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PubMed2002

alpha4beta1 integrin regulates lamellipodia protrusion via a focal complex/focal adhesion-independent mechanism.

alpha4beta1 integrin plays an important role in cell migration. We show that when ectopically expressed in Chinese hamster ovary cells, alpha4beta1 is sufficient and required for promoting protrusion of broad lamellipodia in response to scratch-wounding, whereas alpha5beta1 does not have this effect. By time-lapse microscopy of cells expressing an alpha4/green fluorescent protein fusion protein, we show that alpha4beta1 forms transient puncta at the leading edge of cells that begin to protrude lamellipodia in response to scratch-wounding. The cells expressing a mutant alpha4/green fluorescent protein that binds paxillin at a reduced level had a faster response to scratch-wounding, forming alpha4-positive puncta and protruding lamellipodia much earlier. While enhancing lamellipodia protrusion, this mutation reduces random motility of the cells in Transwell assays, indicating that lamellipodia protrusion and random motility are distinct types of motile activities that are differentially regulated by interactions between alpha4beta1 and paxillin. Finally, we show that, at the leading edge, alpha4-positive puncta and paxillin-positive focal complexes/adhesions do not colocalize, but alpha4beta1 and paxillin colocalize partially in ruffles. These findings provide evidence for a specific role of alpha4beta1 in lamellipodia protrusion that is distinct from the motility-promoting functions of alpha5beta1 and other integrins that mediate cell adhesion and signaling events through focal complexes and focal adhesions.

Animals↗
PubMed2006

Activation pathways of alpha4beta1 integrin leading to distinct T-cell cytoskeleton reorganization, Rac1 regulation and Pyk2 phosphorylation.

Alpha4beta1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. Alpha4beta1 can be activated by intracellular signals elicited upon T-cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside-out activation). Divalent cations or certain anti-beta1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside-in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post-adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of 41 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, alpha-CD3 mAb or the chemokine SDF-1alpha followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (alpha4beta1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the alpha4beta1-induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of alpha4beta1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements.

Cell Adhesion↗
PubMed1999

Fibronectin interaction with alpha4beta1 integrin prevents apoptosis in B cell chronic lymphocytic leukemia: correlation with Bcl-2 and Bax.

B cell chronic lymphocytic leukemia (B-CLL) consists of the accumulation of malignant cells that apparently escape normal apoptotic regulation. We have studied the role of alpha4beta1 integrin/fibronectin interaction in preventing apoptosis of these cells in vitro. B cells from 16 patients showed constant expression of alpha4beta1 and little or no alpha5beta1. B-CLL cells cultured on fibronectin or two previously described fibronectin recombinant fragments (H89 and H0) which contain the ligands for alpha4beta1, consistently showed higher viability than control cells cultured on poly-lysine. The H89 fragment, containing the high affinity ligand CS-1, was the most efficient substrate with mean cell viability values of 72, 60 and 35% at days 2, 5 and 8 of culture, respectively. For control cells these values were 40, 27 and 15%, respectively. Parallel cell cycle analysis confirmed these results. The anti-apoptotic effect required direct contact with immobilized substrata since it was not observed when using B-CLL conditioned media alone or when clustering alpha4beta1 with specific mAbs in suspension. Quantitation of the apoptosis regulatory proteins Bcl-2 and Bax revealed that cells cultured on the H89 fragment showed high/moderate levels of Bcl-2 (with some interpatient variation) and low levels of Bax resulting in an elevated Bcl-2/Bax ratio. These results indicate that adhesion of B-CLL cells to fibronectin upregulate the Bcl-2/Bax ratio and this may contribute to the anti-apoptotic effect induced via alpha4beta1 integrin.

Adult↗
PubMed2006

Selection of a 2-azabicyclo[2.2.2]octane-based alpha4beta1 integrin antagonist as an inhaled anti-asthmatic agent.

