Two distinct forms of human BLT2: long-form and short-form BLT2.

BLT2 is a low-affinity leukotriene B4 receptor that plays an essential role in the pathogenesis of various inflammatory diseases, including asthma and cancer. BLT2 is minimally expressed in a normal internal environment but is overexpressed in a stress-induced inflammatory environment. Recent research indicated that human BLT2 has two distinct forms. Although their functions are likely to be different, very few studies investigated these differences. Therefore, this paper will discuss about the two distinct forms of human BLT2; the short-form of BLT2 and the long-form of BLT2.

Discovery of a novel BLT2 antagonist for the treatment of inflammatory airway diseases.

Leukotriene B4 (LTB4) is a potent chemoattractant that can recruit and activate immune cells such as neutrophils, eosinophils, and monocytes to sites of inflammation. Excessive production of LTB4 has been linked to acute and chronic inflammatory diseases, including asthma, rheumatoid arthritis, and psoriasis. Inhibiting the binding of LTB4 to its receptors, BLT1 and BLT2, is a potential strategy for treating these conditions. While several BLT1 antagonists have been developed for clinical trials, most have failed due to efficacy and safety issues. Therefore, discovering selective BLT2 antagonists could improve our understanding of the distinct functions of BLT1 and BLT2 receptors and their pharmacological implications. In this study, we aimed to discover novel BLT2 antagonists by synthesizing a series of biphenyl analogues based on a BLT2 selective agonist, CAY10583. Among the synthesized compounds, 15b was found to selectively inhibit the chemotaxis of CHO-BLT2 cells with an IC50 value of 224 nM without inhibiting the chemotaxis of CHO-BLT1 cells. 15b also inhibited the binding of LTB4 and BLT2 with a Ki value of 132 nM. Furthermore, 15b had good metabolic stability in liver microsomes and moderate bioavailability (F = 34%) in in vivo PK studies. 15b also showed in vivo efficacy in a mouse model of asthma, reducing airway hyperresponsiveness by 59% and decreasing Th2 cytokines by up to 46%. Our study provides a promising lead for the development of selective BLT2 antagonists as potential therapeutics for inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

Oxidative stress in Wernicke's encephalopathy.

Wernicke's encephalopathy (WE) is a severe life-threatening disease that occurs due to vitamin B1 (thiamine) deficiency (TD). It is characterized by acute mental disorder, ataxia, and ophthalmoplegia. TD occurs because of the following reasons: insufficient intake, increased demand, and long-term drinking due to corresponding organ damage or failure. Recent studies showed that oxidative stress (OS) can damage organs and cause TD in the brain, which further leads to neurodegenerative diseases, such as WE. In this review, we discuss the effects of TD caused by OS on multiple organ systems, including the liver, intestines, and brain in WE. We believe that strengthening the human antioxidant system and reducing TD can effectively treat WE.

Elucidation of Mechanism for Ligand Efficacy at Leukotriene B4 Receptor 2 (BLT2).

G protein-coupled receptors (GPCRs) have always been important drug targets in the pharmaceutical industry. One major question for the current GPCR drug discovery is how drugs have distinct efficacies at the same GPCR target. Related to this question, we studied how different ligands can have disparate efficacies at Leukotriene B4 receptor (BLT2). By using molecular modeling studies, we predicted that Tyr2716.51 located at TM6 of BLT2 performs as a key trigger for its activation and verified the prediction by site-directed mutagenesis, chemotactic motility studies, which included a chemical derivative of agonist CAY10583. We further identified Asn2756.55 located at TM6 as a weak activation trigger in BLT2 and performed double mutation studies to confirm our computational results. Our results provide strong evidence for the exact mechanism of ligand efficacy at BLT2.

Wogonin suppresses the LPS­enhanced invasiveness of MDA­MB­231 breast cancer cells by inhibiting the 5­LO/BLT2 cascade.

Wogonin, a naturally occurring bioactive monoflavonoid isolated from Scutellariae radix (roots of Scutellariae baicalensis Georgi), has known anticancer effects. However, the molecular signaling mechanism by which wogonin inhibits invasiveness in breast cancer cells remains unclear. In the present study, it was observed that wogonin exerted an inhibitory effect on the lipopolysaccharide (LPS)­enhanced invasiveness of MDA­MB­231 cells. In addition, wogonin inhibited the synthesis of interleukin­8 (IL­8) and matrix metallopeptidase­9 (MMP­9), which are critical for promoting invasiveness in MDA­MB­231 cells. Wogonin also suppressed the expression of leukotriene B4 receptor 2 (BLT2) and the synthesis of its ligand, by inhibiting 5­lipoxygenase (5­LO) in LPS­stimulated MDA­MB­231 cells. Notably, wogonin attenuated the production of IL­8 and MMP­9 by inhibiting the BLT2/extracellular signal­regulated kinase (ERK)­linked cascade. Finally, in vivo, LPS­driven MDA­MB­231 cell metastasis was markedly suppressed by wogonin administration. Overall, the present results suggested that wogonin inhibited the 5­LO/BLT2/ERK/IL­8/MMP­9 signaling cascade and demonstrated that this cascade may be an important target through which wogonin exerts its anticancer effects in breast cancer.

