Additive effect of recombinant Mycobacterium tuberculosis ESAT-6 protein and ESAT-6/CFP-10 fusion protein in adhesion of macrophages through fibronectin receptors.

BACKGROUND/PURPOSE: Tuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion. METHODS: Differentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5ß1 and α4ß1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy. RESULTS: Our data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4ß1 integrin. An increased expression level of α4ß1 integrin in comparison with α5ß1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells. CONCLUSION: Increased expression level of α4ß1 in differentiated THP-1 cells could suggest the important role of α4ß1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens.

Human embryonic stem cell-derived cardiomyocytes migrate in response to gradients of fibronectin and Wnt5a.

An improved understanding of the factors that regulate the migration of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) would provide new insights into human heart development and suggest novel strategies to improve their electromechanical integration after intracardiac transplantation. Since nothing has been reported as to the factors controlling hESC-CM migration, we hypothesized that hESC-CMs would migrate in response to the extracellular matrix and soluble signaling molecules previously implicated in heart morphogenesis. To test this, we screened candidate factors by transwell assay for effects on hESC-CM motility, followed by validation via live-cell imaging and/or gap-closure assays. Fibronectin (FN) elicited a haptotactic response from hESC-CMs, with cells seeded on a steep FN gradient showing nearly a fivefold greater migratory activity than cells on uniform FN. Studies with neutralizing antibodies indicated that adhesion and migration on FN are mediated by integrins α-5 and α-V. Next, we screened 10 soluble candidate factors by transwell assay and found that the noncanonical Wnt, Wnt5a, elicited an approximately twofold increase in migration over controls. This effect was confirmed using the gap-closure assay, in which Wnt5a-treated hESC-CMs showed approximately twofold greater closure than untreated cells. Studies with microfluidic-generated Wnt5a gradients showed that this factor was chemoattractive as well as chemokinetic, and Wnt5a-mediated responses were inhibited by the Frizzled-1/2 receptor antagonist, UM206. In summary, hESC-CMs show robust promigratory responses to FN and Wnt5a, findings that have implications on both cardiac development and cell-based therapies.

Cell-Matrix interactions, the role of fibronectin and integrins. A survey.

In this review, we present several aspects of cell-matrix interactions, especially the role of fibronectin and integrins in the mediation of these interactions. As this field of investigations literally exploded over the last decades, we had to limit this review to some aspects of this field. We cited experiments giving details on the modifications of fibronectin molecules during their interactions with cells as well as on recent progress of the molecular mechanisms of fibronectin-integrin interactions. We insisted on the molecular details which were shown to play a role in the bi-directional signals "sent" by cells to the surrounding matrix (inside-out and outside-in). A number of recent publications confirmed the physiopathological importance of these messages both for the normal function of tissues as well as for the understanding of their pathological modifications. We insist also on the importance of fibronectin-fragments during some pathologies.

Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development.

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

[Effect of epidermal growth factor signal pathway on integrin alpha5 beta1 in prostate cancer cell line DU145].

OBJECTIVE: To investigate the molecular mechanism of epidermal growth factor (EGF) signal pathway on the expression of integrin alpha5 beta1 in prostate cancer cell line DU145. METHODS: Using flow-cytometry, the effects of EGF and the mitogen-activated protein kinase (MAPK) signal pathway inhibitor PD98059 on the expression of integrin alpha5 and beta1 subunits on DU145 cell surface were analyzed. RT-PCR and Western blot methods were used to examined the expression of mRNA and cell total protein of integrin alpha5 and beta1 subunits. And the metastatic phenotypes in DU145 cell were investigated. RESULTS: The expression levels of integrin alpha5 beta1, which was the receptor for fibronectin, were changed. EGF up-regulated the protein and mRNA expression of beta1 subunit on DU145 cell surface, 231% and 248% (P < 0.01) compared to the control respectively, and it could significantly promote the ability of DU145 cell adhesion to fibronectin and migration. However PD98058, which was the inhibitor of MAPK signal pathway, down-regulated the protein and mRNA expression of beta1 subunit, 60% and 63% (P < 0.01) compared to the control respectively, and it had the contrary function on the adhesion and migration ability of DU145 cell. But both had no effect the expression of alpha5 subunit. CONCLUSIONS: EGF might promote the metastatic ability mainly by up-regulating the expression of beta1 subunit by activating MAPK signal pathway in DU145 cells. Their regulation effects are on the mRNA transcriptional level.