The alpha4beta1 integrin, expressed on eosinophils and neutrophils, induces inflammation in the lung by facilitating cellular infiltration and activation. From a number of potent alpha4beta1 antagonists that we evaluated for safety and efficacy, 1 was selected as a lead candidate for anti-asthma therapy by the inhalation route. We devised an optimized stereoselective synthesis to facilitate the preparation of a sufficiently large quantity of 1 for assessment in vivo. Administration of 1 to allergen-sensitive sheep by inhalation blocked the late-phase response of asthma and abolished airway hyper-responsiveness at 24h following the antigen challenge. Additionally, the recruitment of inflammatory cells into the lungs was inhibited. Administration of 1 to ovalbumin-sensitized guinea pigs intraperitoneally blocked airway resistance and inhibited the recruitment of inflammatory cells.

Administration, Inhalation↗
PubMed1997

IL-7 activates alpha4beta1 integrin in murine thymocytes.

IL-7, a cytokine produced by thymic epithelium, was shown to induce adhesion of murine thymocytes to gelatin-coated membranes. A major binding component of gelatin was identified as fibronectin. IL-7-induced adhesion was observed for all of the major thymocyte subsets, including double-negative, double-positive, and single-positive cells, and specific IL-7R were verified on each subset. Fibronectin binding was mediated via alpha4beta1 integrin (VLA-4), which is expressed at high levels on thymocytes. VLA-4 surface expression was not increased following IL-7 treatment, but was shown to undergo rapid tyrosine phosphorylation on the beta1 subunit. This tyrosine phosphorylation was blocked by genistein, which also blocked IL-7-induced adhesion. IL-7 was detected on the extracellular matrix of the thymus, suggesting that it could promote matrix association through an integrin pathway.

Animals↗
PubMed2006

MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by alpha4beta1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration.

B-cell chronic lymphocytic leukemia (B-CLL) progression is determined by malignant cell extravasation and lymphoid tissue infiltration. We have studied the role and regulation of matrix metalloproteinase-9 (MMP-9) in B-CLL cell migration and invasion. Adhesion of B-CLL cells to the fibronectin fragment FN-H89, VCAM-1, or TNF-alpha-activated human umbilical vein endothelial cells (HUVECs) up-regulated MMP-9 production, measured by gelatin zymography. This effect was mediated by alpha4beta1 integrin and required PI3-K/Akt signaling. The chemokine CXCL12 also up-regulated MMP-9, independently of alpha4beta1 and involving ERK1/2 but not Akt activity. Accordingly, alpha4beta1 engagement activated the PI3-K/Akt/NF-kappaB pathway, while CXCL12/CXCR4 interaction activated ERK1/2/c-Fos signaling. Anti-MMP-9 antibodies, the MMP-9 inhibitor TIMP-1, or transfection with 3 different MMP-9 siRNAs significantly blocked migration through Matrigel or HUVECs. Cell-associated MMP-9 was mainly at the membrane and contained the proactive and mature forms. Moreover, B-CLL cells formed podosomes upon adhesion to FN-H89, VCAM-1, or fibronectin; MMP-9 localized to podosomes in a PI3-K-dependent manner and degraded a fibronectin/gelatin matrix. Our results are the first to show that MMP-9 is physiologically regulated by alpha4beta1 integrin and CXCL12 and plays a key role in cell invasion and transendothelial migration, thus contributing to B-CLL progression. MMP-9 could therefore constitute a target for treatment of this malignancy.

Cell Adhesion↗
PubMed2005

Vav1 and Rac control chemokine-promoted T lymphocyte adhesion mediated by the integrin alpha4beta1.

The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.

Blotting, Western↗
PubMed2003

CXCR4 chemokine receptors (CD184) and alpha4beta1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis).

Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell-stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord-blood-derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi-1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte-colony-stimulating-factor-mobilized CD34+ cells for pseudoemperipolesis (28.7 +/- 12%vs 18.1 +/- 6.1% of input cells within 24 h, mean +/- SD, n = 8), whereas 9.4 +/- 12.6% (mean +/- SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks alpha4beta1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that alpha4beta1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment.

Antigens, CD34↗
PubMed2004

alpha4beta1-integrin blockade and cyclosporine decreases the prevalence and severity of transplant vasculopathy in a rat transplant model.

