Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Co-ligation of alpha4beta1 integrin and TCR rescues human thymocytes from steroid-induced apoptosis

Maturation of thymocytes represents a sequence of events during which thymocytes expressing TCR with moderate avidity for self antigen/MHC are positively selected, whereas those with high or insufficient TCR avidity die. Glucocorticoids are produced intrathymically and can contribute to apoptosis of unselected thymocytes. Thymocytes differentiate in a close contact with epithelial cells, expressing vascular adhesion molecule-1 (VCAM-1) and secreting glucocorticoids, with bone marrow-derived macrophages, and with extracellular matrix containing fibronectin (FN) and collagen. Their contact with FN is mediated by alpha4beta1 and alpha5beta1 integrins. We examined the contribution of TCR and integrin signaling to the survival of thymocytes from dexamethasone (Dex)-induced apoptosis. We demonstrate that FN and VCAM-1 (both of which bind alpha4beta1 integrin), but not collagen, considerably augment TCR-mediated protection of thymocytes from Dex-induced apoptosis. This 'survival' signal is transduced through the alphabeta1, but not through the alpha5beta1 integrin. The observed protection from Dex-induced apoptosis correlated with an increase in bcl-2 protein levels. FN-alpha4beta1 and VCAM-1-alpha4beta1 engagement induced up-regulation bcl-2 protein, while alpha5beta1 binding to FN induced a negative signal that was blocked by anti-alpha5beta1 antibody. These data suggest that alpha4beta1 integrin may contribute to protection of thymocytes with moderate avidity TCR from glucocorticoid-induced death during intrathymic maturation.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Analysis of the mechanism of tumor metastasis by the transfection of integrin cDNA

Integrin plays an important role in tumor metastasis through its interaction with extracellular matrix and endothelial cell. We have examined the role of each integrin in tumor metastasis by using transfection of integrin cDNA into various cells. Transfection of integrin alpha 2 subunit into RD cells, human rhabdomyosarcoma cells which do not express integrin alpha 2 beta 1, potentiated the frequency of metastases in various organs; lung, bone, adrenal gland, lymph node. alpha 4-transfectant of Chinese hamster ovary (CHO) cells, which do not have alpha 4 beta 1 on the cell surface, metastasized to bone through its interaction with VCAM-1 in the bone marrow stroma cells. On the other hand, alpha 5-transfectant of CHO cells was much less tumorgenic than parent CHO cells. These data suggest integrin influence tumor metastasis sometimes favorably and sometimes unfavorably according to the activity and the balance of various integrins.     

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.     

The role of adhesion molecules in human leukocyte attachment to porcine vascular endothelium: implications for xenotransplantation

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-alpha-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the beta2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and beta2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas beta2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent beta2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.     

The selective inhibition of beta 1 and beta 7 integrin-mediated lymphocyte adhesion by bacitracin

Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment. The regulation of such adhesion often involves changes in the functional state of the integrins rather than alterations in their expression levels. Although the functional basis for such transitions is unknown, a possible role for disulfide exchange might be postulated based on the observations that integrin function can be activated by bifunctional reducing agents or by Abs that react with areas adjacent to predicted long-range disulfide bonds in integrins. Recently, it has been reported that enzymes that catalyze disulfide exchanges such as protein disulfide isomerase (PDI) are present on the surface of lymphoid cells, raising the possibility that such enzymes might be involved in the control of lymphocyte adhesion. A number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of T cells. The results did not support role for PDI in the regulation of integrin function, as the inhibitors somatostatin A, tocinoic acid, dithiobisnitrobenzoic acid, and anti-PDI mAb did not interfere with adherence. However, one of the PDI inhibitors, bacitracin, selectively interfered with the beta1 integrin-mediated adherence of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and with alpha4beta7-dependent adherence to fibronectin and to VCAM-1. In contrast, alpha(v)beta3- and alpha(L)beta2-mediated adherence were not inhibited. Thus, it appears that bacitracin may be a selective inhibitor of beta1 and beta7 integrin functions by an as yet unknown mechanism.     