Impaired migration of NOD mouse thymocytes: a fibronectin receptor-related defect.

We previously showed intrathymic alterations in non-obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)-4 and VLA-5] and laminin (VLA-6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA-5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double-negative cells, and more pronounced in mature CD4(+) and CD8(+) thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin-containing transwell chambers. In situ, intra-perivascular space thymocytes were VLA-5-negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA-5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.

Fibronectin receptor reduction in skin and fibroblasts of patients with Ullrich's disease.

Ullrich's disease is a congenital muscular dystrophy characterized clinically by generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recent studies have demonstrated that collagen VI is deficient in the muscles of patients with Ullrich's disease, and some cases result from recessive mutations of the collagen VIalpha2 gene (COL6A2). Fibronectin is one of the main components of the extracellular matrix (ECM) and associates with a variety of other matrix molecules including collagen. The behavior of fibronectin on cells is mediated by fibronectin receptors, members of the integrin family. We studied the expression of fibronectin receptors and fibronectin in patients with Ullrich's disease, and found a marked reduction of fibronectin receptors in the ECM of skin and cultured fibroblasts of these patients. These results suggest that collagen VI deficiency may lead to the reduction of fibronectin receptors and that an abnormality of cell adhesion may be involved in the pathogenesis of Ullrich's disease.

Interaction of fibronectin with human colon adenocarcinoma cells: effect on the in vivo tumorigenic capacity.

Fibronectin (FN) modulates the behavior of the poorly differentiated, human colon adenocarcinoma-derived BCS-TC2 cells by promoting adhesion through the alpha(5)beta(1)-integrin, as this effect is blocked by anti-alpha(5) and beta(1 )chain antibodies. BCS-TC2 cells are not tumorigenic in vivo, but are able to form tumors when coinjected with FN in nude mice. From these tumors, a tumorigenic cell subline (BCS-TC2.FN) was established. In vivo passaging of BCS-TC2.FN cells in the absence of FN allowed the selection of another tumorigenic subline (BCS-TC2.FN2). The new sublines are characterized by: (1) increased differentiation, (2) slightly higher adhesion to FN, and (3) a higher uptake of [(3)H]thymidine, less dependent on the presence of serum or FN. No significant modifications in alpha(5)-chain surface levels were observed in the tumor-derived sublines, suggesting that the amount of alpha(5)beta(1)-integrin is not related to tumorigenicity. Within the heterogeneous parental cells, FN seems to favor the selection of a cell subpopulation that presents phenotypic and genotypic alterations that are stably maintained throughout in vitro culture and in vivo passaging. These cell lines constitute a model system that may help to extend our knowledge on the events underlying tumor progression and malignancy of colorectal cancer, and the influence of extracellular matrix components and their receptors in these processes.

Overexpression of Betaig-h3 gene downregulates integrin alpha5beta1 and suppresses tumorigenicity in radiation-induced tumorigenic human bronchial epithelial cells.

