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Identification of Jak PTK-regulated rho-specific GEFs involved in activation of lymphocyte adhesion
No full textLa rapida induzione dell’affinità integrinica è un processo dinamico cruciale nel reclutamento leucocitario, che è controllato da complessi meccanismi molecolari di segnale intracellulare indotti da chemochine. Le small GTPasi della famiglia di rho e rap sono certamente le molecole di segnale più studiate in questo contesto; dati recenti da noi ottenuti hanno evidenziato inoltre un ruolo importante delle proteine tirosin-chinasi della famiglia delle Jak (Jak PTKs) che agiscono da regolatori a monte delle small GTPasi.
Gli scambiatori di nucleotidi guanosinici (GEFs-Guanosine Exchange Factors) sono i principali attivatori diretti delle small GTPasi e quindi rappresentano le molecole canditate più probabili per chiarire il legame funzionale tra Jak PTKs e il modulo della rho.
In questo studio abbiamo dimostrato il ruolo regolatorio concorrente di quattro differenti rho-GEFs Vav1, Sos1, Arhgef1 e Dock2 nella modulazione dell’affinità dell’integrina LFA-1 e nella conseguente adesione cellulare di linfociti T umani primari stimolati con la chemochina CXCL12. La ridotta espressione di queste quattro molecole porta ad una minore induzione dell’affinità dell’integrina LFA-1 e ad una ridotta adesione all’ICAM-1 in condizioni statiche e sotto flusso. Da notare, l’attivazione di queste quattro proteine, indotta da CXCL12, è mediata dalle Jak PTKs e avviene in un intervallo di tempo coerente con la rapida induzione dell’affinità integrinica da chemochine. Inoltre l’attivazione di RhoA e Rac1 è strettamente dipendente dall’attività di Vav1, Sos1, Arhgef1 e Dock2.
Complessivamente in questo studio abbiamo identificato e caratterizzato dettagliatamente il ruolo regolatorio di quattro rho-GEFs nell’adesione mediata da LFA-1 indotta da CXCL12, fornendo una descrizione completa dei meccanismi molecolari di segnale esistenti tra Jaks e modulo della rho. Analizzando i nostri dati da un punto di vista quantitativo, abbiamo riscontrato alcune differenze tra queste proteine osservando un ruolo più marcato per Vav1 e Sos1 in confronto a quello di Arhgef1 e Dock2.
Questo diverso coinvolgimento di molteplici rho-GEFs con apparentemente la stessa funzione può avvalorare la nuova interpretazione quantitativa dei meccanismi di trasduzione del segnale, dove la complessità a livello molecolare è essenziale per generare un sistema flessibile in grado di rispondere efficientemente a differenti condizioni ambientali.The rapid integrin affinity up-regulation is a crucial dynamic process in leukocyte recruitment that is controlled by a complex inside-out signalling pathway induced by chemokines.
Small GTP binding proteins of rap and rho family are certainly the most studied signaling molecules involve in this pathway; in addition our recent data identified Jak PTKs as new upstream regulator of these small GTPases. Considering that Guanosine Exchange Factors (GEFs) are the main direct activators of small GTPases, they represent obvious molecule candidates to fill out the functional gap between Jak PTKs and rho-module.
