Urokinase receptor-dependent and -independent p56/59(hck) activation state is a molecular switch between myelomonocytic cell motility and adherence.

Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.     

IL-2 signaling in human monocytes involves the phosphorylation and activation of p59hck

The activating properties of IL-2 and the structure of the IL-2R on human monocytes are well characterized. However, relatively little is known about the biochemical mechanisms involved in IL-2 signal transduction in these cells. We investigated the role of protein tyrosine kinases (PTKs) in the activation of monocytes by IL-2. Incubation of monocytes with the PTK inhibitor herbimycin A (HA) resulted in the dose-dependent suppression of IL-2-induced monocyte tumoricidal activity. This inhibition was rather potent, as a concentration of HA as low as 0.5 microM caused a complete abrogation of cytolytic activity. Furthermore, HA markedly suppressed the ability of IL-2 to induce IL-1 beta, TNF-alpha, IL-6, and IL-8 mRNA expression and protein secretion by monocytes. Anti-phosphotyrosine immunoblotting demonstrated that IL-2 induced a rapid and time-dependent increase in tyrosine phosphorylation of several cellular proteins of molecular masses ranging from 35 to 180 kDa. Interestingly, IL-2 caused a significant up-regulation of the constitutive levels of hck PTK mRNA and protein relative to medium-treated cells as well as an increase in p59hck tyrosine phosphorylation. Finally, we demonstrated by in vitro kinase assay that the specific activity of p59hck PTK was also induced by IL-2 in monocytes. Thus, these data show that the activation of PTKs is required for the triggering of monocyte effector and secretory functions by IL-2 and strongly suggest that p59hck is a key participant in IL-2 signaling in human monocytes.     

Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions

Cross-linking of the neutrophil-beta 2- or beta 3-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosin phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61hck and p58c-fgr, we examined adhesion- dependent respiratory burst in PMNs isolated from hck -/-, fgr -/-, and hck -/- fgr -/- knockout mice. Purified bone marrow PMNS from wild-type mice released significant amounts of O2- when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl- methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck-/-fgr-/- double-mutant mic, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck-/-fgr-/-PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMN plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck-/-fgr-/- PMNs, failed to respond. Direct cross-linking of the subunits of beta 2 and beta 2 integrins by surface-bound mAbs was elicited O2- production by wild-type PMNs, while the double-mutant hck- /-fgr-/- cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck-/-fgr-/- PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck-/-or fgr-/-single mutant cells produced O2- at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61 hck and p 58c-fgr is required for signaling through leukocyte beta 2 and beta 3 integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.     

Variability in the expression of urokinase receptor (CD87) mutants on cells: relevance to cell adhesion

The urokinase-type plasminogen activator receptor (uPAR, CD87) is a glycosylphosphatidylinositol (GPI)-anchored protein, containing three homologous Ly-6 domains, that mediates integrin-independent cell adhesion by directly binding to extracellular matrix protein vitronectin (VN). To elucidate the structural requirements for the uPAR-dependent cell adhesion on VN, several glycolipid-anchored variants of uPAR were expressed in BAF3 cells, (mouse pre B-lymphocytes) followed by functional analysis. The individual domains of uPAR were expressed at very low levels, the two domain mutants were expressed to a higher level and the wild type uPAR was expressed highly. Point mutations in domain 2 of uPAR have been shown to diminish cellular binding of the ligand urokinase and we observed a lack of VN binding to this mutant. Flow cytometry with a number of monoclonal antibodies indicated that the domain-specific antigenic determinants in these mutants were well preserved. Only the cells expressing the intact uPAR with all three domains adhered strongly to a VN substrate, whereas none of the other transfected cells showed significant cell adhesion. Hence, any alterations in the domain structure of uPAR reduce its expression and only the intact receptor can sustain the direct cell adhesion on VN-rich matrices found at sites of inflammation and injury.     

Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn)

Engagement of the clonotypic antigen receptor (TCR) on T lymphocytes provokes an activation response leading to cell proliferation and lymphokine secretion. To examine the molecular basis of T cell signaling, we generated transgenic animals in which a lymphocyte-specific nonreceptor protein-tyrosine kinase p59fyn(T) is 20-fold overexpressed in developing T lineage cells. Thymocytes from these mice, analyzed using both cellular and biochemical assays, were remarkably hyperstimulable. Moreover, the responsiveness of normal thymocytes to TCR-derived signals correlated well with the extent to which p59fyn was expressed in these cells. Overexpression of a catalytically inactive form of p59fyn substantially inhibited TCR-mediated activation in otherwise normal thymocytes. These effects are unique to p59fyn; overexpression of a closely related T cell-specific tyrosine kinase, p56lck, elicits dramatically different phenotypes. Our results suggest that p59fyn is a critically important component of the TCR signal transduction apparatus.     

