Expression of a Soluble Functional Form of the Integrin α4β1 in Mammalian Cells

The integrin α₄β₁(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human α₄ and β₁ subunits were fused to the genomic DNA encoding the human γ1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote α₄β₁ heterodimer formation. The soluble α₄β₁ Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent K_d for VCAM-1 binding were similar for both the soluble and native forms of α₄β₁. In addition, the integrin–Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble α₄β₁ should be generally applicable to a range of integrins.     

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.     

Expression of α4 Integrin mRNA and Protein and Fibronectin in the Early Chicken Embryo

α₄ integrins (α₄β₁ and α₄β₇) have been shown to mediate both cell-matrix adhesion to fibronectin and cell-cell adhesion to VCAM-1. These interactions have been suggested to contribute to hematopoiesis, lymphocyte homing, recruitment of inflammatory cells, neural crest cell migration and myogenesis. We report here the cloning of chicken α₄ cDNA and its use to define the patterns of expression of α₄ mRNA and protein in early chicken embryos (19–22 somite pairs), a stage at which neural crest cells can be examined at various points in their migration and somitic development and differentiation can also be observed at various stages. We observe widespread expression of both α₄ mRNA and protein, although the patterns of steady state expression do not conform precisely. Many neural crest cells contain significant levels of α₄ mRNA. Some neural crest cells express α₄ protein but its expression is transient and/or limited to a subset of these cells. α₄ is strongly expressed at both mRNA and protein levels by somitic cells and their derivatives in the sclerotome, dermatome and myotome and is also expressed in neural tube, otic placode, heart, gut endoderm and some other tissues. Comparison with the distributions of fibronectin shows that, although some α₄ expression occurs in locations consistent with a role in cell-matrix adhesion to fibronectin, α₄ is also expressed in other places where fibronectin is low or absent and a role for α₄ in cell-cell interactions appears more likely.     

BMP-7 Enhances Cell Migration and αvβ3 Integrin Expression via a c-Src-Dependent Pathway in Human Chondrosarcoma Cells

Bone morphogenic protein (BMP)-7 is a member of the transforming growth factor (TGF)-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvβ3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvβ3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.     

Transforming Growth Factor β1 (TGF-β1) Enhances Expression of Profibrotic Genes through a Novel Signaling Cascade and MicroRNAs in Renal Mesangial Cells

Increased expression of transforming growth factor-β1 (TGF-β1) in glomerular mesangial cells (MC) augments extracellular matrix accumulation and hypertrophy during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. MicroRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF-β1 to enhance the expression of profibrotic genes like collagen. In this study, we found a significant decrease in the expression of miR-130b in mouse MC treated with TGF-β1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-β receptor 1 (TGF-βR1) was identified as a target of miR-130b. Interestingly, the RIK promoter contains three NF-Y binding sites and was regulated by NF-YC. Furthermore, NF-YC expression was inhibited by TGF-β1, suggesting that a signaling cascade, involving TGF-β1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-βR1 to augment expression of TGF-β1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-βR1, as well as the profibrotic genes collagen type IV α 1 (Col4a1), Col12a1, CTGF, and PAI-1 were up-regulated not only in mouse MC treated with TGF-β1 but also in the glomeruli of streptozotocin-injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA- and host gene-mediated amplifying cascade initiated by TGF-β1 that results in the up-regulation of profibrotic factors, such as TGF-βR1 and collagens associated with the progression of DN.     

Physiological and Pathological Roles of α3β1 Integrin

α3β1 integrin has been considered to be a mysterious adhesion molecule due to the pleiotropy in its ligand-binding specificity. However, recent studies have identified laminin isoforms as high-affinity ligands for this integrin, and demonstrated that α3β1 integrin plays a number of essential roles in development and differentiation, mainly by mediating the establishment and maintenance of epithelial tissues. Furthermore, α3β1 integrin is also implicated in many other biological phenomena, including cell growth and apoptosis, angiogenesis and neural functions. This integrin receptor forms complexes with various other membrane proteins, such as the transmembrane-4 superfamily proteins (tetraspanins), cytoskeletal proteins and signaling molecules. Recently, lines of evidence have been reported showing that complex formation regulates integrin functions in cell adhesion and migration, signal transduction across cell membranes, and cytoskeletal organization. In addition to these roles in physiological processes, α3β1 integrin performs crucial functions in various pathological processes, especially in wound healing, tumor invasion and metastasis, and infection by pathogenic microorganisms.This revised version was published online in June 2005 with a corrected cover date.     