Interaction between cell and extracellular matrix plays a crucial role in tumour invasion and metastasis. Using an immortalised human bronchial epithelial (BEP2D) cell model, the study here shows that expression of Betaig-h3 gene, which encodes a secreted adhesion molecule induced by transforming growth factor-beta, is markedly decreased in several independently generated, radiation-induced tumour cell lines (TL1-TL5) relative to parental BEP2D cells. Transfection of Betaig-h3 gene into tumour cells resulted in a significant reduction in tumour growth. While integrin receptor alpha5beta1 was overexpressed in tumour cells, its expression was corrected to the level found in control BEP2D cells after Betaig-h3 transfection. These data suggest that Betaig-h3 gene is involved in tumour progression by regulating integrin receptor alpha5beta1. The findings provide strong evidence that the Betaig-h3 gene has tumour suppressor function in human BEP2D cell model and suggest a potential target for interventional therapy.

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.

Insulin-like growth factor 2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis.

Hemangiomas are benign tumors of the vascular endothelium and are the most common tumors of infancy. These tumors are characterized by an initial phase of rapid proliferation, which is followed, in most cases, by spontaneous involution. Although most lesions resolve without complication, there are some cases in which hemangiomas can be life threatening when occurring near a vital structure. Treatment for these aggressive tumors represents an unmet clinical need. In addition, this characteristic progression of hemangiomas through distinct phases provides a unique opportunity for studying endothelial cell biology and angiogenesis. Using DNA microarrays representing approximately 10,000 human genes, we identified insulin-like growth factor 2 (IGF-2) as a potentially important regulator of hemangioma growth. IGF-2 was highly expressed during the proliferative phase and substantially decreased during involution. This finding was confirmed at the message level by quantitative reverse transcription-PCR and at the protein level by immunohistochemistry. IGF-2 protein was localized primarily to tumor vessels or vascular channels. Using a human hemangioma explant model, we show that IGF-2 promotes sprouting from intact hemangioma tissue. In addition, several angiogenesis-related factors, including integrins alpha(v)beta3 and alpha5beta1, are present in proliferating hemangiomas. During the involuting phase, an increase in several IFN-induced genes was observed. These studies identify potential regulators of hemangioma growth and involution and provide a foundation on which to build further mechanistic investigations into angiogenesis, using hemangiomas as a model.

Adhesion of a chondrocytic cell line (USAC) to fibronectin and its regulation by proteoglycan.

BACKGROUND: Chondrocytes produce various extracellular matrices during chondrogenesis. Fibronectin and proteoglycan are major extracellular matrix proteins in cartilage tissue, but the interactions between them are not clear. METHODS: Recently, we succeeded in establishing a cell line (USAC) with phenotypes of chondrocytes from a human osteogenic sarcoma of the mandible. Using this cell line, cell adhesion to fibronectin, the effect of proteoglycan on the cell adhesion and expression of integrin alpha5beta1 were investigated. RESULTS: Cells immediately adhered to fibronectin and then spread. Proteoglycan inhibited cell adhesion to fibronectin dose-dependently, whereas collagen did not. The expression of both mRNAs of alpha5 and beta1 subunits was detected 12 h after treatment with proteoglycan, but the expression of beta1 subunit mRNA had diminished by 24 h after treatment. CONCLUSIONS: These findings suggest that proteoglycan might modulate signal transduction from fibronectin by decreasing the expression of alpha5beta1 integrin.

Integrin alphavbeta1 is a receptor for foot-and-mouth disease virus.

Infection by field strains of Foot-and-mouth disease virus (FMDV) is initiated by binding to certain species of arginine-glycine-aspartic acid (RGD)-dependent integrin including alphavbeta3 and the epithelial integrin alphavbeta6. In this report we show that the integrin alphavbeta1, when expressed as a human/hamster heterodimer on transfected CHOB2 cells, is a receptor for FMDV. Virus binding and infection mediated by alphavbeta1 was inefficient in the presence of physiological concentrations of calcium and magnesium but were significantly enhanced by reagents that activate the integrin and promote ligand binding. The ability of chimeric alpha5/alphav integrin subunits, in association with the beta1 chain, to bind FMDV and mediate infection matched the ligand binding specificity of alphavbeta1, not alpha5beta1, thus providing further evidence for the receptor role of alphavbeta1. In addition, data are presented suggesting that amino acid residues near the RGD motif may be important for differentiating between the binding specificities of alphavbeta1 and alphavbeta6.

