Src-family kinases mediate an outside-in signal necessary for β2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin–IgG fusion protein, by a mechanism that involved β2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(β2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an ‘outside-in’ signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented β2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin–IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck−/−fgr−/− and hck−/−fgr−/−lyn−/− mice showed significant impairment of β2-integrin-mediated adhesion to CHO-E. Moreover, the expression of β2 integrin activation epitopes at the sites of cell–cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a β2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for β2 integrin function in PMN tethered by E-selectin.

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Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability

AJP Cell Physiology ◽

10.1152/ajpcell.00223.2017 ◽

2018 ◽

Vol 314 (2) ◽

pp. C233-C241 ◽

Cited By ~ 4

Author(s):

John D. Tompkins ◽

Todd A. Clason ◽

Thomas R. Buttolph ◽

Beatrice M. Girard ◽

Anne K. Linden ◽

...

Keyword(s):

G Protein ◽

Neuronal Excitability ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Src Family Kinases ◽

Receptor Internalization ◽

Src Family Kinase ◽

Pac1 Receptor ◽

Neuron Excitability ◽

Endosomal Signaling

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

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SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells

Biochemical Journal ◽

10.1042/bj20040151 ◽

2004 ◽

Vol 380 (1) ◽

pp. 57-65 ◽

Cited By ~ 31

Author(s):

Paola PICCARDONI ◽

Stefano MANARINI ◽

Lorenzo FEDERICO ◽

Zsuzsa BAGOLY ◽

Romina PECCE ◽

...

Keyword(s):

Tyrosine Kinase ◽

Actin Polymerization ◽

Protein Tyrosine Kinase ◽

Polymorphonuclear Cells ◽

High Avidity ◽

Β2 Integrin ◽

High Affinity ◽

Tyrosine Kinase 2 ◽

Oncogenic Protein ◽

Β2 Integrins

In human PMN (polymorphonuclear cells), challenged by P-selectin, the β2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108–116]. This suggested that an SRC-dependent outside-in signalling strengthens the β2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock β2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor β2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common β2 chain, by a combination of antibodies against αL and αM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by β2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by β2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize β2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.

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Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

Blood ◽

10.1182/blood-2005-05-1933 ◽

2005 ◽

Vol 106 (10) ◽

pp. 3410-3414 ◽

Cited By ~ 80

Author(s):

Analia Garcia ◽

Todd M. Quinton ◽

Robert T. Dorsam ◽

Satya P. Kunapuli

Keyword(s):

Platelet Aggregation ◽

Phospholipase C ◽

Platelet Activation ◽

Kinase Inhibitor ◽

Thromboxane A2 ◽

Receptor Activation ◽

Src Family Kinases ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Src Family Kinase

AbstractThe binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non–aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.

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β1 and β2 integrins activate different signalling pathways in monocytes

Biochemical Journal ◽

10.1042/bj3630273 ◽

2002 ◽

Vol 363 (2) ◽

pp. 273-280 ◽

Cited By ~ 31

Author(s):

Merit REYES-REYES ◽

Nancy MORA ◽

Gerardo GONZALEZ ◽

Carlos ROSALES

Keyword(s):

Luciferase Reporter ◽

Signalling Pathways ◽

Cross Linking ◽

Β1 Integrin ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Monocytic Cells ◽

Β2 Integrin ◽

Natural Ligands ◽

Β2 Integrins

Integrin-mediated signals play an important but poorly understood role in regulating many leucocyte functions. In monocytes and macrophages, integrins of the β2 subfamily are involved in cell—cell interactions that are important for migration of the cells through the endothelium and also for phagocytosis. On the other hand, in the same cells, β1 integrin-mediated adhesion to extracellular matrix proteins results in a strong induction of immediate early genes that are important in inflammation. To investigate the signalling pathways from these two types of integrin in monocytic cells, THP-1 cells were selectively stimulated via β1 or β2 integrins by cross-linking each type of receptor with specific monoclonal antibodies or their natural ligands. The involvement of extracellular signal-regulated kinase (ERK), Syk and phosphoinositide 3-kinase (PI-3K) was then analysed. Nuclear factor κB (NF-κB) activation was also detected in THP-1 cells transiently transfected with an NF-κB-driven luciferase reporter gene. We found that binding of both types of integrin to their natural ligands activated ERK in a Syk- and PI-3K-dependent manner. Yet, cross-linking of integrins by anti-β1 antibodies caused activation of ERK while that by anti-β2 antibodies did not. Also both types of integrin activated NF-κB. However, PI-3K was required for β1 integrin-, but not β2 integrin-, mediated NF-κB activation. In addition, inhibition of PI-3K with wortmannin and LY294002 blocked β1 integrin-mediated NF-κB activation, but did not affect that mediated by β2 integrin. These data suggest that distinct integrins activate different signalling pathways in monocytic cells.

