segments (reendothelialized area at 2 weeks, 12.3+/-1.8 versus 5.4+/-1.1 mm2, P< 0.01) and resulted in a dose-dependent (0.2 or 1 mg/kg IP) significant reduction in neointimal thickening at 2, 3, and 4 weeks compared with saline-injected controls (n=18). To elucidate the mechanism, we investigated the contribution of bone marrow-derived endothelial progenitor cells (EPCs) by bone marrow transplantation from Tie2/lacZ mice to background mice or nude rats. X-gal staining of mouse carotid artery specimens revealed a 2.9-fold increase in the number of beta-gal-positive cells per square millimeter appearing on the carotid artery luminal surface at 2 weeks, and double-fluorescence immunohistochemistry disclosed a significant 5-fold increase in the number of double-positive cells (beta-gal, isolectin B4) on the luminal surface in carotid arteries of statin-treated nude rats (20+/-3 versus 4+/-1 cells/mm surface length, P<0.005). Statins increased circulating rat EPCs (2.4-fold at 2 weeks and 2.5-fold at 4 weeks, P<0.001) and induced adhesiveness of cultured human EPCs by upregulation of the integrin subunits alpha5, beta1, alpha(v), and beta5 of human EPCs as shown by reverse transcription-polymerase chain reaction and fluorescence-activated cell sorting. These findings establish additional mechanisms by which statins may specifically preempt disordered vascular wall pathology and constitute physiological evidence that EPC mobilization represents a functionally relevant consequence of statin therapy.

Collagen suppresses the proliferative phenotype of allylamine-injured vascular smooth muscle cells

Repeated cycles of oxidative injury by allylamine induce proliferative rat vascular smooth muscle cell (vSMC) phenotypes characterized by enhanced secretion of osteopontin (OPN). The present study was designed to evaluate the role of extracellular matrix (ECM) interactions in the induction of proliferative phenotypes in this model of oxidant injury. Because OPN is involved in ECM/integrin signaling, and may participate in proliferative control, the proliferation profiles of control and allylamine vSMCs seeded on different matrices were compared. Allylamine cells exhibited a proliferative advantage over controls when seeded on plastic, Pronectin, or fibronectin, but not type I collagen. Addition of GRGDS peptide selectively enhanced [ 3H]-thymidine incorporation in allylamine vSMCs, while anti-OPN antibodies nullified their proliferative advantage. Allylamine cells exhibited altered expression of α1, α5 and β3 integrin subunits and enhanced downstream integrin-coupled increases in focal adhesion kinase, AP-1 and NF-κB binding activity. Inhibition of NF-κB by pyrrolidine dithiocarbamate selectively compromised proliferation of allylamine vSMCs, while seeding on a non-permissive collagen matrix ablated enhancement of NF-κB inducibility. These results implicate ECM interactions in the deregulation of vSMC proliferation following repeated cycles of oxidative chemical injury.

Laminins and TGF-β maintain cell polarity and functionality of human gastric glandular epithelium

The human gastric glandular epithelium produces a gastric lipase enzyme (HGL) that plays an important role in digestion of dietary triglycerides. To assess the involvement of extracellular matrix components and transforming growth factor-beta1 (TGF-beta1) in the regulation of this enzymic function, normal gastric epithelial cells were cultured on collagen type I, Matrigel, and laminins (LN)-1 and -2 with or without TGF-beta1. Epithelial morphology and HGL expression were evaluated using microscopy techniques, enzymic assays, Western blot, Northern hybridization, and RT-PCR. A correlation was observed between the cell polarity status and the level of HGL expression. TGF-beta1 alone or individual matrix components stimulated cell spreading and caused a downfall of HGL activity and mRNA. By contrast, Matrigel preserved the morphological features of differentiated epithelial cells and maintained HGL expression. The combination of LNs with TGF-beta1 (two constituents of Matrigel) exerted similar beneficial effects on epithelial cell polarity and evoked a 10-fold increase of HGL levels that was blunted by a neutralizing antibody against the alpha(2)-integrin subunit and by mitogen-activated protein kinase (MAPK) inhibitors PD-98059 (p42/p44) or SB-203580 (p38). This investigation demonstrates for the first time that a powerful synergism between a growth factor and basement membrane LNs positively influences cell polarity and functionality of the human gastric glandular epithelium through an activation of the alpha(2)beta(1)-integrin and effectors of two MAPK pathways.

Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.

Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the alpha5 integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on alpha(v) or beta3 integrin subunits. In mouse embryos lacking alpha5 integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in alpha5-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for alpha5 integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking alpha5 integrin or FN compared with wild-type EBs or EBs lacking alpha(v) or beta3 integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for alpha5 and FN in early blood vessel development not dependent on embryo nutrition or alpha(v) or beta3 integrin subunits. Thus, successful early vasculogenesis and angiogenesis require alpha5-FN interactions.

Association of the tetraspanin CD151 with the laminin-binding integrins alpha3beta1, alpha6beta1, alpha6beta4 and alpha7beta1 in cells in culture and in vivo.

CD151 is a cell surface protein that belongs to the tetraspanin superfamily. It forms complexes with the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha6beta4 and is codistributed with these integrins in many tissues at sites of cell-matrix interactions. In this study we show that CD151 can also form stable complexes with the laminin-binding integrin alpha7beta1. The strength of this interaction is comparable to that between CD151 and alpha3beta1. Complexes of alpha3beta1, alpha6beta1 and alpha7beta1 with CD151 are equally well formed with all splice variants of the alpha3, alpha6 and alpha7 subunits, and complex formation is not affected by mutations that prevent the cleavage of the integrin alpha6 subunit. Like the expression of alpha3beta1 and alpha6beta1, expression of alpha7beta1 in K562 cells results in increased levels of CD151 at its surface. Two non-integrin laminin receptors, dystroglycan and the polypeptide on which the Lutheran blood group antigens are expressed, are also often colocalized with CD151, but no association with CD151-alpha3beta1 complexes was found with biochemical analysis. The anti-CD151 antibody TS151R detects an epitope at a site at which CD151 interacts with integrins, and therefore it cannot react with CD151 when it is bound to an integrin. Comparison of the straining patterns produced by TS151R with that by of an anti-CD151 antibody recognizing an epitope outside the binding site (P48) revealed that most tissues expressing one or more laminin-binding integrins reacted with P48 but not with TS151R. However, smooth muscle cells that express alpha7beta1 and renal tubular epithelial cells that express alpha6beta1 were stained equally well by TS151R and P48. These results suggest that the interactions between CD151 and laminin-binding integrins are subject to cell-type-specific regulation.

An Alternative Processing of Integrin αv Subunit in Tumor Cells by Membrane Type-1 Matrix Metalloproteinase

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alpha(v)beta(3) integrin are both essential to cell invasion. Maturation of integrin pro-alpha(v)chain (pro-alpha(v)) involves its cleavage by proprotein convertases (PC) to form the disulfide-bonded 125-kDa heavy and 25-kDa light alpha chains. Our report presents evidence of an alternative pathway of pro-alpha(v) processing involving MT1-MMP. In breast carcinoma MCF7 cells deficient in MT1-MMP, pro-alpha(v) is processed by a conventional furin-like PC, and the mature alpha(v) integrin subunit is represented by the 125-kDa heavy chain and the 25-kDa light chain commencing from the N-terminal Asp(891). In contrast, in cells co-expressing alpha(v)beta(3) and MT1-MMP, MT1-MMP functions as an integrin convertase. MT1-MMP specifically cleaves pro-alpha(v), generating a 115-kDa heavy chain with the truncated C terminus and a 25-kDa light chain commencing from the N-terminal Leu(892). PC-cleavable alpha(3) and alpha(5) but not the PC-resistant alpha(2) integrin subunit are also susceptible to MT1-MMP cleavage. These novel mechanisms involved in the processing of integrin alpha subunits underscore the significance and complexity of interactions between MT1-MMP and adhesion receptors and suggest that regulation of integrin functionality may be an important role of MT1-MMP in migrating tumor cells.