Activated blood platelets and macrophages metabolize prostaglandin H2 into thromboxane A2 and 12(S)-hydroxyheptadeca-5Z, 8E, 10E–trienoic acid (12-HHT) in an equimolar ratio through the action of thromboxane synthase. Although it has been shown that 12-HHT is abundant in tissues and bodily fluids, this compound has long been viewed as a by-product lacking any specific function. We show that 12-HHT is a natural ligand for leukotriene B4 (LTB4) receptor-2 (BLT2), a G protein–coupled receptor that was originally identified as a low-affinity receptor for LTB4. BLT2 agonistic activity in lipid fractions from rat small intestine was identified as 12-HHT using high-performance liquid chromatography and mass spectrometry. Exogenously expressed BLT2 in mammalian cells was activated by synthetic 12-HHT, as assessed by guanosine 5′-O-(3-thio) triphosphate binding, the activation of intracellular signaling pathways, and chemotaxis assay. Displacement analysis using [3H]LTB4 showed that 12-HHT binds to BLT2 with a higher affinity than LTB4. Lipid extracts from cyclooxygenase 1–deficient mice failed to activate BLT2. Bone marrow–derived mast cells (BMMCs) isolated from wild-type mice migrated toward a low concentration of 12-HHT, whereas BMMCs from BLT2-deficient mice did not. We conclude that 12-HHT is a natural lipid agonist of BLT2 in vivo and induces chemotaxis of mast cells.

BLT2 is a low-affinity leukotriene B(4) (LTB(4)) receptor that is activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB(4). Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. To clarify the role of BLT receptors in intestinal inflammation, we assessed susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice lacking either BLT1 or BLT2. BLT2(-/-) mice exhibited increased sensitivity to DSS as compared to wild-type and BLT1(-/-) mice, with more severe body weight loss and inflammation. Expression of inflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-1β, and IL-6, chemokines such as CXC chemokine ligand 9 (CXCL9) and C-C motif chemokine 19 (CCL19), and metalloproteinases was highly up-regulated in the colons of DSS-treated BLT2(-/-) mice, and there was an enhanced accumulation of activated macrophages. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also markedly accelerated in the crypts of DSS-treated BLT2(-/-) mice. Madin-Darby canine kidney II (MDCKII) cells transfected with BLT2 exhibited enhanced barrier function as measured by transepithelial electrical resistance (TER) and FITC-dextran leakage through MDCK monolayers. Thus, BLT2 is expressed in colon cryptic cells and appears to protect against DSS-induced colitis, possibly by enhancing barrier function in epithelial cells of the colon. These novel results suggest a direct anti-inflammatory role of BLT2 that is distinct from the proinflammatory roles of BLT1.