Tumorigenicity of the mixed spindle-epithelioid SP6.5 and epithelioid TP17 uveal melanoma cell lines is differentially related to alpha5beta1 integrin expression.

PURPOSE: It has been suggested that the epithelioid morphology and high aggressiveness that is typical of the uveal melanoma cell line TP17 is dependent on the loss of alpha5beta1 integrin expression at the cell surface. The purpose of the current study was to test this hypothesis in the TP17 cell line and investigate the role this integrin may play in the tumorigenicity of the SP6.5 cells, a mixed spindle-epithelioid culture-type human uveal melanoma that shows tumorigenic properties clearly distinct from that of TP17 cells. METHODS: Expression of the alpha5 integrin subunit was restored in the alpha5-TP17 cell line by stably transfecting the cells with a recombinant plasmid encoding the integrin subunit. Flow cytometry and adhesion assays on fibronectin (FN)-coated culture plates were used to monitor alpha5 expression in the cells. The effect of alpha5 expression on both tumorigenicity and cell proliferation was evaluated in vivo in nude mice. In vitro growth properties of the alpha5(+) TP17 cells was evaluated by cell counting and compared with that of the alpha5 parental TP17 cell line. The influence exerted by the alpha5 integrin subunit on the tumorigenic and proliferative properties of the SP6.5 cells was evaluated in vivo in nude mice by exposing the cells to increasing doses of a blocking antibody directed against the alpha5-subunit before subcutaneous injection, and compared with the results obtained with untreated SP6.5 cells. RESULTS: Expression of the alpha5 integrin subunit in the alpha5-TP17 cells was successfully achieved, as evidenced by both flow cytometry and adhesion assays on FN-coated culture plates. Restoring expression of alpha5 in TP17 cells enhanced epithelioid cell morphology and increased the growth properties of this cell line in vivo. The ability of the SP6.5 cells to yield subcutaneous tumors was found to be concentration dependent and was reduced in a dose-dependent manner when the cells were exposed to the anti-alpha5 blocking antibody. CONCLUSIONS: Restoring expression of alpha5 in the alpha5-negative TP17 uveal melanoma cell line influenced the proliferative properties of these cells but did not alter its tumorigenic potential. In contrast, the ability of the SP6.5 cells to yield tumors in vivo in nude mice appeared to be related to expression of this integrin.

All-trans-retinoic acid increased the expression of integrin alpha5beta1 and induced "anoikis" in SMMC-7721 hepatocarcinoma cell.

It is extensively shown that integrin can regulate various cellular functions, including apoptosis, probably by contributing to signal transduction processes through interaction with extracellular matrix (ECM) proteins. In the present study, DNA flow cytometric analysis demonstrated that SMMC-7721 hepatocarcinoma cells treated with 80 microM all-trans-retinoic acid (atRA) showed an increased expression of the integrin alpha5beta1, which was associated with the growth inhibition of the cells. We found that atRA treated cells showed obvious apoptosis. Then, it was postulated that the enhanced content of integrin alpha5beta1 in the absence of ligation with fibronectin (Fn) would stop transducing survival signals, and lead to decreased cell growth and apoptosis. To elucidate this hypothesis, we cultured the atRA treated cell in L-poly-lysine-coated and Fn-coated flask, respectively. The results indicated that Fn binding prevented the cells from apoptosis induced by atRA, in contrast to L-poly-lysine binding. When the transfectant with enhanced expression of integrin alpha5beta1 at the same level of atRA treated cell was cultured in L-poly-lysine-coated flask, apoptosis was triggered. However, apoptotic cell was not detected when those cells were cultured in Fn-coated flask. Meanwhile, culturing the transfectant in the antibody (against integrin alpha5 subunit)-coated flask induced 18.33% of the cells into apoptosis, which is far more than the control group. Our study suggests that increased expression of integrin alpha5beta1 on the surface of SMMC 7721 hepatocarcinoma cell treated by atRA, when unbound to Fn, would stop transducing survival signals to lead to "anoikis", and can be reverted by the interaction of integrin alpha5beta1 with Fn.

