Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival.

We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6ß1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.

Matricellular protein Cyr61 bridges lysophosphatidic acid and integrin pathways leading to cell migration.

Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA1) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA1 is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6ß1 and ανß3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.

α6ß1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF.

BACKGROUND: The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. RESULTS: Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and ß1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. ß1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. CONCLUSIONS: In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of ß1-, α6- and αV-integrins.

Strain-induced differentiation of fetal type II epithelial cells is mediated via the integrin α6ß1-ADAM17/tumor necrosis factor-α-converting enzyme (TACE) signaling pathway.

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and ß1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and ß1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and ß1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6ß1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.

Cell surface structures influence lung clearance rate of systemically infused mesenchymal stromal cells.

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.

Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6ß1 integrin receptor.

Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6ß1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis.

Netrin-4 activates endothelial integrin {alpha}6{beta}1.

RATIONALE: Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3ß1, α6ß1, and α6ß4) in nonendothelial cells. OBJECTIVE: We tested whether integrins are netrin-4 receptors in the endothelium. METHODS AND RESULTS: The α6ß1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or ß1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or ß1 inhibition. Finally, netrin-4 and α6ß1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature. CONCLUSIONS: The α6ß1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.

CD151 amplifies signaling by integrin α6ß1 to PI3K and induces the epithelial-mesenchymal transition in HCC cells.

BACKGROUND & AIMS: Overexpression of CD151 is associated with poor prognosis for hepatocellular carcinoma (HCC), yet its role in pathogenesis is not known. METHODS: We analyzed the expression of the integrin subunit α6 by quantitative, real-time polymerase chain reaction and immunoblot analyses of 120 HCC tissue samples; its clinical significance was investigated using tissue microarray (TMAs) analysis of samples from 335 patients with HCC. Immunoprecipitation was used to assess the relationship between α6 and CD151. The molecular effects of high expression levels of α6 and CD151 in HCC cells were determined using RNA interference and pharmacologic approaches. RESULTS: Overexpression of α6 correlated with poor prognosis of patients with HCC; α6 formed a complex with endogenous CD151 in HCC cells. In cells that expressed high levels of α6 and CD151, laminin-5 promoted cell spreading by inducing the epithelial-mesenchymal transition (EMT); this effect was not observed in cells that expressed high levels of only α6 or CD151. Cells that expressed high levels of α6 and CD151 underwent the EMT in response to laminin-5, through hyperactivation of phosphatidylinositol-3-kinase (PI3K), primarily induced via the PI3K-protein kinase B (Akt)-Snail-phosphatase and tensin homolog feedback pathway. The EMT was reversed by PI3K inhibitors and antibodies against CD151 or α6 in vitro, and was delayed by specific interference with CD151 and α6 in vivo. CONCLUSIONS: High expression levels of CD151 and α6 promote invasiveness of HCC cells. Either of these proteins, or PI3K signaling, might be targets for therapeutics for subgroups of patients with HCC.

Lethal junctional epidermolysis bullosa with pyloric atresia due to compound heterozygosity for two novel mutations in the integrin ß4 gene.

BACKGROUND: Junctional epidermolysis bullosa with pyloric atresia (JEB-PA) is a rare autosomal recessive disease with blister formation within the lamina lucida due to mutations in the integrin ß4 (ITGB4) and α6 (ITGA6) genes. CASE REPORT: A female preterm infant, first child of healthy non-consanguineous parents, was born at 26 + 4 weeks of gestation by caesarean section, following polyhydramnion and abruption of placenta. She presented with extensive areas of denuded skin on both lateral sides of the head, neck and extremities. Auricles were hypoplastic. Abdominal ultrasound and X-ray were suggestive of pyloric atresia which was revised surgically on the 4th day of life. Further course was complicated by progressive skin detachment, sepsis, and renal insufficiency with fatal outcome at 18 days of age. Immunofluorescence mapping of cryopreserved skin showed junctional cleft formation with negative staining for integrin α6 and integrin ß4. Mutational analysis disclosed compound heterozygosity for two novel nonsense mutations in the ITGB4 gene: c.600dupC/p.F201fsX14 and c.2533C>T/p.Q845X. 2 subsequent pregnancies were terminated following prenatal diagnosis disclosing the same ITGB4 mutations, a 4th pregnancy was unaffected. CONCLUSION: We describe a case of lethal JEB-PA with negative immunoreactivity to integrin α6 and integrin ß4 predicting a poor outcome. Identification of compound heterozygosity for two novel ITGB4 mutations in the affected preterm infant permitted prenatal diagnosis and finally birth of a healthy sibling.