BACKGROUND: Transplant vasculopathy leads to neointimal proliferation of allograft arteries, and alpha4beta1-integrin (very late antigen-4 [VLA-4]) seems to play an important role in the pathogenesis. This study evaluates the effect of a new, synthetic, VLA-4 blocker (S3429) on transplant vasculopathy in a rat cardiac transplant model. METHODS: After transplantation (Lewis to Fisher), rats were divided randomly into 6 therapy groups: Group 1, n = 14, saline solution (vehicle); Group 2, n = 14, 3 mg/kg/day cyclosporine; Group 3, n = 21, 10 mg/kg/day S3429 + 3 mg/kg/day cyclosporine; Group 4, n = 21, 5 mg/kg/day S3429 + 3 mg/kg/day cyclosporine; Group 5: n = 21, 10 mg/kg/day S3429; Group 6, n = 21, 5 mg/kg/day S3429. Cyclosporine was given continuously until rats were killed. S3429 was either given for the entire study time or was discontinued after 20 days and animals were killed at Day 80. Twenty-eighty days after grafting, we assessed vasculopathy prevalence and mean vessel occlusion in coronary arteries. RESULTS: Cyclosporine decreased the prevalence of vasculopathy and mean vessel occlusion compared with controls. We observed a further decrease in prevalence and mean vessel occlusion with 80 days of therapy with S3429 and cyclosporine. After discontinuing S3429 therapy at Day 20, prevalence and mean vessel occlusion increased to values seen in cyclosporine-treated animals at Day 80. S3429 alone decreased mean vessel occlusion only within the first 20 days compared with controls but had no effect on the prevalence of vasculopathy. CONCLUSION: Because of the further decrease with S3429 therapy and the dramatic increase after discontinuation of S3429 therapy, we conclude that blocking VLA-4 receptors may prevent the development of transplant vasculopathy.

Animals↗
PubMedDate not supplied

Fibronectin-alpha4beta1 integrin interactions modulate p42/44 MAPK phosphorylation in steatotic liver cold ischemia-reperfusion injury.

We investigated the effects of the connecting segment-1 (CS1) peptide, which blocks fibronectin (FN)-alpha4beta1 integrin interactions upon cell signaling, leukocyte migration, and secretion of proinflammatory cytokines, in a well-established steatotic rat liver model using ex vivo cold ischemia followed by isotransplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers prior and after cold ischemic storage. Lean Zucker recipients of fatty orthotopic liver transplantation (OLT) received an additional 3-day course of CS1 peptides post-OLT. CS1 peptide-treated steatotic OLTs harvested at 1, 3, and 7 days showed moderated levels of p42/44 mitogen-activated protein kinase (MAPK) phosphorylation, comparable to those observed in steatotic naive livers. In contrast, p42/44 MAPK phosphorylation was found up-regulated in 1- to 3-day damaged control OLTs. However, 7-day control OLTs were characterized by virtually lack of p42/44 MAPK phosphorylation. Lack of p42/44 MAPK phosphorylation in 7-day control OLTs was correlated with massive presence of leukocytes in the grafts and elevated levels of proinflammatory cytokines. CS1 peptide-treated OLTs at 7 days showed a profound decrease in T-cell (10 +/- 3 vs 56 +/- 20, P < .03) and monocyte/macrophage (+/++ vs +++) infiltration and significantly reduced levels of cytokine expression, such as IL-2 (approximately sixfold), and IFN-gamma (approximately three- to fourfold), as compared with controls.

Animals↗
PubMed1997

The alpha4beta1 integrin can mediate leukocyte adhesion to casein and denatured protein substrates.

An understanding of the binding specificity of leukocyte integrins is important to determine the range of ligands that interact with these receptors during inflammatory processes. In this study we show that the alpha4beta1 integrin can interact with casein and denatured albumin and promote leukocyte adhesion through these interactions. This was demonstrated with the use of blocking antibodies directed to alpha4beta1 and peptide adhesion competitors containing the alpha4beta1 binding tripeptide, Leu-Asp-Val (LDV). Consistent with this data, the adhesion is completely divalent cation-dependent and is stimulated by known activators of leukocyte integrin function, namely phorbol ester and the beta1 integrin activating antibody, 8A2. It is interesting to note that neither bovine alpha-casein or human albumin contain an LDV site (present in the CS-1 site of alternatively spliced fibronectin) or an IDS site (present in VCAM-1) yet they promote adhesion through this integrin. Furthermore, alpha4beta1 directly binds to Sepharose columns containing casein, casein fragments, or denatured albumin but does not bind columns containing native albumin. These data suggest that the binding specificity for the alpha4beta1 integrin is considerably broader than previously realized. This work has implications for how subsets of leukocytes may interact with damaged proteins during tissue injury and inflammation.

