Beta2 integrin-dependent phosphorylation of protein-tyrosine kinase Pyk2 stimulated by tumor necrosis factor alpha and fMLP in human neutrophils adherent to fibrinogen.

Tumor necrosis factor alpha and fMLP can activate a broad range of cellular functions in neutrophils adherent to biological surfaces. These functions are mediated by integrins and involve the activation of tyrosine kinases. Here, we report that Pyk2, a member of the focal adhesion kinase family, was present in human neutrophils and was rapidly phosphorylated and activated following tumor necrosis factor alpha and fMLP stimulation in an adhesion-dependent manner. Tyrosine phosphorylation of Pyk2 was attenuated by beta2 integrin blocking with specific antibodies. The tyrosine phosphorylation of Pyk2 was downstream of protein kinases Lyn, Syk and protein kinase C and cytoskeletal organization. The activation of Pyk2 may play a role in adhesion/cytoskeleton-associated neutrophils function.     

A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils

Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.     

Cocoa procyanidins attenuate 4-hydroxynonenal-induced apoptosis of PC12 cells by directly inhibiting mitogen-activated protein kinase kinase 4 activity

Neurodegenerative disorders such as Alzheimer's disease (AD) are associated with oxidative stress, and it has been suggested that apoptosis is a crucial pathway in neuronal cell death in AD patients. 4-Hydroxynonenal (HNE), one of the aldehydic products of membrane lipid peroxidation, is reported to be elevated in the brains of AD patients and mediates the induction of neuronal apoptosis in the presence of oxidative stress. In this study, we investigated the HNE-induced apoptosis mechanism and the protective effects of the cocoa procyanidin fraction (CPF) and its major antioxidant procyanidin B2 against the apoptosis induced by HNE in rat pheochromocytoma (PC12) cells. HNE-induced nuclear condensation and increased sub-G1 fraction, both of which are markers of apoptotic cell death, were inhibited by CPF and procyanidin B2. Intracellular reactive oxygen species (ROS) accumulation was attenuated by pretreatment with CPF and procyanidin B2. CPF and procyanidin B2 also prevented HNE-induced poly(ADP-ribose) polymerase cleavage, antiapoptotic protein (Bcl-2 and Bcl-X_L) down-regulation, and caspase-3 activation. Activation of c-Jun N-terminal protein kinase (JNK) and mitogen-activated protein kinase kinase 4 (MKK4) was attenuated by CPF and procyanidin B2. Moreover, CPF and procyanidin B2 bound directly to MKK4 and inhibited its activity. Data obtained with SP600125, a selective inhibitor of JNK, revealed that JNK is involved in HNE-induced apoptosis through the inhibition of PARP cleavage and caspase-3 activation in PC12 cells. Collectively, these results indicate that CPF and procyanidin B2 protect PC12 cells against HNE-induced apoptosis by blocking MKK4 activity as well as ROS accumulation.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

TNFalpha elicits association of PI 3-kinase with the p60TNFR and activation of PI 3-kinase in adherent neutrophils

Tumor necrosis factor (TNFalpha) is an incomplete secretagogue in neutrophils and requires the engagement of beta integrins to trigger secretion of superoxide anion (O(-)(2)). The p60 TNF receptor (p60TNFR) is responsible for signal transduction for activation of O(-)(2) generation. Activation of TNFalpha-triggered O(-)(2) generation in neutrophils adherent to fibrinogen-coated surfaces involves the beta2 integrin receptor CD11b/CD18. Phosphoinositide 3-kinase (PI 3-kinase) is essential for activation of O(-)(2) generation; wortmannin, an inhibitor of PI 3-kinase, inhibited TNFalpha-elicited O(-)(2) generation. p60TNFR immunoprecipitated from neutrophils was associated with immunoreactivity to PI 3-kinase in adherent neutrophils exposed to TNFalpha, but not in TNFalpha-treated neutrophils in suspension. In addition, PI 3-kinase immunoprecipitated from TNFalpha-activated neutrophils showed enhanced activity in adherent but not in nonadherent neutrophils. These findings suggest that synergism between CD11b/CD18 and p60TNFR in the presence of TNFalpha is required to elicit assembly of a signaling complex involving association of p60TNFR with PI 3-kinase, activation of PI 3-kinase, and generation of O(-)(2).     

Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1.     

Cutting edge: functional role for proline-rich tyrosine kinase 2 in NK cell-mediated natural cytotoxicity

Protein tyrosine kinase activation is one of the first biochemical events in the signaling pathway leading to activation of NK cell cytolytic machinery. Here we investigated whether proline-rich tyrosine kinase 2 (Pyk2), the nonreceptor protein tyrosine kinase belonging to the focal adhesion kinase family, could play a role in NK cell-mediated cytotoxicity. Our results demonstrate that binding of NK cells to sensitive target cells or ligation of beta2 integrins results in a rapid induction of Pyk2 phosphorylation and activation. By contrast, no detectable Pyk2 tyrosine phosphorylation is found upon CD16 stimulation mediated by either mAb or interaction with Ab-coated P815 cells. A functional role for Pyk2 in natural but not Ab-mediated cytotoxicity was demonstrated by the use of recombinant vaccinia viruses encoding the kinase dead mutant of Pyk2. Finally, we provide evidence that Pyk2 is involved in the beta2 integrin-triggered extracellular signal-regulated kinase activation, supporting the hypothesis that Pyk2 plays a role in the natural cytotoxicity by controlling extracellular signal-regulated kinase activation.     

Activation of the c-Jun N-terminal kinase pathway by MST1 is essential and sufficient for the induction of chromatin condensation during apoptosis

Chromatin condensation is the most recognizable nuclear hallmark of apoptosis. Cleavage and activation of MST1 by caspases induce chromatin condensation. It was previously reported that, during apoptosis, activated MST1 induced c-Jun N-terminal kinase (JNK) activation and also phosphorylated histone H2B. However, which of these mechanisms underlies MST1's induction of chromatin condensation has yet to be clarified. Here, we report that MST1-mediated activation of JNK is both essential and sufficient for chromatin condensation. MST1 activation did not result in chromatin condensation in mitogen-activate protein kinase kinase 4 (MKK4)/MKK7 double knockout (MKK4/7 DKO) embryonic stem (ES) cells, which genetically lack the ability to activate JNK. On the other hand, constitutively active JNK was able to induce chromatin condensation in MKK4/7 DKO ES cells. In contrast, histone H2B phosphorylation did not correlate with chromatin condensation in wild-type ES cells. Finally, inhibition of JNK as well as inhibitor of caspase-activated DNase blocked chromatin condensation during Fas-mediated apoptosis of Jurkat cells. Taken together, our results indicate that caspase-mediated cleavage of MST1, followed by MST1-mediated activation of the JNK pathway, is the mechanism responsible for inducing chromatin condensation during apoptosis.     

Titanium dioxide nanoparticles induce apoptosis through the JNK/p38-caspase-8-Bid pathway in phytohemagglutinin-stimulated human lymphocytes

We investigated the signaling pathways underlying nano-TiO₂-induced apoptosis in cultured human lymphocytes. Nano-TiO₂ increased the proportion of sub-G1 cells, activated caspase-9 and caspase-3, and induced caspase-3-mediated PARP cleavage. Nano-TiO₂ also induced loss of mitochondrial membrane potential, which suggests that nano-TiO₂ induces apoptosis via a mitochondrial pathway. A time-sequence analysis of the induction of apoptosis by nano-TiO₂ revealed that nano-TiO₂ triggered apoptosis through caspase-8/Bid activation. We also observed that inhibition of caspase-8 by z-IETD-fmk suppressed the caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and apoptosis. Nano-TiO₂ activated two MAPKs, p38 and JNK. In addition, the selective p38 inhibitor SB203580 and selective JNK inhibitor SP600125 suppressed nano-TiO₂-induced apoptosis and caspase-8 activation to moderate and significant extents, respectively. Knockdown of protein levels of JNK1 and p38 using an RNA interference technique also suppressed caspase-8 activation. Our results suggest that nano-TiO₂-induced apoptosis is mediated by the p38/JNK pathway and the caspase-8-dependent Bid pathway in human lymphocytes.     