In this study we show the concurrent regulatory role of four rho specific GEFs Vav1, Sos1, Arhgef1 and Dock2 in CXCL12-induced LFA-1 affinity triggering and mediated-adhesion in human T lymphocytes. A reduced expression of these four molecules resulted in an impaired chemokine-induced LFA-1 affinity up-regulation and in a reduced cell adhesion to ICAM-1 in static and under-flow conditions. Importantly, CXCL12-activation of these four proteins is mediated by Jak PTKs and occurs in a time frame coherent with LFA-1 affinity triggering by chemokine. Moreover the activation of RhoA and Rac1 is strictly dependent on Vav1, Sos1, Arhgef1 and Dock2 activity. Collectively in this study we identified and fully characterized the role of four rho-GEFs in CXCL12-induced LFA-1 mediated adhesion providing a comprehensive signalling link between Jak PTKs and rho-module. Considering our results from a quantitative point of view, we observed some variability in the relative regulatory role of these proteins, with a major role for Vav1 and Sos1 with respect to Arhgef1 and Dock2 activity. This variable involvement of multiple rho-GEFs with apparently the same function may support the new emergent quantitative-concurrency view of signal transduction in which this complexity in mechanisms controlling integrin activation is essential to generate a very flexible signalling system able to efficiently respond to a variety of environmental conditions
ESAT-6 and HspX improve the effectiveness of Bacille Calmette-Guerin to induce human dendritic cells-dependent T cells and natural killer cells activation.
No full textThe limited efficacy of Bacillus Calmette-Guerin (BCG) vaccination against some forms of tuberculosis is partly due to a missing expression of critical immunogenic proteins. We hypothesized that addition of ESAT-6 and HspX Mycobacterium Tuberculosis (Mtb) antigens could ameliorate the BCG ability to activate human dendritic cells (DC), that play an essential role in immune response. Here we report that BCG showed a weak ability to induce DC maturation, cytokine release and the subsequent CD4+ lymphocytes and NK cells activation. Addition of single ESAT-6 or HspX to BCG-stimulated DC did not significantly improve these processes. However, simultaneous addition of ESAT-6 and HspX enhanced BCG-dependent DC maturation and IL-12, IL-1β, IL-23, IL-6 and TNFα release. Moreover, DC incubated with BCG in presence of both ESAT-6 and HspX elicited IFN-γ release by CD4+ lymphocytes, and increased IFN-γ secretion and CD69 cytolysis marker expression in NK cells. These effects were inhibited by IL-12-blocking antibodies. A specific TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DC incubated with ESAT-6 and HspX, as well as IFN-γ secretion by CD4+ lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DC to induce the expression of the memory phenotype marker CD45RO in naïve CD4+ T cells. Our results indicate that ESAT-6 and HspX cooperation enhances the ability of BCG to stimulate human DC, that become able to induce T lymphocytes and NK cells-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of vaccination with BCG
ESAT-6 and HspX improve the effectiveness of BCG to induce human Dendritic Cells-dependent T cells and NK cells activation
No full textThe limited efficacy of Bacillus Calmette-Guerin (BCG) vaccination against some forms of tuberculosis is partly due to a missing expression of critical immunogenic proteins. We hypothesized that addition of ESAT-6 and HspX Mycobacterium Tuberculosis (Mtb) antigens could ameliorate the BCG ability to activate human dendritic cells (DC), that play an essential role in immune response. Here we report that BCG showed a weak ability to induce DC maturation, cytokine release and the subsequent CD4+ lymphocytes and NK cells activation. Addition of single ESAT-6 or HspX to BCG-stimulated DC did not significantly improve these processes. However, simultaneous addition of ESAT-6 and HspX enhanced BCG-dependent DC maturation and IL-12, IL-1β, IL-23, IL-6 and TNFα release. Moreover, DC incubated with BCG in presence of both ESAT-6 and HspX elicited IFN-γ release by CD4+ lymphocytes, and increased IFN-γ secretion and CD69 cytolysis marker expression in NK cells. These effects were inhibited by IL-12-blocking antibodies. A specific TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DC incubated with ESAT-6 and HspX, as well as IFN-γ secretion by CD4+ lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DC to induce the expression of the memory phenotype marker CD45RO in naïve CD4+ T cells. Our results indicate that ESAT-6 and HspX cooperation enhances the ability of BCG to stimulate human DC, that become able to induce T lymphocytes and NK cells-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of vaccination with BCG
ESAT-6 and HspX improve the effectiveness of BCG to induce human dendritic cells-dependent Th1 and NK cells activation.