A cyclic hexapeptide is a potent antagonist of alpha 4 integrins

The alpha 4 integrins mediate leukocyte adhesion to specific counter-receptors, including vascular cell adhesion molecule-1 (VCAM-1), the fibronectin splice variant containing connecting segment 1 (CS1), and mucosal addressin cell adhesion molecule-1. A series of cyclized peptides based on the LDV sequence of CS1 were synthesized and assayed for inhibition of alpha 4 integrin binding. The most potent peptide, C*WLDVC* (where * indicates disulfide-linked residues), inhibited alpha 4 beta 1-dependent binding of lymphocytes to VCAM-1 and CS1 with half-maximal inhibition achieved at 1 to 3 microM of peptide. The peptide proved more potent when the lymphocytes were activated with 1 mM MnCl2; half-maximal inhibition was reached at 0.4 and 0.05 microM for VCAM-1 and CS1, respectively. This represents a 100- to 800-fold increase in potency over a linear CS1 peptide in these same assays. C*WLDVC* also inhibited alpha 4 beta 7-dependent lymphocyte binding to the ligands mucosal addressin cell adhesion molecule-1, VCAM-1 and CS1. Immunoprecipitation of radiolabeled integrin indicated that the peptide could bind alpha 4 beta 1 and alpha 4 beta 7 directly and elute alpha 4 beta 1 from a CS1-conjugated agarose resin. The peptide showed selectivity for alpha 4 integrins in that it effectively inhibited alpha 4 beta 1-dependent, but not alpha 5 beta 1-dependent, binding of cells to intact fibronectin. Due to its small size and potency, C*WLDVC* may serve as a useful tool for the study of alpha 4 integrin biology and the development of small molecule therapeutics.     

Phenotype, and migration properties of three major subsets of tissue homing T cells in sheep

T cells show a bias in their migration pathways: some T cells preferentially migrate to peripheral lymph nodes (LN), some to mucosal tissues, and some to peripheral tissues such as skin. These recirculation pathways were examined in sheep by collecting lymph draining into and out of peripheral and intestinal LN, and using fluorescent dyes to trace the recirculation of the lymph cells. Monoclonal antibodies (mAb) to alpha 4, beta 1, and beta 7 integrins, and L-selectin, were used to define three major populations of recirculating T cells. Naive-type T cells (L-selectin+, alpha 4 beta 1lo beta 7lo) migrated preferentially through peripheral LN. Two memory populations could be defined: alpha 4 beta 1hi beta 7- and alpha 4 beta 7hi beta 1lo. alpha 4 beta 1hi beta 7- T cells were present in lymph draining from the skin. T cells migrating preferentially through intestinal LN were alpha 4 beta 7hi beta 1lo. Consistent with this migration pattern, the endothelial receptor for alpha 4 beta 7, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was detected on high endothelial venules within intestinal LN and Peyer's patches, but only weakly on high endothelial venules within peripheral LN. Thus, there are at least three easily definable subsets of T cells, based on integrin expression, which show distinct migration preferences.     

Down-regulation of monocytic VLA-4 leads to a decreased adhesion to VCAM-1

The alpha 4 beta 1 integrin VLA-4 is expressed on practically all leukocytes, except on mature granulocytes. Here we show that in vitro treatment of monocytic cells with phorbol-12-myristate-13-acetate (PMA) leads to a selective decrease in the VLA-4 alpha-chain expression, both at the RNA and protein level. Meanwhile the expression of beta 1 and that of alpha 5, another alpha-chain associating with beta 1, was seen to increase. The decrease of alpha 4 expression was restricted to monocytic cells, and was not observed on other VLA-4-positive cells tested (MOLT-4 T cells and HOS sarcoma cells). The down-regulation of the VLA-4 alpha-chain was followed by a decreased binding capacity of the cells to recombinant VCAM-1. This data indicates that while previous findings show that the integrin-dependent adhesion may rapidly be regulated by altering the avidity of the interacting molecules, their quantitative modulation also has a clear impact on adhesion.     

Expression of the beta 7 integrin by human endothelial cells

Integrin adhesion receptors mediate fundamental intercellular interactions of many cell types as well as cellular interactions with specific extracellular matrix molecules. To date, the beta 7 integrin has been shown to be expressed by leukocyte subsets and to mediate interactions of these cells with extracellular matrix molecules as well as with endothelial and epithelial cells. The data presented here indicate that human endothelial cells also express the beta 7 integrin both in vitro and in situ. Analysis of cDNA indicated that endothelial beta 7 was identical to that expressed by leukocytes. Cell surface expression of beta 7 was increased by exposure of the endothelium to the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1 beta. In leukocytes, beta 7 complexes with alpha 4 or alpha E integrin chains. Endothelial cells also expressed a number of alpha-integrin chains, including alpha 4, but not alpha E. The expression and utilization of beta 7, presumably complexed with alpha 4, by endothelial cells may be instrumental in the maintenance of the function or phenotype of endothelial cells.     