Mechanosensitive collaboration between integrins and connexins allows nutrient and antioxidant transport into the lens.

The delivery of glucose and antioxidants is vital to maintain homeostasis and lens transparency. Here, we report a new mechanism whereby mechanically activated connexin (Cx) hemichannels serve as a transport portal for delivering glucose and glutathione (GSH). Integrin α6ß1 in outer cortical lens fiber activated by fluid flow shear stress (FFSS) induced opening of hemichannels. Inhibition of α6 activation prevented hemichannel opening as well as glucose and GSH uptake. The activation of integrin ß1, a heterodimeric partner of α6 in the absence of FFSS, increased Cx50 hemichannel opening. Hemichannel activation by FFSS depended on the interaction of integrin α6 and Cx50 C-terminal domain. Moreover, hemichannels in nuclear fiber were unresponsive owing to Cx50 truncation. Taken together, these results show that mechanically activated α6ß1 integrin in outer cortical lens fibers leads to opening of hemichannels, which transport glucose and GSH into cortical lens fibers. This study unveils a new transport mechanism that maintains metabolic and antioxidative function of the lens.

The interaction of HAb18G/CD147 with integrin alpha6beta1 and its implications for the invasion potential of human hepatoma cells.

BACKGROUND: HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin alpha3beta1. However, it has never been investigated whether alpha3beta1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells. METHODS: Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin alpha6beta1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin alpha6beta1. Invasion potential was evaluated with an invasion assay and gelatin zymography. RESULTS: We found that integrin alpha6beta1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin alpha6beta1 antibodies (P < 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P < 0.05). Importantly, no additive effect between Wortmannin and alpha6beta1 antibodies was observed, indicating that alpha6beta1 and PI3K transmit the signal in an upstream-downstream relationship. CONCLUSION: These results suggest that alpha6beta1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

Laminin isoforms of lymph nodes and predominant role of alpha5-laminin(s) in adhesion and migration of blood lymphocytes.

During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains and that the same chains, in addition to alpha2, are found in RFs. In functional studies with laminin isoforms covering all Lm alpha chains, alpha5-laminin (Lm-511) was the most adhesion- and migration-promoting isoform for human blood lymphocytes, followed by alpha3- (Lm-332) and alpha4- (Lm-411) laminins, and the lymphocytes used the alpha6beta1 integrin as the primary receptor for the alpha5-laminin. Moreover, Lm-511 strongly co-stimulated T cell proliferation, and blood lymphocytes were able to secrete alpha4- and alpha5-laminins following stimulation. The LN cell number in laminin alpha4-deficient mice compared with wild-type did not differ significantly. This study demonstrates a predominant role for alpha5-laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.

Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro.

A prerequisite for axon regeneration is the interaction between the growth cone and the extracellular matrix (ECM). Laminins are prominent constituents of ECM throughout the body, known to support axon growth in vitro and in vivo. The regenerative capacity of adult neurons is greatly diminished compared to embryonic or early postnatal neurons. Since most lesions in the nervous system occur in the adult, we have examined neurite outgrowth from adult mouse DRG neurons on four laminin isoforms (laminin-1/LM-111, laminin-2/LM-211, laminin-8/LM-411 and laminin-10/LM-511) in vitro. The growth on laminin-1 and -10 was trophic factor-independent and superior to the one on laminin-2 and -8, where growth was very poor in the absence of neurotrophins. Among other ECM proteins, laminins were by far the most active molecules. Using function-blocking antibodies to laminin-binding integrins, we identified non-overlapping functions of integrins alpha3beta1, alpha7beta1 and alpha6beta1 on different laminin isoforms, in that alpha3beta1 and alpha7beta1 integrins appeared to be specific receptors for both laminin-1 and-2, whereas integrin alpha6beta1 was a receptor for laminin-8 and-10. Lastly, by use of immunohistochemistry, expression of subunits of laminin-1, -2, -8 and -10 in sensory organs in the human epidermis could be demonstrated, supporting an important role for these laminins in relation to primary sensory axons.

The laminin binding α3 and α6 integrins cooperate to promote epithelial cell adhesion and growth.