Positive regulation of ASK1-mediated c-Jun NH(2)-terminal kinase signaling pathway by the WD-repeat protein Gemin5

Gemin5 is a 170-kDa WD-repeat-containing protein that was initially identified as a component of the survival of motor neurons (SMN) complex. We now show that Gemin5 facilitates the activation of apoptosis signal-regulating kinase 1 (ASK1) and downstream signaling. Gemin5 physically interacted with ASK1 as well as with the downstream kinases SEK1 and c-Jun NH(2)-terminal kinase (JNK1), and it potentiated the H(2)O(2)-induced activation of each of these kinases in intact cells. Moreover, Gemin5 promoted the binding of ASK1 to SEK1 and to JNK1, as well as the ASK1-induced activation of JNK1. In comparison, Gemin5 did not physically associate with MKK7, MKK3, MKK6, or p38. Furthermore, depletion of endogenous Gemin5 by RNA interference (RNAi) revealed that Gemin5 contributes to the activation of ASK1 and JNK1, and to apoptosis induced by H(2)O(2) and tumor necrosis factor-alpha (TNFalpha) in HeLa cells. Together, our results suggest that Gemin5 functions as a scaffold protein for the ASK1-JNK1 signaling module and thereby potentiates ASK1-mediated signaling events.     

PYK2 is an adhesion kinase in macrophages, localized in podosomes and activated by beta(2)-integrin ligation

Pyk2 is a member of the focal adhesion kinase (FAK) family, highly expressed in the central nervous system and haemopoietic cells. Although Pyk2 is homologous to FAK, its role in signaling pathways was shown to be distinct from that of FAK. We show here that Pyk2 is highly expressed in peritoneal IC-21 macrophage and is tyrosine phosphorylated in response to cell attachment to fibronectin and fibrinogen. Upon IC-21 cell adhesion, Pyk2 tyrosine phosphorylation is inhibited by blocking antibodies to the integrin subunits alpha(M) and beta(2). Furthermore, Pyk2 is rapidly tyrosine phosphorylated in response to ligation of beta(2) integrins by antibodies. In migrating macrophages, Pyk2 localizes to perinuclear regions and to podosomes, where it is clustered with tyrosine phosphorylated proteins. Furthermore, in the podosomal ring structure, which surrounds the central actin core, Pyk2 co-localizes with vinculin, talin, and paxillin. In the podosomes, Pyk2 also co-localizes with the integrin alpha(M)beta(2). Lastly, reduction of Pyk2 expression in macrophages leads to inhibition of cell migration. We propose that Pyk2 is functionally linked to the formation of podosomes where it mediates the integrin-cytoskeleton interface and regulates cell spreading and migration.     

Differential role of the JNK and p38 MAPK pathway in c-Myc- and s-Myc-mediated apoptosis

The s-Myc is similar to c-Myc in its ability to induce apoptosis requiring caspase activation. However, s-Myc is distinct from c-Myc in that it has activity to suppress tumor growth and does not require wild-type p53 to induce apoptosis. These facts suggest differential regulation between s-Myc and c-Myc. Here we showed that s-Myc-mediated apoptosis triggered by UV was not inhibited by the inactive form mutant JNK (APF), though c-Myc-mediated apoptosis was. Moreover, we found that JNK did not affect the transactivation activity of s-Myc, but stimulated that of c-Myc. In contrast, both Myc-mediated apoptosis and caspase-3-like protease activation were suppressed by kinase-negative MKK6 and an inactive form mutant p38(AGF). Our results indicate that s-Myc does not require the JNK signaling unlike c-Myc during UV-triggered apoptosis, but the MKK6/p38MAPK pathway might regulate common apoptotic machinery for both s-Myc and c-Myc upstream of caspase.     