Vascular endothelial cell expression of ICAM-1 and VCAM-1 at the onset of eliciting contact hypersensitivity in mice: evidence for a dominant role of TNF-alpha

We have studied vascular endothelial activation and increased expression of ICAM-1 and VCAM-1 at the onset of the elicitation phase of oxazolone contact hypersensitivity in mice. By measuring the local uptake of i.v. administered radiolabeled anti-ICAM-1 and anti-VCAM-1 mAb, we found that endothelial ICAM-1 and VCAM-1 was increased by 4 h after challenge, 2 h later than the first peak of ear swelling and 125I-labeled human serum albumen uptake. Increased expression of endothelial ICAM-1 and VCAM-1 was significantly greater in sensitized animals than in naive animals. Anti-TNF-alpha antiserum significantly inhibited both the increase in ear thickness (p < 0.01), and the up-regulation of ICAM-1 and VCAM-1 expression (p < 0.01 for both) at 4 h. In contrast, the combination of anti-IL-1alpha and IL-1beta had only a small inhibitory effect on ICAM-1 expression (p < 0.05) and no significant effect on increased ear thickness or on VCAM-1 expression. A mixture of anti-TNF-alpha, anti-IL-1alpha, and IL-1beta was no more inhibitory for endothelial ICAM-1 and VCAM-1 expression than anti-TNF-alpha alone. ICAM-1 and VCAM-1 expression at 4 h was unaffected by a combination of mAb against alpha4 and beta2 integrins, whereas expression at 24 h was significantly inhibited (p < 0.05), suggesting that the release of TNF-alpha and other cytokines involved in the initiation of the response may not require leukocyte traffic or other leukocyte functions involving these integrins. We conclude that the early up-regulation of endothelial ICAM-1 and VCAM-1 during the elicitation of contact hypersensitivity is primarily due to the immune-dependent local release of TNF-alpha.     

alphadbeta2 integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1)

The beta2 family of integrins, CD11a, CD11b, CD11c, and alphad, are expressed on most leukocytes. We show that the newest member of this family, alphad, is expressed on human eosinophils in peripheral blood, and surface expression can be upregulated within minutes by phorbol ester or calcium ionophore A23187. Culture of eosinophils with interleukin 5 (IL-5) leads to a two- to fourfold increase in alphad levels by 3-7 d without a change in alpha4 integrin expression. Eosinophils isolated from late phase bronchoalveolar lavage fluids express alphad at levels similar to that seen after 3 d of IL-5 culture. Regarding alphadbeta2 ligands, in both freshly isolated and IL-5-cultured eosinophils, as well as alphadbeta2-transfected Chinese hamster ovary cells, alphadbeta2 can function as a ligand for vascular cell adhesion molecule 1 (VCAM-1). This conclusion is based on the ability of monoclonal antibodies to alphad, beta2, or VCAM-1 to block cell attachment in static adhesion assays. In experiments with eosinophils, the relative contribution of alphadbeta2 integrin- mediated adhesion is enhanced after IL-5 culture. These experiments demonstrate that alphadbeta2 is an alternative ligand for VCAM-1, and this integrin may play a role in eosinophil adhesion to VCAM-1 in states of chronic inflammation.     

The thrombospondin receptor CD47 (IAP) modulates and associates with alpha2 beta1 integrin in vascular smooth muscle cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a beta1 integrin, rather than alphav beta3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to alpha2 and beta1 integrin subunits and to IAP. A combination of antialpha2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antialphav beta3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between alpha2 beta1 and IAP was detected by the coimmunoprecipitation of both alpha2 and beta1 integrin subunits with IAP. These data suggest that IAP can associate with alpha2 beta1 integrin and modulate its function.     

The development of experimental autoimmune encephalomyelitis in the mouse requires alpha4-integrin but not alpha4beta7-integrin

Because monoclonal antibodies (mAbs) directed against alpha4-integrin and VCAM-1 inhibit the development of experimental autoimmune encephalomyelitis (EAE) in vivo, it has been concluded that the successful therapeutic effect is due to interference with alpha4beta1/VCAM-1-mediated interaction of autoaggressive T cells with the blood-brain barrier. A possible role for alpha4beta7-integrin, or interference with other T cell mediated events during the pathogenesis of EAE, has not been considered. We have compared the effects of mAb therapy on the development of EAE in the SJL/N mouse, using a large panel of mAbs directed against alpha4, beta7, the alpha4beta7-heterodimer, and against VCAM-1. Although encephalitogenic T cells express both alpha4-integrins, mAbs directed against the alpha4beta7-heterodimer or against the beta7-subunit did not interfere with the development of EAE. In contrast, mAbs directed against alpha4 and VCAM-1 inhibited or diminished clinical or histopathological signs of EAE. Our data demonstrate for the first time that alpha4beta7 is not essential for the development of EAE. Furthermore, our in vitro studies suggest that the therapeutic effect of anti-alpha4-treatment of EAE might also be caused by inhibition of antigen-specific T cell proliferation.     

Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium

Disruptions in the mucosal lining of the gastrointestinal tract reseal by epithelial cell migration, a process termed restitution. We examined the involvement of laminin isoforms and their integrin receptors in restitution using the intestinal epithelial cell line T84. T84 cells express primarily laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced in cells adjoining wounds. Because T84 cells stained faintly with MAbs 4C7 (laminin alpha 1 subunit) and with MAbs 4F11 and 1B4 (laminin alpha 2 subunit), we suggest that expression of laminins 6 and 7 is enhanced in response to wounding. The alpha 3 beta 1 integrin and the alpha 6-containing integrins function in wound closure because MAbs specific for the beta 1 integrin subunit (MAb13), the alpha 3 subunit (IVA5), and the alpha 6 subunit (2B7) potently inhibited T84 migration into wounds. Immunofluorescence using UMA9, a beta 4-integrin-specific MAb, revealed that alpha 6 beta 4 integrin exists in a Triton-X-100-insoluble structure at the basal surface and that the staining of this structure is enhanced in cells adjoining wounds. In addition, a Triton-X-100-soluble pool of alpha 6 beta 4, as well as alpha 3 beta 1 and presumably alpha 6 beta 1, was found along lateral surfaces of T84 cells. On flattened cells adjoining wounds, staining for these integrins was distributed diffusely, suggesting a redistribution that accompanies cell migration. Taken together, these data suggest that wound-induced epithelial cell migration is a finely tuned process that is dependent upon the regulated function and localization of specific laminins and their integrin receptors.     

Potent alpha 4 beta 1 peptide antagonists as potential anti-inflammatory agents

The migration, adhesion, and subsequent extravasation of leukocytes into inflamed tissues contribute to the pathogenesis of a variety of inflammatory diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. The integrin adhesion receptor alpha 4 beta 1 expressed on leukocytes binds to the extracellular matrix protein fibronectin and to the cytokine inducible vascular cell adhesion molecule-1 (VCAM-1) at inflamed sites. Binding of alpha 4 beta 1 to VCAM-1 initiates firm adhesion of the leukocyte to the vascular endothelium followed by extravasation into the tissue. Monoclonal antibodies generated against either alpha 4 beta 1 or VCAM-1 can moderate this inflammatory response in a variety of animal models. Recently peptides containing a consensus LDV sequence based on the connecting segment-1 (CS-1) of fibronectin and cyclic peptides containing an RCD motif have shown promise in modulating leukocyte migration and inflammation presumably by blocking the interaction of alpha 4 beta 1 with VCAM-1. Here we describe novel, highly potent, cyclic peptides that competitively inhibit alpha 4 beta 1 binding to VCAM-1 and fibronectin at sub nanomolar concentrations. The structure of a representative analog was determined via NMR spectroscopy and used to facilitate optimization of peptide leads. The peptides discussed here utilize similar functional groups as the binding epitope of VCAM-1, inhibit lymphocyte migration in vivo, and are highly selective for alpha 4 beta 1. Furthermore the structure--activity relationships described here have provided a template for the structure-based design of small molecule antagonists of alpha 4 beta 1-mediated cell adhesion processes.     

Transforming growth factor beta 1 suppresses transformation in hepatocytes by regulating alpha 1 beta 1 integrin expression

We previously reported that (a) treatment of the ras-transformed hepatocyte cell line NR4 with transforming growth factor (TGF) beta 1 suppresses many characteristics associated with the transformed phenotype including altered morphology, actin cytoskeleton reorganization, and anchorage-independent growth such that the cells more closely resemble the immortalized CWSV1 parent cell line; (b) transformed NR4 cells expressed significantly less alpha 1 integrin RNA than the immortalized CWSV1 cells; and (c) TGF-beta 1 treatment of NR4 cells stimulated the expression of alpha 1 and beta 1 integrin RNAs. In this report, the role of the alpha 1 beta 1 integrin in TGF-beta 1-mediated suppression of the ras-transformed phenotype was investigated. We determined that (a) the cell surface integrin that increased in response to TGF-beta 1 treatment of NR4 cells was alpha 1 integrin; (b) TGF-beta 1 altered the ability of NR4 cells to attach to collagen and laminin, the extracellular matrix components that interact with the alpha 1 beta 1 integrin receptor; (c) TGF-beta 1 treatment resulted in relocalization of the alpha 1 integrin on the NR4 cell surface; and (d) TGF-beta 1-mediated inhibition of anchorage-independent growth was blocked by the presence of alpha 1 integrin antibody. A cell line that overexpresses alpha 1 integrin was derived from NR4 cells; characterization of these cells indicated that they continued to express H-ras RNA but were less transformed than the parent NR4 cells. Specifically, they had an altered morphology, an organized actin cytoskeleton, and reduced ability to demonstrate anchorage-independent growth.(ABSTRACT TRUNCATED AT 250 WORDS)