Protease-activated receptor (PAR)-induced interleukin-8 production in airway epithelial cells requires activation of MAP kinases p44/42 and JNK

The mechanism underlying protease-activated receptor (PAR)-activation and subsequent interleukin (IL)-8 production in airway epithelial cells is not yet understood. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 airway epithelial cells. We studied the consequence of activation of PARs with simultaneous exposure to LPS. Thrombin, PAR-2-activating peptide and LPS, were tested alone and in combination. They induced significant synthesis of IL-8. However, only activation of PAR triggered phosphorylation of ERK1/2 and JNK. The application of the inhibitors of these two MAPKs resulted in reduction of IL-8 production. Thus, activation of PARs but not stimulation with LPS leads to ERK1/2 and JNK-mediated production of IL-8.     

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.     

Integrin-induced epidermal growth factor (EGF) receptor activation requires c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosines.

Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c-Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.     

TCR-induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56(lck) residues

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56(lck) ) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho-epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56(lck) phosphorylation, using a multiparameter flow-cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56(lck) or cognate phospho-tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H(2) O(2) or TCR-induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56(lck) . This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56(lck) -specific inhibitor, PP2, abolished phosphorylation at either residue in a dose-dependent manner. Taken together, these observations show that TCR-induced stimulation of T cells led to simultaneous phosphorylation of p56(lck) residues. This implies that dephosphorylation of Y505 is not crucial for p56(lck) activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56(lck) signaling pathways in T cells at single cell level. ? 2012 International Society for Advancement of Cytometry.     

Occupancy of the cancer cell urokinase receptor (uPAR): effects of acid elution and exogenous uPA on cell surface urokinase (uPA).

The development of a simple, sensitive fluorimetric assay for the measurement of cell surface-associated urokinase plasminogen activator (uPA) on viable, adherent HCT116 cells in microtitre plates, after a preincubation with purified human plasminogen is described. The assay determines plasmin activity by the cleavage of H-D-Val-Leu-Lys 4-aminomethyl coumarin under near physiological pH and ionic conditions with a sensitivity in the range of 5-100 mIU uPA/well at excitation 355 nm and emission 460 nm. Plasmin generated during the assay converted all cell-surface sc-uPA to tc-uPA, allowing the determination of total uPA activity. Inhibitor studies (PAI-2, amiloride or Glu-Gly-Arg chloromethylketone) confirmed the specificity of the uPA assay. Removal of these agents prior to assay allowed determination of the cell surface sc-uPA:tc-uPA ratio. Cell surface activity was only partially removed by acid elution. This corresponded with the loss of a number of proteins and uPA-containing species as detected by SDS-PAGE, gelatin enzymography and Western blotting. Although the major protein species eluted had a M(r) of 55 kDa, reacted with a commercial anti-human uPA mAb and correlated with the main lytic zone, other higher M(r) species were also eluted from HCT116 cells. Exogenous uPA increased cell-surface activity markedly on cells previously treated with acid. Following acid elution, cell surface uPA activity was restored after 30h in culture suggesting either de novo synthesis or release of pre-formed uPA with subsequent secretion and binding to uPAR. The assay has enabled studies on adherent cells to address questions about the regulation and expression of cell-surface uPA.     

TNF type 2 receptor (p75) lowers the threshold of T cell activation.

T cell activation requires a threshold amount of TCR-mediated signals, an amount that is reduced by signals mediated through costimulatory molecules expressed on the T cell surface. Here the role of TNFR2 (p75) as a putative costimulatory receptor for T cell activation was examined. It was found that p75 deficiency in CD8(+) T cells increased the requirements for TCR agonist approximately 5-fold. Furthermore, p75(-/-) T cells display a marked reduction in the proliferative response to TCR agonist. This hypoproliferative response was associated with delayed kinetics of induction of the acute activation markers CD25 and CD69 as well as a marked decrease in the production of IL-2 and IFN-gamma. The net result is that very few cells are recruited into the dividing population. Interestingly, CD28 costimulation was only partially effective in rescuing the proliferative defect of p75(-/-)CD8(+) T cells. Thus, p75 provides an important costimulatory signal in addition to that provided by CD28 toward optimal T cell proliferation.     

Cancer/testis antigen cancer-associated gene (CAGE) promotes motility of cancer cells through activation of focal adhesion kinase (FAK)

The cancer-associated gene (CAGE) is a novel cancer/testis antigen. Over-expression of it increased phosphorylation of focal adhesion kinase (FAK) and enhanced motility of SNU387 cells. Focal adhesion, kinase-related non-kinase (FRNK), an endogenous inhibitor of FAK, was significantly suppressed. This suggests that CAGE-promoted motility requires FAK. The inhibition of Rho-Associated coiled-coil-containing protein kinase (ROCK), an activator of FAK, also suppressed CAGE-promoted motility.     