Amino Acid Sequence↗
PubMedDate not supplied

Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity.

A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.

Antibodies, Monoclonal↗
PubMed1997

Cross talk between alpha(v)beta3 and alpha4beta1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1.

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand alpha4beta1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between alpha4beta1 and VCAM-1 in vivo. Here we show that the lymphocyte alpha(v)beta3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte alpha(v)beta3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of alpha4beta1 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This alpha(v)beta3-mediated enhanced migration of lymphocytes via alpha4beta1 is likely to depend on the interaction of alpha(v)beta3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between alpha(v)beta3 integrin and alpha4beta1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.

Animals↗
PubMed1998

Analysis of ligand-induced and ligand-attenuated epitopes on the leukocyte integrin alpha4beta1: VCAM-1, mucosal addressin cell adhesion molecule-1, and fibronectin induce distinct conformational changes.

The leukocyte integrin alpha4beta1 is a receptor for both cell surface ligands (VCAM-1 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)) and extracellular matrix components (fibronectin). Through regulated interactions with these molecules, alpha4beta1 mediates leukocyte migration from the vasculature at sites of inflammation. Regulation of integrin activity plays a key role in controlling leukocyte-adhesive events and appears to be partly determined by changes in integrin conformation. Several mAbs that recognize ligand-induced binding site epitopes on integrins have been characterized, and a subset of these mAbs are capable of stimulating integrin-ligand binding. Conversely, some mAbs recognize epitopes that are attenuated by ligand engagement and allosterically inhibit ligand binding. To gain insight into ligand-specific effects on integrin conformation, we have examined the ability of different ligands to modulate the binding of four distinct classes (A, B1, B2, and C) of anti-alpha4 Abs to alpha4beta1. VCAM-1 attenuated B (antifunctional) class epitopes via an allosteric mechanism and also allosterically inhibited the binding of the function-blocking anti-beta1 mAb 13. Additional alpha4beta1 ligands (fibronectin fragments, MAdCAM-1, and the CS1 peptide) also inhibited mAb 13-integrin binding; however, the epitopes of the class B anti-alpha4 mAbs were attenuated by the fibronectin fragments, but not by MAdCAM-1 or the CS1 peptide. Of the two anti-alpha4 class A mAbs examined, one recognized an epitope that was induced uniquely by VCAM-1. Taken together, these data suggest that overlapping but distinct binding mechanisms exist for different alpha4beta1 ligands and that distinct conformational changes are induced upon integrin engagement by different ligands.

Animals↗
PubMed2006

Marked strain differences in the pharmacokinetics of an alpha4beta1 integrin antagonist, 4-[1-[3-Chloro-4-[N-(2-methylphenyl)-ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]-methoxybenzoic Acid (D01-4582), in Sprague-Dawley rats are associated with albumin genetic polymorphism.

Strain differences in pharmacokinetics of an alpha4beta1 integrin antagonist, 4-[1-[3-chloro-4-[N-(2-methylphenyl)-ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), in Sprague-Dawley rat strains (SD rat and CD rat) and their mechanism were investigated. Total plasma clearances of D01-4582 were 31.5 and 5.23 ml/min/kg in SD and CD rats, respectively. From in vivo studies, hepatic uptake process was thought to be involved in the strain differences. Differences in the uptake of D01-4582 by isolated hepatocytes prepared from the both strains were not observed when hepatocytes were incubated with simple buffer, but marked differences were observed when hepatocytes were incubated with plasma. When the dissociation constants (Kd) for the plasma protein binding of D01-4582 were examined in six rat strains, each strain was classified into two groups: a high-Kd group, which included SD rats, Brown Norway rats, and Wistar rats; and a low-Kd group, which included CD rats, Lewis rats, and Eisai hyperbilirubinemic rats. Since all rat strains in the low-Kd group showed higher area under the concentration-time curve for D01-4582 than rats in the high-Kd group, it was considered that the strain differences in the pharmacokinetics of D01-4582 were due to differences in the binding affinity. Purified albumin also showed strain differences in Kd. The cDNA sequence of the albumin was analyzed, and 11 substitutions were observed. V238L and T293I were found only in the high-Kd group, suggesting that these amino acid changes reduced the binding affinity of albumin for D01-4582. In conclusion, the strain differences in D01-4582 pharmacokinetics were suggested to be caused by an alteration in Kd, associated with albumin genetic polymorphism.