Leukotriene B4 receptor 2 gene polymorphism (rs1950504, Asp196Gly) leads to enhanced cell motility under low-dose ligand stimulation.

Recently, single-nucleotide polymorphisms (SNPs) in G-protein-coupled receptors (GPCRs) have been suggested to contribute to physiopathology and therapeutic effects. Leukotriene B4 receptor 2 (BLT2), a member of the GPCR family, plays a critical role in the pathogenesis of several inflammatory diseases, including cancer and asthma. However, no studies on BLT2 SNP effects have been reported to date. In this study, we demonstrate that the BLT2 SNP (rs1950504, Asp196Gly), a Gly-196 variant of BLT2 (BLT2 D196G), causes enhanced cell motility under low-dose stimulation of its ligands. In addition, we demonstrated that Akt activation and subsequent production of reactive oxygen species (ROS), both of which act downstream of BLT2, are also increased by BLT2 D196G in response to low-dose ligand stimulation. Furthermore, we observed that the ligand binding affinity of BLT2 D196G was enhanced compared with that of BLT2. Through homology modeling analysis, it was predicted that BLT2 D196G loses ionic interaction with R197, potentially resulting in increased agonist-receptor interaction. To the best of our knowledge, this report is the first to describe a SNP study on BLT2 and shows that BLT2 D196G enhances ligand sensitivity, thereby increasing cell motility in response to low-dose ligand stimulation.

RanBPM inhibits BLT2-mediated IL-8 production and invasiveness in aggressive breast cancer cells.

RanBPM is a scaffolding protein that regulates several cellular processes by interacting with various proteins. Previously, we reported that RanBPM acts as a negative regulator of BLT2, a low-affinity leukotriene B4 receptor; thus, it interferes with BLT2-mediated cell motility. In the present study, we observed that the expression levels of RanBPM were markedly reduced in the highly aggressive MDA-MB-435 and MDA-MB-231 human breast cancer cell lines compared with those in non-invasive MCF-7 cells. Additionally, we found that the restoration of RanBPM levels suppressed the invasiveness of these aggressive breast cancer cells in a manner dependent on BLT2 activation. In contrast, the knockdown of endogenous RanBPM by shRNA strongly promoted invasiveness in non-invasive MCF-7 cells. We also observed that RanBPM suppressed the invasiveness of aggressive breast cancer cells by inhibiting BLT2-mediated reactive oxygen species (ROS) generation and IL-8 production. Taken together, our results suggest that RanBPM acts as a negative regulator of BLT2, thus attenuating the invasiveness of aggressive breast cancer cells.

BLT2 is a pro-tumorigenic mediator during cancer progression and a therapeutic target for anti-cancer drug development.

Cancer is a leading cause of death worldwide and has been linked to inflammation. Leukotriene B4 (LTB4) is synthesized from arachidonic acid via the 5-lipoxygenase pathway and is a potent chemoattractant for inflammatory cells. LTB4 was recently shown to be associated with the pathogenesis of inflammatory diseases, including cancer. Of the two known LTB4 receptors, BLT1 and BLT2, the biological roles of the low-affinity LTB4 receptor 2, BLT2, have only recently been elucidated. This review focuses on recent discoveries regarding BLT2 and its roles in cancer progression and the downstream signaling mechanisms of the BLT2-linked signaling cascade in cancer cells. We believe that these findings will facilitate the development of new cancer treatments.

RanBPM protein acts as a negative regulator of BLT2 receptor to attenuate BLT2-mediated cell motility.