Integrins, the major receptors for cell-extracellular matrix (ECM) interactions, regulate multiple cell biological processes including adhesion, migration, proliferation and growth factor-dependent signaling. The principal laminin (LM) binding integrins α3ß1, α6ß1 and α6ß4 are usually co-expressed in cells and bind to multiple laminins with different affinities making it difficult to define their specific function. In this study, we generated kidney epithelial collecting duct (CD) cells that lack both the α3 and α6 integrin subunits. This deletion impaired cell adhesion and migration to LM-332 and LM-511 more than deleting α3 or α6 alone. Cell adhesion mediated by both α3ß1 and α6 integrins was PI3K independent, but required K63-linked polyubiquitination of Akt by the ubiquitin-modifying enzyme TRAF6. Moreover, we provide evidence that glial-derived neurotrophic factor (GDNF) and fibroblast growth factor 10 (FGF10)- mediated cell signaling, spreading and proliferation were severely compromised in double integrin α3/α6- but not single α3- or α6-null CD cells. Interestingly, these growth factor-dependent cell functions required both PI3K- and TRAF6-dependent Akt activation. These data suggest that expression of the integrin α3 or α6 subunit is sufficient to mediate GDNF- and FGF10-dependent spreading, proliferation and signaling on LM-511. Thus, our study shows that α3 and α6 containing integrins promote distinct functions and signaling by CD cells on laminin substrata.

Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization.

BACKGROUND: Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization. RESULTS: Using Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process. CONCLUSION: These findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported.

Mechanistic basis for the recognition of laminin-511 by α6ß1 integrin.

Laminins regulate diverse cellular functions through interaction with integrins. Two regions of laminins-three laminin globular domains of the α chain (LG1-3) and the carboxyl-terminal tail of the γ chain (γ-tail)-are required for integrin binding, but it remains unclear how the γ-tail contributes to the binding. We determined the crystal structure of the integrin binding fragment of laminin-511, showing that the γ-tail extends to the bottom face of LG1-3. Electron microscopic imaging combined with biochemical analyses showed that integrin binds to the bottom face of LG1-3 with the γ1-tail apposed to the metal ion-dependent adhesion site (MIDAS) of integrin ß1. These findings are consistent with a model in which the γ-tail coordinates the metal ion in the MIDAS through its Glu residue.

Integrin alpha6 maintains the structural integrity of the kidney collecting system.

Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.

Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton's jelly mesenchymal stem cells (WJ-MSC).

BACKGROUND: Angiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context. METHODS: Mesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton's jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation. RESULTS: The paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10-200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin α6ß1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway. CONCLUSIONS: Thus, here we show that stromal production of Netrin-1 is a critical component of the vascular regulatory machinery. This signaling event may have deep implications in the modulation of several processes related to a number of diseases where angiogenesis plays a key role in vascular homeostasis.

Interleukin-1alpha enhances the aggressive behavior of pancreatic cancer cells by regulating the alpha6beta1-integrin and urokinase plasminogen activator receptor expression.

BACKGROUND: In human pancreatic cancer progression, the alpha6beta1-integrin is expressed on cancer cell surface during invasion and metastasis formation. In this study, we investigated whether interleukin (IL)-1alpha induces the alterations of integrin subunits and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) expression in pancreatic cancer cells. We hypothesize that the alterations of integrin subunits and uPA/uPAR expression make an important role in signaling pathways responsible for biological behavior of pancreatic cancer cells. RESULTS: IL-1alpha upregulated the expression of alpha6 and beta1 integrins without any alterations of alpha5 and alphav integrins expression. IL-1alpha also induced enhancement in the expression of uPA/uPAR in pancreatic cancer cells. IL-1alpha enhanced the proliferation, adhesion, and migration in pancreatic cancer cells, and IL-1alpha-induced alterations of uPA/uPAR expression correlated with the increased the migration of pancreatic cancer cells. Upregulation of alpha6 integrin subunit and uPA/uPAR correlated with the activation of Ras and downstream extracellular signal-regulated kinase (ERK) pathways. IL-1alpha-induced activation of Ras and downstream ERK can be inhibited by using inhibitory antibodies against alpha6 and beta1 integrin and uPAR, consistent with the inhibition of proliferation, adhesion and migration of pancreatic cancer cells. Immunohistochemical analysis demonstrated a significant association between strong expressions of alpha6 integrin with uPAR in pancreatic cancer specimens. Furthermore, the strong expression of alpha6 integrin and uPAR was found to be independent prognosticator in pancreatic cancer patients. CONCLUSION: Based on these findings, we conclude that IL-1alpha can induce selective upregulation of alpha6beta1-integrin and uPA/uPAR in pancreatic cancer cells and these changes may modulate the aggressive functions of pancreatic cancer.

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.