Leukotriene B 4 is a potent chemoattractant known to be involved mainly in inflammation, immune responses, and host defense against infection, although the exact signaling mechanisms by which it exerts its effects are not well understood. Here we show that exogenous leukotriene B 4 induces reactive oxygen species (ROS) generation via a Rac-dependent pathway, and that stable expression of Rac N17 , a dominant negative Rac1 mutant, completely blocks leukotriene B 4 -induced ROS generation. In addition, leukotriene B 4 -induced ROS generation is selectively blocked by inhibition of ERK or cytosolic phospholipase A 2 , but not p38 kinase, which is indicative of its dependence on ERK activation and synthesis of arachidonic acid. Consistent with those findings, leukotriene B 4 Rac-dependently stimulates ERK and cytosolic phospholipase A 2 activity, and transient transfection with plasmid expressing Rac V12 , a constitutively activated Rac1 mutant, also dose-dependently stimulates ERK activity. Our findings suggest that ERK and cytosolic phospholipase A 2 are situated downstream of Rac, and we conclude that Rac, ERK, and cytosolic phospholipase A 2 all play pivotal roles in mediating the ROS generation that appears to be a prerequisite for leukotriene B 4 -induced chemotaxis and cell proliferation. LTs1 are potent biological mediators of inflammation generated from arachidonic acid via the 5-LO pathway (1, 2). Among them, LTB 4 is one of the most potent chemoattractants known, acting mainly on neutrophils and eosinophils, but also on mast cells and endothelial cells (3)(4)(5). LTB 4 stimulates a number of cellular functions in addition to chemotaxis, including release of lysosomal enzymes and production of ROS (6 -8); it also promotes cell adhesion to vascular endothelial cells and transmigration, which amplifies inflammatory responses. Although LTB 4 -induced leukocyte recruitment is thought to play a protective role in the host defense against various pathogens, it is also involved in the pathogenesis of such inflammatory diseases as bronchial asthma (9, 10), inflammatory bowel diseases (11, 12), and psoriasis (13,14).Despite many reports on the cellular functions of LTB 4 , the exact signaling pathway along which its biological activities are transduced remains largely unknown. It is known, however, that LTB 4 acts via two G protein-coupled receptors, . The former is a high affinity LTB 4 receptor expressed mainly in polymorphonuclear leukocytes, whereas the latter is a ubiquitous, low affinity receptor whose expression is highest in spleen (17, 18). The details of the cellular functions of BLT1 and BLT2 are still largely unknown. Recently, however, LTB 4 -induced chemotaxis was shown to be completely inhibited in cells pretreated with PTX (100 ng/ml), indicating the participation of a PTX-sensitive G protein in LTB 4 signaling to chemotaxis (16). LTB 4 also elicits increases in intracellular free Ca 2ϩ and inositol 1,4,5-triphosphate, but these are apparently not involved in the chemotactic response by LTB 4 (15). ...
The neuregulin 1 (NRG1) fusion is a recently identified novel driver oncogene in invasive mucinous adenocarcinoma of the lung (IMA). After identification of a case of SLC3A2-NRG1 in a patient with IMA, we verified this fusion gene in a cohort of 59 patients with IMA. Targeted cancer panel sequencing and RT-PCR identified the possible coexistence of other driver oncogenes. Among 59 IMAs, we found 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Of 16 patients with NRG1 fusions, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusions. When comparing overall survival (OS) according to the presence of NRG1 fusions showed that patients harboring NRG1 fusions had significantly inferior OS than those without NRG1 fusions (hazard ratio = 0.286; 95% confidence interval, .094 to .865). Ectopic expression of the SLC3A2-NRG1 fusion in lung cancer cells increased cell migration, proliferation and tumor growth in vitro and in xenograft models, suggesting oncogenic function for the fusion protein. We found that the SLC3A2-NRG1 fusion promoted ERBB2-ERBB3 phosphorylation and heteroduplex formation and activated the downstream PI3K/AKT/mTOR pathway through paracrine signaling. These findings suggested that the SLC3A2-NRG1 fusion was a driver in IMA with an important prognostic impact. SLC3A2-NRG1 should be considered a therapeutic target for patients with IMA.
The epithelial cells that form a barrier lining the lung airway are key regulators of neutrophil trafficking into the airway lumen in a variety of lung inflammatory diseases. Although the lipid mediator leukotriene B4 (LTB4) is known to be a principal chemoattractant for recruiting neutrophils to inflamed sites across the airway epithelium, the precise signaling mechanism involved remains largely unknown. In the present study, therefore, we investigated the signaling pathway through which LTB4 induces transepithelial migration of neutrophils. We found that LTB4 induces concentration-dependent transmigration of DMSO-differentiated HL-60 neutrophils and human polymorphonuclear neutrophils across A549 human lung epithelium. This effect was mediated via specific LTB4 receptors and was inhibited by pretreating the cells with N-acetylcysteine (NAC), an oxygen free radical scavenger, with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase-like flavoproteins, or with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Consistent with those findings, LTB4-induced ERK phosphorylation was completely blocked by pretreating cells with NAC or DPI. Taken together, our observations suggest LTB4 signaling to transepithelial migration is mediated via generation of reactive oxygen species, which leads to downstream activation of ERK. The physiological relevance of this signaling pathway was demonstrated in BALB/c mice, in which intratracheal instillation of LTB4 led to acute recruitment of neutrophils into the airway across the lung epithelium. Notably, the response to LTB4 was blocked by NAC, DPI, PD98059, or CP105696, a specific LTB4 receptor antagonist.
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