Expression of the c-Met Proto-Oncogene and Integrin α5β1 in Human Gastric Cardia Adenocarcinoma

The hepatocyte growth factor receptor c-Met, a receptor tyrosine kinase, and Integrin α5β1, one of the main ECM receptors of hepatocytes have been reported to play important roles in tumor growth by activating mitogenic signaling pathways. In human gastric cardia adenocarcinoma, however, expression of the c-Met and Integrin α5β1 have not been reported. Here we examined the mRNA levels and protein expressions of these two genes and their relationship in human normal gastric cardia mucosa and primary carcinomas. Quantitative real-time PCR was used to analyze the mRNA expression of both c-Met and Integrin α5β1. The relationship between c-Met and Integrin α5β1 expression and the histologic characteristics of tumors were studied. Western blot analysis was performed to investigate the presence of c-Met and Integrin α5β1. The expression patterns of c-Met and Integrin α5β1 in 45 frozen slides of cardia adenocarcinoma were identified by immunohistochemistry. Our results indicate that the expression of c-Met and of Integrin α5β1 was significantly associated with tumor differentiation, TNM, and metastasis via the lymphogenic route. A significant positive correlation was also found between c-Met and Integrin α5β1 mRNA expression, suggesting that expression of c-Met and Integrin α5β1 has mechanical significance in the early stages of human gastric cardia adenocarcinoma.     

High-Level Expression and Purification of a Recombinant Human α-1,3-Fucosyltransferase in Baculovirus-Infected Insect Cells

A human α-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 μg/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG–Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed α-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal α-2,3-linked sialic acid among various acceptors. The apparentK_mvalues of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.     

PAR-3 Knockdown Enhances Adhesion Rate of PANC-1 Cells via Increased Expression of Integrinαv and E-Cadherin

The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells'' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells'' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.     

Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of α1 Integrin Expression

Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7⁺hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7⁺hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7⁺hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1β1 integrin was strongly up-regulated with the attenuation of muscle-specific α7β1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7⁺hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.     

Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes

Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.     

Reduction of Tumorigenicity by α3 Integrin in a Rhabdomyosarcoma Cell Line

The expression levels of integrin adhesion receptors have often been correlated with neoplastic transformation and invasiveness. To investigate more definitively the role of the integrin VLA-3 (α³β₁) in tumor cell behavior, we transfected α³ subunit cDNA into human rhabdomyosarcoma (RD) cells. Transi ectants expressing high levels of α³β₁, on their cell surface displayed an altered morphology and decreased anchorage-dependent growth in vitro. Cells expressing α³ also displayed marked reduction in anchorage-independent growth in soft agar and in their ability to form tumors when injected subcutaneously into athymic nude mice. Thus, VLA-3 can repress the transformed phenotype of rhabdomyosarcoma tumor cells. Similar changes in morphology and growth characteristics were observed in cells expressing a chimeric molecule X3C4 in which the α³ cytoplasmic domain had been exchanged with that of the α⁴ integrin subunit. Therefore, α³ inhibitory effects in RD cells appear not to require specific signalling through the α³ cytoplasmic domain.     

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.     

ZEB1 Coordinately Regulates Laminin-332 and β4 Integrin Expression Altering the Invasive Phenotype of Prostate Cancer Cells

Metastasis involves the invasion of cancer cells across both the extracellular matrix and cellular barriers, and an evolving theme is that epithelial-to-mesenchymal transition (EMT) may mediate invasive cellular behavior. Previously, we isolated and analyzed a subpopulation of PC-3 prostate cancer cells, TEM4-18, and found that these cells both invaded an endothelial barrier more efficiently and exhibited enhanced metastatic colonization in vivo. Transendothelial migration of these cells depended on expression of ZEB1, a known regulator of EMT. Surprisingly, these cells were much less invasive than parental PC-3 cells in assays that involve matrix barriers. Here, we report that TEM4-18 cells express significantly reduced levels of two subunits of laminin-332 (β3 and γ2) and that exogenous laminin-332, or co-culture with laminin-332-expressing cells, rescues the in vitro invasion phenotype in these cells. Stable knockdown of ZEB1 in prostate cancer cells up-regulated LAMC2 and ITGB4 mRNA and protein and resulted in a concomitant increase in Transwell migration. Using chromatin immunoprecipitation (ChIP), we show that ZEB1 directly interacts with the promoters of LAMC2 and ITGB4. These results provide a novel molecular basis for reduced laminin-332 observed in clinical prostate cancer specimens and demonstrate a context-dependent role for EMT in invasive cellular behavior.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.     

Dual Control by Divalent Cations and Mitogenic Cytokines of α4β1 and α5β1 Integrin Avidity Expressed by Human Hemopoietic Cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function α4β1 and α5β1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by “inside-out” mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn²⁺, Mg²⁺ and Ca²⁺ for α4β1 and α5β1 activation by “inside-out” mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-β₁ integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of β₁ integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, α4β1 and α5β1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca²⁺, Mg²⁺ or Mn²⁺ were able to restore adhesive functions of α4β1 and α5β1 when activated by 8A2, only Mg²⁺ and Mn²⁺ were able to support activation of α5β1 and α5β1 by cytokines. Furthermore, high concentrations of Ca²⁺ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn²⁺ and cytokines but not by 8A2. On the contrary, in the presence of both Ca²⁺ and Mg²⁺, Mn²⁺ had an additive effect on the activation of α5β1 and α5β1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca²⁺, Mg²⁺ and Mn²⁺ may modulate in vivo α4β1 and α5β1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.