Integrin-specific activation of Rac controls progression through the G(1) phase of the cell cycle.

Adhesion to fibronectin through the alpha5beta1 integrin enables endothelial cells to proliferate in response to growth factors, whereas adhesion to laminin through alpha2beta1 results in growth arrest under the same conditions. On laminin, endothelial cells fail to translate Cyclin D1 mRNA and activate CDK4 and CDK6. Activated Rac, but not MEK1, PI-3K, or Akt, rescues biosynthesis of cyclin D1 and progression through the G(1) phase. Conversely, dominant negative Rac prevents these events on fibronectin. Mitogens promote activation of Rac on fibronectin but not laminin. This process is mediated by SOS and PI-3K and requires coordinate upstream signals through Shc and FAK. These results indicate that Rac is a crucial mediator of the integrin-specific control of cell cycle in endothelial cells.

Cell adhesion-mediated drug resistance (CAM-DR) protects the K562 chronic myelogenous leukemia cell line from apoptosis induced by BCR/ABL inhibition, cytotoxic drugs, and gamma-irradiation.

Integrin-mediated cellular adhesion to extracellular matrix (ECM) components is an important determinant of chemotherapeutic response of human myeloma cells. Here, we demonstrate that when K562 chronic myelogenous leukemia (CML) cells are adhered to fibronectin (FN), they become resistant to apoptosis induced by the BCR/ABL inhibitors AG957 and STI-571, as well as DNA damaging agents and gamma-irradiation. This phenomenon, termed cell adhesion-mediated drug resistance (CAM-DR), was induced by adhesion through the alpha5beta1 (VLA-5) integrin. Phosphotyrosine analysis demonstrates that anti-apoptotic signaling through integrins in K562 cells is independent of the tyrosine kinases activated by BCR/ABL, with the possible exception of an unknown 80 kDa protein. Cytoprotection of FN-adhered CML cells indicates that tumor-ECM interactions may be critical for the emergence of drug-resistant tumor populations and treatment failure in this disease. Antagonists of beta1 integrin-mediated adhesion or corresponding signal transduction elements may sensitize CML cells to chemotherapy and prevent resistance to the novel BCR/ABL kinase inhibitors being used for the treatment of this disease.

Generation of a minimal alpha5beta1 integrin-Fc fragment.

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377-390), a series of recombinant soluble human alpha(5)beta(1) integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional alpha(5)-subunit truncation contained the N-terminal 613 residues, whereas the shortest beta(1)-subunit was a fragment containing residues 121-455. Each of these minimally truncated integrins displayed the antibody binding characteristics of alpha(5)beta(1) purified from human placenta and bound ligand with the same apparent affinity as the native receptor.

Insulin-like growth factor-binding protein 1 stimulates human trophoblast migration by signaling through alpha 5 beta 1 integrin via mitogen-activated protein Kinase pathway.

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.

Adult neuronal regeneration induced by transgenic integrin expression.

In a variety of adult CNS injury models, embryonic neurons exhibit superior regenerative performance when compared with adult neurons. It is unknown how young neurons extend axons in the injured adult brain, in which adult neurons fail to regenerate. This study shows that cultured adult neurons do not adapt to conditions that are characteristic of the injured adult CNS: low levels of growth-promoting molecules and the presence of inhibitory proteoglycans. In contrast, young neurons readily adapt to these same conditions, and adaptation is accompanied by an increase in the expression of receptors for growth-promoting molecules (receptors of the integrin family). Surprisingly, the regenerative performance of adult neurons can be restored to that of young neurons by gene transfer-mediated expression of a single alpha-integrin.