Albumins↗
PubMed2003

Activation of Rac and tyrosine phosphorylation of cytokine receptors induced by cross-linking of integrin alpha4beta1 and cell adhesion in hematopoietic cells.

Adhesion of hematopoietic cells, mainly through alpha4beta1 and alpha5beta1 integrins, to the bone marrow microenvironment may play important roles in regulation of hematopoiesis. However, the mechanisms for signaling, outside-in signaling, have largely remained to be established. We demonstrate here that cross-linking of alpha4beta1 by anti-alpha4 antibody induces tyrosine phosphorylation of Pyk2, Shc, and Cbl as well as binding of the adaptor protein CrkL with Cbl in a murine hematopoietic cell line, 32D/EpoR-Wt. Furthermore, cross-linking of alpha4beta1 induced activation of the Rho family small GTPase Rac, which was enhanced by induced overexpression of CrkL and was inhibited by the phosphatidylinositol 3(')-kinase (PI3K) inhibitor LY294002. In addition, adhesion of 32D/EpoR-Wt cells to immobilized H-296, a recombinant fibronectin peptide specific for alpha4beta1, induced tyrosine phosphorylation of Jak2, the erythropoietin receptor (EpoR), and the IL-3 receptor beta subunit as well as Pyk2, Shc, and Cbl. Tyrosine phosphorylation of Jak2 and EpoR was also induced in a human leukemic cell line, UT-7, by adhesion to immobilized H-296. However, adhesion of 32D/EpoR-PM4 cells, expressing the W282R mutant EpoR defective in coupling with Jak2, to immobilized H-296 failed to induce tyrosine phosphorylation of the mutant EpoR. These results implicate CrkL in PI3K-dependent activation of Rac by outside-in signaling from alpha4beta1 and suggest that adhesion through alpha4beta1 further activates cytokine receptor-associated Jak2 to induce phosphorylation of these receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Identification of ligand binding sites on integrin alpha4beta1 through chemical cross-linking.

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.

Amino Acid Sequence↗
PubMed2004

Expression and function of integrin alpha4beta1 and vascular cell adhesion molecule-1 (VCAM-1) during sympathetic innervation of the heart.

The interaction between the integrin alpha4beta1 receptor on superior cervical ganglion (SCG) neurons and vascular cell adhesion molecule-1 (VCAM-1) in cardiac tissue has been implicated in proper development of the sympathetic innervation of the heart (Wingerd et al. [2002] J Neurosci 22:10772-10780). In this study, we examined the expression and function of alpha4beta1 and VCAM-1 in developing rat SCG and heart. In vitro, the alpha4beta1-dependent neurite outgrowth on VCAM-1 decreased by approximately 50% from postnatal day 1 to 6. This down-regulation was correlated with a shift in alpha4 isoform and a shift in alpha4 localization from neurites to cell bodies. This altered localization was also observed in vivo but on a different time scale. alpha4 was detected on most developing SCG neurons and on macrophages and blood vessels. In the heart, alpha4 was detected on sympathetic axons, but the percentage of alpha4-positive fibers decreased with age. VCAM-1 immunoreactivity was abundant in heart tissue throughout development, in close proximity to sympathetic axons. The regulation of alpha4beta1 function, and localization of alpha4 and VCAM-1, are consistent with a role for the alpha4beta1--VCAM-1 interaction in extension of sympathetic axons into the myocardium.

Animals↗