Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation

The involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase-1 (JNK1) has never been investigated in hemostasis and thrombosis. Using two JNK inhibitors (SP600125 and 6o), we have demonstrated that JNK1 is involved in collagen-induced platelet aggregation dependent on ADP. In these conditions, JNK1 activation requires the coordinated signaling pathways of collagen receptors (alpha2beta1 and glycoprotein (GP)VI) and ADP. In contrast, JNK1 is not required for platelet adhesion on a collagen matrix in static or blood flow conditions (300-1500 s(-1)) involving collagen receptors (alpha2beta1 and GPVI). Importantly, at 1500 s(-1), JNK1 acts on thrombus formation on a collagen matrix dependent on GPIb-von Willebrand factor (vWF) interaction but not ADP receptor activation. This is confirmed by the involvement of JNK1 in shear-induced platelet aggregation at 4000 s(-1). We also provide evidence during rolling and adhesion of platelets to vWF that platelet GPIb-vWF interaction triggers alphaIIbbeta3 activation in a JNK1-dependent manner. This was confirmed with a Glanzmann thrombastenic patient lacking alphaIIbbeta3. Finally, in vivo, JNK1 is involved in arterial but not in venular thrombosis in mice. Overall, our in vitro studies define a new role of JNK1 in thrombus formation in flowing blood that is relevant to thrombus development in vivo.     

Phosphorylation of protein phosphatase 2Czeta by c-Jun NH2-terminal kinase at Ser92 attenuates its phosphatase activity

The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Czeta, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH 2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser (92), Thr (202), and Thr (205) of PP2Czeta. Phosphorylation of these Ser and Thr residues of PP2Czeta ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Czeta by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser (92) but not at Thr (202) or Thr (205) suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Czeta at Ser (92) by stress-activated JNK attenuates its phosphatase activity in cells.     

c-Jun NH2-terminal kinase mediates interleukin-1beta-induced inhibition of lacrimal gland secretion

Sjögren's syndrome, an inflammatory disease affecting the lacrimal and salivary glands, is the leading cause of aqueous tear‐deficient type of dry eye. We previously showed that interleukin‐1β (IL‐1β) protein is up regulated in the lacrimal gland of a murine model of Sjögren's syndrome and that exogenous addition of this cytokine inhibits neurotransmitter release and lacrimal gland protein secretion. In the present study we investigated the role of c‐Jun NH₂‐terminal kinase (JNK) in IL‐1β‐mediated inhibition of lacrimal gland secretion and tear production. In vitro, IL‐1β induced a time‐dependent activation of JNK with a maximum 7.5‐fold at 30 min. SP600125, a JNK inhibitor, inhibited, in a concentration‐dependent manner, IL‐1β‐induced activation of JNK with a maximum of 87% at 10^?4 m . In vivo, IL‐1β stimulated JNK and the expression of the inducible isoform of nitric oxide synthase (iNOS). IL‐1β inhibited high KCl and adrenergic agonist induced protein secretion by 85% and 66%, respectively. SP600125 alleviated the inhibitory effect of IL‐1β on KCl‐ and agonist‐induced protein secretion by 79% and 47%, respectively, and completely blocked the expression of iNOS. Treatment for 7 days with SP600125 increased tear production in a murine model of Sjögren's syndrome dry eye. We conclude that JNK plays a pivotal role in IL‐1β‐mediated inhibition of lacrimal gland secretion and subsequent dry eye.     

Role of Gab1 in UV-induced c-Jun NH2-terminal kinase activation and cell apoptosis

Exposure of mammalian cells to UV irradiation leads to activation of the c-Jun NH(2)-terminal protein kinase (JNK) pathway, which is associated with cell apoptosis. However, the molecular mechanism for JNK activation by UV exposure is not fully understood. We show here an essential role of a multisubstrate adapter, Gab1, in this signaling cascade. Gab1-deficient mouse fibroblast cells were defective in induction of JNK activity by UV exposure or heat shock, and this defect was rescued by reintroduction of Gab1 into Gab1(-/-) cells. Consistently, Gab1(-/-) cells displayed reduced caspase 3 induction and apoptotic cell death in response to UV irradiation. Gab1 was constitutively complexed with JNK and became tyrosine phosphorylated in UV-irradiated cells. Genetic and pharmaceutical analyses suggest the involvement of c-Met and the Src family tyrosine kinases in mediating UV-induced Gab1 phosphorylation as well as JNK activation. In aggregate, these observations identify a new function of Gab1 in the response of mammalian cells to UV light.     

Inhibition of c-Jun NH2-terminal kinase activity improves ischemia/reperfusion injury in rat lungs

Although c-Jun NH(2)-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-kappaB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4 degrees C for 4 h followed by reperfusion at 37 degrees C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-kappaB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-alpha into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.     