Full text linkThe limited efficacy of the BCG vaccine against tuberculosis is partly due to the missing expression of immunogenic proteins. We analyzed whether the addition to BCG of ESAT-6 and HspX, two Mycobacterium tuberculosis (Mtb) antigens, could enhance its capacity to activate human dendritic cells (DCs). BCG showed a weak ability to induce DC maturation, cytokine release, and CD4(+) lymphocytes and NK cells activation. The addition of ESAT-6 or HspX alone to BCG-stimulated DC did not improve these processes, whereas their simultaneous addition enhanced BCG-dependent DC maturation and cytokine release, as well as the ability of BCG-treated DCs to stimulate IFN-γ release and CD69 expression by CD4(+) lymphocytes and NK cells. Addition of TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DCs incubated with ESAT-6 and HspX, as well as IFN-γ secretion by CD4(+) lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DCs to induce the expression of memory phenotype marker CD45RO in naïve CD4(+) T cells. Our results indicate that ESAT-6 and HspX cooperation enables BCG-treated human DCs to induce T lymphocyte and NK cell-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of BCG vaccination
JAK2 tyrosine kinase mediates integrin activation induced by CXCL12 in B-cell chronic lymphocytic leukemia
No full textChemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment
Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells
No full textThe regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment
Protein Tyrosine Phosphatase Receptor Type γ Is a JAK Phosphatase and Negatively Regulates Leukocyte Integrin Activation
No full textRegulation of signal transduction networks depends on protein kinase and phosphatase activities. Protein tyrosine kinases of the JAK family have been shown to regulate integrin affinity modulation by chemokines and mediated homing to secondary lymphoid organs of human T lymphocytes. However, the role of s in leukocyte recruitment is still elusive. In this study, we address this issue by focusing on protein tyrosine phosphatase receptor type γ (PTPRG), a tyrosine phosphatase highly expressed in human primary monocytes. We developed a novel methodology to study the signaling role of receptor type tyrosine phosphatases and found that activated PTPRG blocks chemoattractant-induced β2 integrin activation. Specifically, triggering of LFA-1 to high-affinity state is prevented by PTPRG activation. High-throughput phosphoproteomics and computational analyses show that PTPRG activation affects the phosphorylation state of at least 31 signaling proteins. Deeper examination shows that JAKs are critically involved in integrin-mediated monocyte adhesion and that PTPRG activation leads to JAK2 dephosphorylation on the critical 1007-1008 phosphotyrosine residues, implying JAK2 inhibition and thus explaining the antiadhesive role of PTPRG. Overall, the data validate a new approach to study receptor tyrosine phosphatases and show that, by targeting JAKs, PTPRG downmodulates the rapid activation of integrin affinity in human monocytes, thus emerging as a potential novel critical regulator of leukocyte trafficking
Chemokines and the signaling modules regulating integrin affinity
No full textIntegrin-mediated adhesion is a general concept referring to a series of adhesive phenomena including tethering-rolling, affinity, valency and binding stabilization altogether controlling cell avidity (adhesiveness) for the substrate. Arrest chemokines modulate each aspect of integrin activation, although integrin affinity regulation has been recognized as the prominent event in rapid leukocyte arrest induced by chemokines. A variety of inside-out and outside-in signaling mechanisms have been related to the process of integrin-mediated adhesion in different cellular models, but only few of them have been clearly contextualized to rapid integrin affinity modulation by arrest chemokines in primary leukocytes. Complex signaling processes triggered by arrest chemokines and controlling leukocyte integrin activation have been described for ras-related rap and for rho-related small GTPases. We summarize the role of rap and rho small GTPases in the regulation of rapid integrin affinity in primary leukocytes and provide a modular view of these pro-adhesive signaling events. A potential, albeit still speculative, mechanism of rho-mediated regulation of cytoskeletal proteins controlling the last step of integrin activation is also discussed. We also discuss data suggesting a functional integration between the rho- and rap-modules of integrin activation. Finally we examine the universality of signaling mechanisms regulating integrin triggering by arrest chemokines
