Lung injury in the neonatal piglet caused by hyperoxia and mechanical ventilation

Neonatal lung injury from hyperoxia and mechanical hyperventilation was studied in newborn piglets hyperventilated (arterial PCO2 15-20 Torr) for 24-48 h with 100% O2 and compared with unventilated controls. Pulmonary function testing was performed, and biochemical indicators of lung injury were analyzed from tracheobronchial aspirates at 0, 24, and 48 h. Lung sections were obtained for light and electron microscopy, and bronchoalveolar lavage fluid was analyzed for surfactant composition and activity. At 24 h significant changes in tracheobronchial aspirate albumin concentrations (up 78%) and percent of polymorphonuclear cells (up 16%) were demonstrated. At 48 h a 35% decrease in dynamic lung compliance (P less than 0.05) and a 36% increase in pulmonary resistance (P less than 0.05) were noted. Further biochemical abnormalities occurred with total cell counts increased by 271% (P less than 0.02), albumin 163% (P less than 0.05), total protein 217% (P less than 0.01), and elastase 108% (P less than 0.02). Pathological analyses revealed mild lung injury at 24 h and marked inflammation, abnormal inflation patterns, flattening of Clara cells, fibrinous exudate and edema, early collagen formation, and cell necrosis observed at 48 h. Bronchoalveolar lavage surfactant had normal biophysical activity. Results demonstrate that exposure of neonatal piglets to O2 and mechanical hyperventilation for 48 h cause severe progressive lung injury.     

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.     

PKC-alpha and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.     

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.     

Requirement for protein-tyrosine phosphatase SHP-2 in insulin-induced activation of c-Jun NH(2)-terminal kinase

Mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinases (JNKs), are activated by insulin. Although the mechanism by which the insulin receptor activates extracellular signal-regulated kinases is relatively well defined, the pathway that leads to JNK activation is poorly understood. Overexpression of a catalytically inactive mutant (SHP-2C/S) of the protein-tyrosine phosphatase SHP-2 in Rat-1 fibroblasts that also express human insulin receptors has now revealed that activation of JNKs by insulin and epidermal growth factor, but not that by anisomycin or sorbitol, requires SHP-2. A dominant negative mutant (RasN17) of Ha-Ras blocked insulin-induced JNK activation, whereas a dominant negative mutant (RacN17) of Rac1 or a specific inhibitor (LY294002) of phosphoinositide 3-kinase did not, indicating a role for Ras, but not for Rac or phosphoinositide 3-kinase, in this effect. SHP-2C/S markedly inhibited Ras activation in response to insulin without affecting insulin-induced tyrosine phosphorylation of cellular substrates or the dissociation of the Crk-p130(Cas) complex. In contrast, SHP-2C/S did not inhibit activation of JNKs induced by a constitutively active mutant (RasV12) of Ha-Ras. Furthermore, expression of myristoylated SOS, which functions as a potent activator of Ras, induced JNK activation even when SHP-2 was inactivated. These results suggest that SHP-2 contributes to JNK activation in response to insulin by positively regulating the Ras signaling pathway at the same level as, or upstream from, SOS.     

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.     

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.     

Loss of oxidative stress tolerance in hypertension is linked to reduced catalase activity and increased c-Jun NH2-terminal kinase activation

Hypertension is accompanied by increased levels of reactive oxygen species, which may contribute to progressive renal injury and dysfunction. Here we tested the hypothesis that sensitivity to exogenous hydrogen peroxide (H₂O₂) is enhanced in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rats (SHR) compared to normotensive control Wistar Kyoto rats (WKY). We found that SHR cells were more sensitive to H₂O₂-induced cell death than WKY cells. Lower survival in SHR cells correlated with increased DNA fragmentation, chromatin condensation, and caspase-3 activity, indicating apoptosis. H₂O₂ degradation was slower in SHR than in WKY cells, suggesting that reduced antioxidant enzyme activity might be the basis for their increased sensitivity. In fact, catalase activity was downregulated in SHR cells, whereas glutathione peroxidase activity was similar in both cell types. We next examined whether MAPK signaling pathways contributed to H₂O₂-mediated apoptosis. Inhibition of c-Jun NH₂-terminal kinase (JNK) with SP600125 partially rescued H₂O₂-induced apoptosis in WKY but not in SHR cells. In addition, p54 JNK2 isoform was robustly phosphorylated by H₂O₂, this effect being more pronounced in SHR cells. Together, these results suggest that the survival disadvantage of SHR cells upon exposure to H₂O₂ stems from impaired antioxidant mechanisms and activated JNK proapoptotic signaling pathways.     

Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis

Here, we identified caspase-2, protein kinase C (PKC)delta, and c-Jun NH2-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCdelta as a novel caspase-2 substrate. PKCdelta was cleaved/activated in a caspase-2-dependent manner after doxorubicin treatment both in cells and in vitro. PKCdelta is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCdelta severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCdelta and JNK inhibition show that PKCdelta lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCdelta and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCdelta, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCdelta, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.     

A peptide inhibitor of c-Jun NH2-terminal kinase reduces myocardial ischemia-reperfusion injury and infarct size in vivo

The c-Jun NH(2)-terminal kinase (JNK) pathway of the mitogen-activated protein kinase (MAPK) signaling cascade regulates cell function and survival after stress stimulation. Equally robust studies reported dichotomous results suggesting both protective and detrimental effects of JNK during myocardial ischemia-reperfusion (I/R). The lack of a highly specific JNK inhibitor contributed to this controversy. We recently developed a cell-penetrating, protease-resistant peptide inhibitor of JNK, d-JNKI-1. Here we report on the effects of d-JNKI-1 in myocardial I/R. d-JNKI-1 was tested in isolated-perfused adult rat hearts. Increased activation of JNK, p38-MAPK, and extracellular signal-regulated kinase-1/2 (ERK1/2), as assessed by kinase assays and Western blotting, occurred during I/R. d-JNKI-1 delivered before onset of ischemia prevented the increase in JNK activity while not affecting ERK1/2 and p38-MAPK activation. JNK inhibition reduced ischemic injury, as manifested by increased time to contracture (P < 0.05) and decreased left ventricular end-diastolic pressure during ischemia (P < 0.01), and enhanced posthypoxic recovery of systolic and diastolic function (P < 0.01). d-JNKI-1 reduced mitochondrial cytochrome-c release, caspase-3 activation, and the number of apoptotic cells determined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (P < 0.05), indicating suppression of the mitochondrial machinery of apoptosis. d-JNKI-1 delivered at the time of reperfusion did not improve functional recovery but still prevented apoptosis. In vivo, d-JNKI-1 reduced infarct size after coronary artery occlusion and reperfusion by approximately 50% (P < 0.01). In conclusion, d-JNKI-1 is an important compound that can be used in preclinical models to investigate the role of JNK signaling in vivo. Inhibition of JNK during I/R is cardioprotective in anesthetized rats in vivo.     

Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice

Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/- mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1-/- mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1-/- mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1-/- and wild-type mice. Associated with increased mortality, Jnk1-/- mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice.     

Calcineurin promotes protein kinase C and c-Jun NH2-terminal kinase activation in the heart. Cross-talk between cardiac hypertrophic signaling pathways

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiomyocytes. Both necessary and sufficient roles have been described for the mitogen activated protein kinase(1) (MAPK) signaling pathway, specific protein kinase C (PKC) isoforms, and calcineurin. Here we investigate the interdependence between calcineurin, MAPK, and PKC isoforms in regulating cardiomyocyte hypertrophy using three separate approaches. Hearts from hypertrophic calcineurin transgenic mice were characterized for PKC and MAPK activation. Transgenic hearts demonstrated activation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2), but not p38 MAPK factors. Calcineurin transgenic hearts demonstrated increased activation of PKCalpha, beta(1), and theta, but not of epsilon, beta(2), or lambda. In a second approach, cultured cardiomyocytes were infected with a calcineurin adenovirus to induce hypertrophy and the effects of pharmacologic inhibitors or co-infection with a dominant negative adenovirus were examined. Calcineurin-mediated hypertrophy was prevented with PKC inhibitors, Ca(2+) chelation, and attenuated with a dominant negative SEK-1 (MKK4) adenovirus, but inhibitors of ERK or p38 activation had no effect. In a third approach, we examined the activation of MAPK factors and PKC isoforms during the progression of load-induced hypertrophy in aortic banded rats with or without cyclosporine. We determined that inhibition of calcineurin activity with cyclosporine prevented PKCalpha, theta, and JNK activation, but did not affect PKCepsilon, beta, lambda, ERK1/2, or p38 activation. Collectively, these data indicate that calcineurin hypertrophic signaling is interconnected with PKCalpha, theta, and JNK in the heart, while PKCepsilon, beta, lambda, p38, and ERK1/2 are not involved in calcineurin-mediated hypertrophy.