The proline-rich tyrosine kinase Pyk2 modulates integrin-mediated neutrophil adhesion and reactive oxygen species generation

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMβ2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation.     

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.     

An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway

Inhibition of NF-kappaB activation increases susceptibility to tumor necrosis factor (TNF)alpha-induced cell death, concurrent with caspases and prolonged c-Jun N-terminal kinase (JNK) activation, and reactive oxygen species (ROS) accumulation. However, the detailed mechanisms are unclear. Here we show that cellular FLICE-inhibitory protein (c-FLIP) is rapidly lost in NF-kappaB activation-deficient, but not wild-type fibroblasts upon TNFalpha stimulation, indicating that NF-kappaB normally maintains the cellular levels of c-FLIP. The ectopic expression of the long form of c-FLIP (c-FLIPL) inhibits TNFalpha-induced prolonged JNK activation and ROS accumulation in NF-kappaB activation-deficient fibroblasts. Conversely, TNFalpha induces prolonged JNK activation and ROS accumulation in c-Flip-/- fibroblasts. Moreover, c-FLIPL directly interacts with a JNK activator, MAP kinase kinase (MKK)7, in a TNFalpha-dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1, apoptosis-signal-regulating kinase 1, and TGFbeta-activated kinase 1. This stimuli-dependent interaction of c-FLIPL with MKK7 might selectively suppress the prolonged phase of JNK activation. Taken that ROS promote JNK activation and activation of the JNK pathway may promote ROS accumulation, c-FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation.     

Differential roles for alpha(M)beta(2) integrin clustering or activation in the control of apoptosis via regulation of akt and ERK survival mechanisms

The role of integrins in leukocyte apoptosis is unclear, some studies suggest enhancement, others inhibition. We have found that beta(2)-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alpha(M)beta(2) delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alpha(M)beta(2) resulted in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins did not show this proapoptotic effect and continued to inhibit apoptosis. This discrepancy was attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alpha(M)beta(2) was capable of stimulating the kinases ERK and Akt. Activated alpha(M)beta(2) stimulated Akt, but not ERK. When proapoptotic stimuli were combined with either alpha(M)beta(2) clustering or activation, Akt activity was blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alpha(M)beta(2) continued to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, beta(2)-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes.     

Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.     

Regulation of myostatin signaling by c-Jun N-terminal kinase in C2C12 cells

Myostatin, a member of the transforming growth factor beta (TGF-beta) superfamily, is a negative regulator of skeletal muscle growth. We found that myostatin could activate c-Jun N-terminal kinase (JNK) signaling pathway in both proliferating and differentiating C2C12 cells. Using small interfering RNA (siRNA) mediated activin receptor type IIB (ActRIIB) knockdown, the myostatin-induced JNK activation was significantly reduced, indicating that ActRIIB was required for JNK activation by myostatin. Transfection of C2C12 cells with TAK1-specific siRNA reduced myostatin-induced JNK activation. In addition, JNK could not be activated by myostatin when the expression of MKK4 was suppressed with MKK4-specific siRNA, suggesting that TAK1-MKK4 cascade was involved in myostatin-induced JNK activation. We also found that blocking JNK signaling pathway by pretreatment with JNK specific inhibitor SP600125, attenuated myostatin-induced upregulation of p21 and downregulation of the differentiation marker gene expression. Furthermore, it was also observed that the presence of SP600125 almost annulled the growth inhibitory role of myostatin. Our findings provide the first evidence to reveal the involvement of JNK signaling pathway in myostatin's function as a negative regulator of muscle growth.