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A Src family kinase maintains latent sensitization in rats, a model of inflammatory and neuropathic pain

10.1101/2020.02.18.954859 ◽

2020 ◽

Cited By ~ 1

Author(s):

Wenling Chen ◽

Juan Carlos Marvizón

Keyword(s):

Chronic Pain ◽

Nerve Injury ◽

Mechanical Allodynia ◽

Kinase Inhibitors ◽

Src Family Kinases ◽

Opioid Antagonist ◽

Spared Nerve Injury ◽

Src Family Kinase ◽

Intracellular Signals

AbstractLatent sensitization is a long-term model of chronic pain in which hyperalgesia is continuously suppressed by opioid receptors. This is demonstrated by the induction of mechanical allodynia by opioid antagonists. Different intracellular signals may mediate the initiation, maintenance and expression of latent sensitization. Our criterion for the involvement of a signal in the maintenance of latent sensitization is that it inhibitors should permanently eliminate the allodynia produced by an opioid antagonist. We hypothesized that Src family kinases (SFKs) maintain latent sensitization and tested this hypothesis in rats with latent sensitization induced by complete Freund’s adjuvant (CFA) or by spared nerve injury. After measures of mechanical allodynia returned to baseline, the SFK inhibitor PP2 or vehicle were injected intrathecally. The opioid antagonist naltrexone injected intrathecally 15 min later produced allodynia in control rats but not in rats injected with PP2. PP2 or vehicle were injected daily for two more days and naltrexone was injected five days later. Again, naltrexone induced allodynia in the control rats but not in the rats injected with PP2. Results were similar when latent sensitization was induced either with CFA or spared nerve injury. We concluded that an SFK, likely Fyn, maintains latent sensitization induced by inflammation or nerve injury.PerspectiveThis article presents evidence that a Src family kinase, likely Fyn, maintains latent sensitization induced by inflammation or nerve injury. If latent sensitization is a valid model of chronic pain, inhibiting its maintenance with Src family kinase inhibitors may cure chronic pain.

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Combination of subtherapeutic anti-TNF dose with dasatinib restores clinical and molecular arthritogenic profiles better than standard anti-TNF treatment

Journal of Translational Medicine ◽

10.1186/s12967-021-02764-y ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Lydia Ntari ◽

Christoforos Nikolaou ◽

Ksanthi Kranidioti ◽

Dimitra Papadopoulou ◽

Eleni Christodoulou-Vafeiadou ◽

...

Keyword(s):

Gene Expression ◽

Therapeutic Effect ◽

Expression Analysis ◽

Mode Of Action ◽

Gene Expression Analysis ◽

Kinase Inhibitors ◽

High Dose ◽

Dependent Manner ◽

Combination Therapies ◽

Number Of Patients

Abstract Background New medications for Rheumatoid Arthritis (RA) have emerged in the last decades, including Disease Modifying Antirheumatic Drugs (DMARDs) and biologics. However, there is no known cure, since a significant proportion of patients remain or become non-responders to current therapies. The development of new mode-of-action treatment schemes involving combination therapies could prove successful for the treatment of a greater number of RA patients. Methods We investigated the effect of the Tyrosine Kinase inhibitors (TKIs) dasatinib and bosutinib, on the human TNF-dependent Tg197 arthritis mouse model. The inhibitors were administered either as a monotherapy or in combination with a subtherapeutic dose of anti-hTNF biologics and their therapeutic effect was assessed clinically, histopathologically as well as via gene expression analysis and was compared to that of an efficient TNF monotherapy. Results Dasatinib and, to a lesser extent, bosutinib inhibited the production of TNF and proinflammatory chemokines from arthritogenic synovial fibroblasts. Dasatinib, but not bosutinib, also ameliorated significantly and in a dose-dependent manner both the clinical and histopathological signs of Tg197 arthritis. Combination of dasatinib with a subtherapeutic dose of anti-hTNF biologic agents, resulted in a synergistic inhibitory effect abolishing all arthritis symptoms. Gene expression analysis of whole joint tissue of Tg197 mice revealed that the combination of dasatinib with a low subtherapeutic dose of Infliximab most efficiently restores the pathogenic gene expression profile to that of the healthy state compared to either treatment administered as a monotherapy. Conclusion Our findings show that dasatinib exhibits a therapeutic effect in TNF-driven arthritis and can act in synergy with a subtherapeutic anti-hTNF dose to effectively treat the clinical and histopathological signs of the pathology. The combination of dasatinib and anti-hTNF exhibits a distinct mode of action in restoring the arthritogenic gene signature to that of a healthy profile. Potential clinical applications of combination therapies with kinase inhibitors and anti-TNF agents may provide an interesting alternative to high-dose anti-hTNF monotherapy and increase the number of patients responding to treatment.