SOS1, ARHGEF1, and DOCK2 rho-GEFs mediate JAK-Dependent LFA-1 activation by chemokines
No full textJAK-dependent activation of the rho module of integrin affinity triggering mediates chemokine-induced leukocyte adhesion. However, the signaling events linking JAKs to rho small GTPase activation by chemokines is still incompletely described. In this study, we show that son of sevenless 1 (SOS1), rho guanine nucleotide exchange factor (GEF)1 (ARHGEF1), and dedicator of cytokinesis (DOCK)2 GEFs mediate CXCL12-induced LFA-1 activation in human primary T lymphocytes. Downregulated expression of SOS1, ARHGEF1, and DOCK2 impairs LFA-1-mediated rapid T lymphocyte adhesion as well as underflow arrest on ICAM-1 induced by CXCL12. Moreover, LFA-1 affinity triggering by CXCL12 is impaired by SOS1, ARHGEF1, and DOCK2 downregulation. Notably, the three GEFs are all critically involved in chemokine-induced RhoA and Rac1 activation, thus suggesting the occurrence of a SOS1 specificity shift in the context of chemokine signaling. Accordingly, SOS1, ARHGEF1, and DOCK2 are tyrosine phosphorylated upon chemokine signaling with timing coherent with rapid LFA-1 affinity activation. Importantly, chemokine-induced tyrosine phosphorylation of these GEFs is fully mediated by JAK protein tyrosine kinases. Unexpectedly, and differently from VAV1, tyrosine phosphorylation of SOS1, ARHGEF1, and DOCK2 is completely inhibited by pertussis toxin pretreatment, thus suggesting different routes of rho-GEF triggering upon CXCR4 engagement. Taken together, these findings reveal a deeper level of complexity in the rho-signaling module, with at least four different rho-GEFs cooperating in the regulation of chemokine-induced integrin activation, possibly suggesting the emergence of stochastic concurrency in signaling mechanisms controlling leukocyte trafficking
CXCR4- and BCR-triggered integrin activation in B-cell chronic lymphocytic leukemia cells depends on JAK2-activated Bruton's tyrosine kinase
No full textBruton's tyrosine kinase (BTK) regulates the B-cell receptor (BCR) signaling pathway, which, in turn, plays a critical role in B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis. The BTK-specific inhibitor Ibrutinib blocks BCR signaling and is now approved as effective B-CLL therapy. Chemokines, such as the homeostatic chemokine CXCL12, play a central role in B-CLL pathogenesis and progression, by regulating CLL cell interaction with the stromal microenvironment, leading to cells survival and proliferation. In this study, we investigated, in normal versus CLL B-lymphocytes, the role of BTK in signal transduction activated by the CXCL12-CXCR4 signaling axis and its involvement in rapid integrin activation. We show that BTK is rapidly activated by CXCL12 in healthy as well as CLL B-lymphocytes, with a kinetic of tyr-phosphorylation coherent with rapid adhesion triggering. BTK inhibition prevents CXCL12-induced triggering of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. Furthermore, BTK inhibition blocks the activation of the small GTP-binding protein RhoA, controlling integrin affinity. Very importantly, we show that BTK tyr-phosphorylation and activation by CXCL12 depends on upstream activation of JAK2 tyrosine kinase. A comparative analysis of 36 B-CLL patients demonstrates that JAK2-dependent BTK regulatory role on integrin activation by CXCL12 is fully conserved in CLL cells. Finally, we show that the JAK2-BTK axis also regulates signaling to integrin activation by BCR. Thus, BTK and JAK protein tyrosine kinases (PTKs) manifest a hierarchical activity both in chemokine- as well as BCR-mediated integrin activation and dependent adhesion, potentially suggesting the possibility of combined therapeutic approaches to B-CLL treatment
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