Epidermal growth factor stimulation of trophoblast differentiation requires MAPK11/14 (p38 MAP kinase) activation

Cultured human term villous cytotrophoblasts (CT) have been reported to be nonproliferating but differentiate when exposed to epidermal growth factor (EGF). Here we show that CT differentiate into chorionic gonadotropin (beta-hCG/CGB)-expressing cells when cultured with medium alone. The addition of EGF decreases CGB secretion and prolongs production for up to 13 days. EGF stimulates the phosphorylation (activation) of the signaling intermediate p38 (MAPK11/14), and blocking phosphorylation pharmacologically with either SB203580 or SB202190 strongly inhibited spontaneous and EGF-stimulated secretion of CGB. In addition, EGF-stimulated fusion of cytotrophoblasts into syncytial units was strongly inhibited by SB203580. EGF upregulated trophoblast proliferation (measured by bromodeoxyuridine uptake) and SB203580 increased this proliferation after 5 days. In agreement with these observations, EGF and SB203580 increased expression of the G1-phase-specific gene cyclin-D1 (CCND1) and SB203580 downmodulated its inhibitor p21 (CDKN1A). When added to villous explant cultures, EGF did nothing to the pattern of CGB secretion, but addition of SB203580 prevented the normal surge in secretion during syncytial regeneration over Days 3-7. These data support the hypothesis that EGF-stimulated cytotrophoblast differentiation to syncytium requires MAPK11/14 activation, and that cytotrophoblast proliferation can be stimulated in culture by EGF and enhanced by MAPK11/14 inhibition with a consequent reduction of differentiation.     

Class A scavenger receptor-mediated cell adhesion requires the sequential activation of Lyn and PI3-kinase

Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A–/– macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting G_i/o activation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading. macrophage; inflammation; intracellular signaling     

Cutting edge: CD96 (tactile) promotes NK cell-target cell adhesion by interacting with the poliovirus receptor (CD155)

The poliovirus receptor (PVR) belongs to a large family of Ig molecules called nectins and nectin-like proteins, which mediate cell-cell adhesion, cell migration, and serve as entry receptors for viruses. It has been recently shown that human NK cells recognize PVR through the receptor DNAM-1, which triggers NK cell stimulation in association with beta(2) integrin. In this study, we show that NK cells recognize PVR through an additional receptor, CD96, or T cell-activated increased late expression (Tactile). CD96 promotes NK cell adhesion to target cells expressing PVR, stimulates cytotoxicity of activated NK cells, and mediates acquisition of PVR from target cells. Thus, NK cells have evolved a dual receptor system that recognizes nectins and nectin-like molecules on target cells and mediates NK cell adhesion and triggering of effector functions. As PVR is highly expressed in certain tumors, this receptor system may be critical for NK cell recognition of tumors.     

Class A scavenger receptors mediate cell adhesion via activation of G(i/o) and formation of focal adhesion complexes

Class A macrophage scavenger receptors (SR-A) are multifunctional receptors with roles in modified lipoprotein uptake, innate immunity, and macrophage adhesion. Our previous studies conducted in mouse peritoneal macrophages demonstrated that pertussis toxin (PTX) mediated inhibition of G(i/o) attenuated SR-A-dependent uptake of modified lipoprotein. The finding that SR-A-mediated lipoprotein internalization was PTX-sensitive led us to hypothesize that SR-A-mediated cell adhesion might be similarly regulated by G(i/o)-dependent signaling pathways. To test this hypothesis, SR-A was expressed in HEK cells under inducible control. Relative to HEK cells that lack SR-A, SR-A expressing cells displayed enhanced adhesion to tissue culture dishes. SR-A-mediated adhesion was significantly reduced following PTX treatment and was insensitive to chelating divalent cations with EDTA. SR-A-expressing cells exhibited a distinct cell morphology characterized by fine filopodia-like projections. Both polymerized actin and vinculin were codistributed with SR-A in the filopodia-like projections indicating the formation of focal adhesion complexes. Overall, our results indicate that the ability of SR-A to enhance cell adhesion involves G(i/o) activation and formation of focal adhesion complexes.     

L-plastin peptide activation of alpha(v)beta(3)-mediated adhesion requires integrin conformational change and actin filament disassembly

L-plastin (LPL) is a leukocyte actin binding protein previously implicated in the activation of the integrin alpha(M)beta(2) on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562 cell adhesion to vitronectin via alpha(v)beta(3), a well-studied model for activable integrins, was examined. Cell permeant versions of peptides based on the N-terminal sequence of LPL and the LPL headpiece domain both activated alpha(v)beta(3)-mediated adhesion. In contrast to adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of integrin beta(3) cytoplasmic domain tyrosines and was not inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides synergized with RGD ligand or Mn(2+) for generation of a conformational change in alpha(v)beta(3) associated with the high affinity state of the integrin, as determined by binding of a ligand-induced binding site antibody. Although LPL and ligand showed synergy for ligand-induced binding site expression when actin depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin depolymerization and ligand-induced integrin conformational change are required for LPL peptide-induced adhesion. We hypothesize that the critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.