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Identification of imidazo[4,5-c]pyridin-2-one derivatives as novel Src family kinase inhibitors against glioblastoma

Journal of Enzyme Inhibition and Medicinal Chemistry ◽

10.1080/14756366.2021.1948542 ◽

2021 ◽

Vol 36 (1) ◽

pp. 1541-1552

Author(s):

Lishun Zhang ◽

Zichao Yang ◽

Huiting Sang ◽

Ying Jiang ◽

Mingfeng Zhou ◽

...

Keyword(s):

Kinase Inhibitors ◽

Src Family Kinase

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Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

Journal of Endocrinology ◽

10.1677/joe-08-0104 ◽

2008 ◽

Vol 198 (3) ◽

pp. 561-569 ◽

Cited By ~ 72

Author(s):

Wenbin Shang ◽

Ying Yang ◽

Libin Zhou ◽

Boren Jiang ◽

Hua Jin ◽

...

Keyword(s):

Insulin Receptor ◽

Glucose Uptake ◽

Signaling Pathway ◽

Glucose Transport ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

C2c12 Myotubes ◽

Maximal Effect ◽

Dependent Manner ◽

Active Constituent

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb1 has been shown to regulate peroxisome proliferator-activated receptor γ activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb1 on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb1 significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 μM and a time of 3 h. In adipocytes, Rb1 promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb1 increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb1-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb1 stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.

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Tyrosine kinase inhibitors enhance GHRH-stimulated cAMP accumulation and GH release in rat anterior pituitary cells

Journal of Endocrinology ◽

10.1677/joe.0.1520193 ◽

1997 ◽

Vol 152 (2) ◽

pp. 193-199 ◽

Cited By ~ 10

Author(s):

T Ogiwara ◽

C L Chik ◽

A K Ho

Keyword(s):

Tyrosine Kinase ◽

Cholera Toxin ◽

Tyrosine Kinase Inhibitors ◽

Anterior Pituitary ◽

Kinase Inhibitors ◽

Pituitary Cells ◽

Dependent Manner ◽

Camp Accumulation ◽

Anterior Pituitary Cells ◽

Site Of Action

Abstract In this study, the role of tyrosine phosphorylation in agonist-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. It was found that genistein, a tyrosine kinase inhibitor, while having no effect on its own, potentiated GHRH-stimulated cAMP accumulation in a concentration-dependent manner. In comparison, daidzein, an inactive analogue of genistein, was ineffective and vanadate, a phosphotyrosine phosphatase inhibitor, reduced GHRH-stimulated cAMP accumulation. Additional structurally unrelated tyrosine kinase inhibitors, erbstatin and tyrphostins, also potentiated GHRH-stimulated cAMP accumulation. To determine the site of action of the tyrosine kinase inhibitors, pituitary adenylate cyclase-activating polypeptide (PACAP), cholera toxin and forskolin were used to increase cAMP accumulation. Genistein enhanced the PACAP-, cholera toxin- or forskolin-stimulated cAMP accumulation, suggesting that the site of action is at the post-receptor level. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein did not potentiate and vanadate did not inhibit GHRH-stimulated cAMP accumulation, indicating that phosphodiesterase is a probable site of action for the inhibitor. Genistein and erbstatin also enhanced GHRH-stimulated GH release and the effect of vanadate was inhibitory. These results indicate that tyrosine kinase inhibitors enhance cAMP accumulation through their action on phosphodiesterase activity in rat anterior pituitary cells and the tyrosine kinase pathway appears to be involved in the control of GH release. Journal of Endocrinology (1997) 152, 193–199

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Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.2.c248 ◽

2001 ◽

Vol 280 (2) ◽

pp. C248-C253 ◽

Cited By ~ 90

Author(s):

Stephen C. Dahl ◽

Joseph S. Handler ◽

H. Moo Kwon

Keyword(s):

Transcription Factor ◽

Nuclear Localization ◽

Kinase Inhibitors ◽

Dependent Manner ◽

Binding Assays ◽

P38 Inhibitor ◽

Vitro Binding ◽

Compatible Osmolytes

The accumulation of compatible osmolytes during osmotic stress is observed in virtually all organisms. In mammals, the hypertonicity-induced expression of osmolyte transporters and synthetic enzymes is conferred by the presence of upstream tonicity-responsive enhancer (TonE) sequences. Recently, we described the cloning and initial characterization of TonE-binding protein (TonEBP), a transcription factor that translocates to the nucleus and associates with TonE sequences in a tonicity-dependent manner. We now report that hypertonicity induces an increase in TonEBP phosphorylation that temporally correlates with increased nuclear localization of the molecule. TonEBP phosphorylation is not affected by a number of kinase inhibitors, including the p38 inhibitor SB-203580. In addition, in vitro binding assays show that the association of TonEBP with TonE sequences is not affected by phosphorylation. Thus TonEBP phosphorylation is an early step in the response of cells to hypertonicity and may be required for nuclear import or retention.

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