BLT2, a low affinity receptor for leukotriene B4 (LTB4), is a member of the G protein-coupled receptor family and is involved in many signal transduction pathways associated with various cellular phenotypes, including chemotactic motility. However, the regulatory mechanism for BLT2 has not yet been demonstrated. To understand the regulatory mechanism of BLT2, we screened and identified the proteins that bind to BLT2. Using a yeast two-hybrid assay with the BLT2 C-terminal domain as bait, we found that RanBPM, a previously proposed scaffold protein, interacts with BLT2. We demonstrated the specific interaction between BLT2 and RanBPM by GST pulldown assay and co-immunoprecipitation assay. To elucidate the biological function of the RanBPM-BLT2 interaction, we evaluated the effects of RanBPM overexpression or knockdown. We found that BLT2-mediated motility was severely attenuated by RanBPM overexpression and that knockdown of endogenous RanBPM by shRNA strongly promoted BLT2-mediated motility, suggesting a negative regulatory function of RanBPM toward BLT2. Furthermore, we observed that the addition of BLT2 ligands caused the dissociation of BLT2 and RanBPM, thus releasing the negative regulatory effect of RanBPM. Finally, we propose that Akt-induced BLT2 phosphorylation at residue Thr(355), which occurs after the addition of BLT2 ligands, is a potential mechanism by which BLT2 dissociates from RanBPM, resulting in stimulation of BLT2 signaling. Taken together, our results suggest that RanBPM acts as a negative regulator of BLT2 signaling to attenuate BLT2-mediated cell motility.

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid suppresses UV-induced IL-6 synthesis in keratinocytes, exerting an anti-inflammatory activity.

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid (12- HHT) is an enzymatic product of prostaglandin H(2) (PGH(2)) derived from cyclooxygenase (COX)-mediated arachidonic acid metabolism. Despite the high level of 12-HHT present in tissues and bodily fluids, its precise function remains largely unknown. In this study, we found that 12-HHT treatment in HaCaT cells remarkably down-regulated the ultraviolet B (UVB) irradiation-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation. In an approach to identify the down-stream signaling mechanism by which 12-HHT down-regulates UVB-induced IL-6 synthesis in keratinocytes, we observed that 12-HHT inhibits the UVB-stimulated activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB). In addition, we found that 12-HHT markedly up-regulates MAPK phosphatase-1 (MKP-1), a critical negative regulator of p38 MAPK. When MKP-1 was suppressed by siRNA knock-down, the 12-HHT-mediated inhibitory effects on the UVB-stimulated activation of p38 MAPK and NF-κB, as well as the production of IL-6, were attenuated in HaCaT cells. Taken together, our results suggest that 12-HHT exerts anti-inflammatory effect via up-regulation of MKP-1, which negatively regulates p38 MAPK and NF-κB, thus attenuating IL-6 production in UVB-irradiated HaCaT cells. Considering the critical role of IL-6 in cutaneous inflammation, our findings provide the basis for the application of 12-HHT as a potential anti-inflammatory therapeutic agent in UV-induced skin diseases.

BLT2 phosphorylation at Thr355 by Akt is necessary for BLT2-mediated chemotaxis.

BLT2, a low-affinity leukotriene B(4) (LTB(4)) receptor, is a member of the G protein-coupled receptor family and is involved in multiple cellular responses, including chemotaxis. Despite its biological significance, the mechanisms of BLT2 regulation, especially by protein kinases, are poorly characterised. In this study, we found that Akt phosphorylates BLT2 at its C-terminal Thr(355) residue and that this event is critical for BLT2-mediated chemotactic responses. In addition, we found that Rac1 stimulation and subsequent reactive oxygen species (ROS) production lie downstream of BLT2 phosphorylation, thus mediating chemotaxis.

Proinflammatory cytokine IL-1beta stimulates IL-8 synthesis in mast cells via a leukotriene B4 receptor 2-linked pathway, contributing to angiogenesis.

Recent studies have suggested that mast cells have critical roles in angiogenesis. However, the detailed mechanism by which mast cells contribute to angiogenesis is not yet clearly understood, especially in response to proinflammatory cytokines. In this study, we showed that the proinflammatory cytokine IL-1beta induces the synthesis of IL-8, a potent angiogenic factor, in human mast cells via the leukotriene B(4) receptor (BLT)2. We also characterized the BLT2 downstream signaling pathway and determined that BLT2-mediated IL-8 synthesis involves the upregulation of Nox1, a member of the NADPH oxidase family, Nox1-dependent reactive oxygen species generation and the subsequent activation of the redox-sensitive transcription factor NF-kappaB. For instance, knockdown of BLT2 and Nox1 with specific small interfering RNA, treatment with a specific BLT2 antagonist, LY255283, or treatment with a potential Nox inhibitor, diphenylene iodonium, suppressed IL-1beta-induced IL-8 synthesis. We found that the conditioned media collected from IL-1beta-treated human mast cell line HMC-1 had significantly enhanced angiogenic activity that could be dramatically attenuated by either small interfering RNA knockdown of BLT2 or treatment with neutralizing Ab to IL-8. Finally, the experiments were repeated using human primary cord blood-derived mast cells, and the results were clearly reproduced. Taken together, our results suggest that BLT2-Nox1-reactive oxygen species-dependent pathway plays a role in promoting the secretion of IL-8 from human mast cells in response to the proinflammatory cytokine IL-1beta, thus contributing to angiogenesis.