Poliovirus induces Bax-dependent cell death mediated by c-Jun NH2-terminal kinase

Poliovirus (PV) is the causal agent of paralytic poliomyelitis, a disease that involves the destruction of motor neurons associated with PV replication. In PV-infected mice, motor neurons die through an apoptotic process. However, mechanisms by which PV induces cell death in neuronal cells remain unclear. Here, we demonstrate that PV infection of neuronal IMR5 cells induces cytochrome c release from mitochondria and loss of mitochondrial transmembrane potential, both of which are evidence of mitochondrial outer membrane permeabilization. PV infection also activates Bax, a proapoptotic member of the Bcl-2 family; this activation involves its conformational change and its redistribution from the cytosol to mitochondria. Neutralization of Bax by vMIA protein expression prevents cytochrome c release, consistent with a contribution of PV-induced Bax activation to mitochondrial outer membrane permeabilization. Interestingly, we also found that c-Jun NH(2)-terminal kinase (JNK) is activated soon after PV infection and that the PV-cell receptor interaction alone is sufficient to induce JNK activation. Moreover, the pharmacological inhibition of JNK by SP600125 inhibits Bax activation and cytochrome c release. This is, to our knowledge, the first demonstration of JNK-mediated Bax-dependent apoptosis in PV-infected cells. Our findings contribute to our understanding of poliomyelitis pathogenesis at the cellular level.     

Chlamydophilal antigens induce foam cell formation via c-Jun NH2-terminal kinase

Chlamydophila pneumoniae is known to be associated with atherosclerosis. Recent studies have reported that components of Chlamydophila pneumoniae (chlamydophilal antigens) induce foam cell formation in macrophages. However, the mechanism of foam cell formation induced by chlamydophilal antigens has yet to be elucidated. In this paper, we first found that mitogen-activated protein kinases including extracellular signal-regulated kinase, p38 and c-Jun NH2 terminal kinase are phosphorylated after stimulation by chlamydophilal antigens. We then showed that chlamydophilal antigens induce foam cell formation mainly via c-Jun NH2 terminal kinase. Finally, we demonstrated that foam cell formation and phosphorylation of mitogen-activated protein kinases induced by chlamydophilal antigens are mainly recognized through Toll-like receptor 2. These results collectively indicated that chlamydophilal antigens induce foam cell formation mainly via Toll-like receptor 2 and c-Jun NH2 terminal kinase.     

Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck

p21-activated kinase (PAK) has been shown to be an upstream mediator of JNK in angiotensin II (AngII) signaling. Little is known regarding other signaling molecules involved in activation of PAK and JNK by AngII. Rho family GTPases Rac and Cdc42 have been shown to enhance PAK activity by binding to p21-binding domain of PAK (PAK-PBD). In vascular smooth muscle cells (VSMC) AngII stimulated Rac1 binding to GST-PAK-PBD fusion protein. Pretreatment of VSMC by genistein inhibited AngII-induced Rac1 activation, whereas Src inhibitor PP1 had no effect. Inhibition of protein kinase C by phorbol 12,13-dibutyrate pretreatment also decreased AngII-mediated activation of Rac1. The adaptor molecule Nck has been shown previously to mediate PAK activation by facilitating translocation of PAK to the plasma membrane. In VSMC AngII stimulated translocation of Nck and PAK to the membrane fraction. Overexpression of dominant-negative Nck in Chinese hamster ovary (CHO) cells, stably expressing the AngII type I receptor (CHO-AT1), inhibited both PAK and JNK activation by AngII, whereas it did not affect ERK1/2. Finally, dominant-negative Nck inhibited AngII-induced DNA synthesis in CHO-AT1 cells. Our data provide evidence for Rac1 and Nck as upstream mediators of PAK and JNK in AngII signaling and implicate JNK in AngII-induced growth responses.     

G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.     

Activation mechanism and physiological roles of stress-activated protein kinase/c-Jun NH2-terminal kinase in mammalian cells

Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stress or extracellular signals. Recent studies, including the analysis with knockout cells and mice, have led towards understanding the molecular mechanism of stress-induced SAPK/JNK activation and the physiological roles of SAPK/JNK in embryonic development and immune responses. Two SAPK/JNK activators, SEK1 and MKK7, are required for full activation of SAPK/JNK, which responds to various stimuli in an all-or-none manner in mouse embryonic stem (ES) cells. SAPK/JNK activation plays essential roles in organogenesis during mouse development by regulating cell proliferation, survival or apoptosis and in immune responses by regulating cytokine gene expression. Furthermore, SAPK/JNK is involved in regulation of mRNA stabilization, cell migration, and cytoskeletal integrity. Thus, SAPK/JNK has a wide range of functions in mammalian cells.     

The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307)

Tumor necrosis factor alpha (TNFalpha) inhibits insulin action, in part, through serine phosphorylation of IRS proteins; however, the phosphorylation sites that mediate the inhibition are unknown. TNFalpha promotes multipotential signal transduction cascades, including the activation of the Jun NH(2)-terminal kinase (JNK). Endogenous JNK associates with IRS-1 in Chinese hamster ovary cells. Anisomycin, a strong activator of JNK in these cells, stimulates the activity of JNK bound to IRS-1 and inhibits the insulin-stimulated tyrosine phosphorylation of IRS-1. Serine 307 is a major site of JNK phosphorylation in IRS-1. Mutation of serine 307 to alanine eliminates phosphorylation of IRS-1 by JNK and abrogates the inhibitory effect of TNFalpha on insulin-stimulated tyrosine phosphorylation of IRS-1. These results suggest that phosphorylation of serine 307 might mediate, at least partially, the inhibitory effect of proinflammatory cytokines like TNFalpha on IRS-1 function.