Leukotriene B4 (LTB4) is a potent chemoattractant and activator for granulocytes and macrophages and is considered to be an inflammatory mediator. Two G-protein-coupled receptors for LTB4, BLT1 and BLT2, have been cloned from human and shown to be high and low affinity LTB4 receptors, respectively. To reveal the biological roles of BLT2 using mouse disease models, we cloned and characterized mouse BLT2. Chinese hamster ovary cells stably expressing mouse BLT2 exhibited specific binding to LTB4, LTB4-induced calcium mobilization, inhibition of adenylyl cyclase, and phosphorylation of extracellular signal-regulated kinase. We found that Compound A (4 -{[pentanoyl (phenyl) amino] methyl}-1, 1 -biphenyl-2-carboxylic acid) was a BLT2-selective agonist and induced Ca 2؉ mobilization and phosphorylation of extracellular signal-regulated kinase through BLT2, whereas it had no effect on BLT1. 12-epi LTB4 exhibited a partial agonistic activity against mBLT1 and mBLT2, whereas 6-trans-12-epi LTB4 did not. Northern blot analysis showed that mouse BLT2 is expressed highly in small intestine and skin in contrast to the ubiquitous expression of human BLT2. By in situ hybridization and the reverse transcriptase polymerase chain reaction, we demonstrated that mouse BLT2 is expressed in follicular and interfollicular keratinocytes. Compound A, LTB4, and 12-epi LTB4 all induced phosphorylation of extracellular signal-regulated kinase in primary mouse keratinocytes. Furthermore, Compound A and LTB4 induced chemotaxis in primary mouse keratinocytes. These data suggest the presence of functional BLT2 in primary keratinocytes.Leukotriene B4 (LTB4 1 ; 5[S], 12[R]-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid) (1) is biosynthesized from arachidonic acid released from membrane phospholipids by the action of cytosolic and various types of phospholipase A2s (2). Two enzymes, 5-lipoxygenase (3, 4) and LTA4 hydrolase (5, 6), are required for LTB4 biosynthesis from arachidonic acid (7,8). LTB4 is a potent proinflammatory mediator mainly synthesized by phagocytic cells, principally polymorphonuclear leukocyte and macrophage, which responds to infectious and inflammatory stimuli. LTB4 induces granulocyte migration, degranulation, superoxide generation, and adhesion to vascular endothelial cells. It plays an important role in the initial phase of the inflammatory response and is thought to be involved in a number of inflammatory diseases including psoriasis (9), bronchial asthma (10), rheumatoid arthritis (11) inflammatory bowel diseases (12), and ischemic tissue damage (13). Recently, LTB4 was shown to act as an immunomodulator by recruiting CD4ϩ early effector and CD8ϩ cytotoxic effector T-cells to inflamed tissues (14 -16), suggesting novel roles of LTB4 in immunological disorders. Thus, many BLT antagonists have been currently under development as novel antiinflammatory and anti-allergic drugs.Two types of LTB4 receptors (BLT1 and BLT2) have been isolated. BLT1 (17) and BLT2 (18 -20) are high and low affinity LTB4 receptors, respecti...

Summary Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), play fundamental roles in mammalian physiology. Although PUFA imbalance causes various disorders, mechanisms of the regulation of their systemic levels are poorly understood. Here, we report that hepatic DHA-containing phospholipids (DHA-PLs) determine the systemic levels of PUFAs through the SREBP1-mediated transcriptional program. We demonstrated that liver-specific deletion of Agpat3 leads to a decrease of DHA-PLs and a compensatory increase of ARA-PLs not only in the liver but also in other tissues including the brain. Together with recent findings that plasma lysophosphatidylcholine (lysoPC) is the major source of brain DHA, our results indicate that hepatic AGPAT3 contributes to brain DHA accumulation by supplying DHA-PLs as precursors of DHA-lysoPC. Furthermore, dietary fish oil-mediated suppression of hepatic PUFA biosynthetic program was blunted in liver-specific Agpat3 deletion. Our findings highlight the central role of hepatic DHA-PLs as the molecular rheostat for systemic homeostasis of PUFAs.

BLT2 is a low‐affinity leukotriene B4 (LTB4) receptor that is activated by 12(S)‐hydroxyhep‐tadeca‐5Z,8E,10E‐trienoic acid (12‐HHT) and LTB4. Despite the well‐defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. To clarify the role of BLT receptors in intestinal inflammation, we assessed susceptibility to dextran sodium sulfate (DSS)‐induced colitis in mice lacking either BLT1 or BLT2. BLT2–/– mice exhibited increased sensitivity to DSS as compared to wild‐type and BLT1_/_ mice, with more severe body weight loss and inflammation. Expression of inflammatory cytokines such as interferon (IFN)‐γ, interleukin (IL)‐1β, and IL‐6, chemokines such as CXC chemokine ligand 9 (CXCL9) and C‐C motif chemokine 19 (CCL19), and metalloproteinases was highly up‐regulated in the colons of DSS‐treated BLT2–/– mice, and there was an enhanced accumulation of activated macrophages. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also markedly accelerated in the crypts of DSS‐treated BLT2_/_ mice. Madin‐Darby canine kidney II (MDCKII) cells transfected with BLT2 exhibited enhanced barrier function as measured by transepithelial electrical resistance (TER) and FITC‐dextran leakage through MDCK monolayers. Thus, BLT2 is expressed in colon cryptic cells and appears to protect against DSS‐induced colitis, possibly by enhancing barrier function in epithelial cells of the colon. These novel results suggest a direct anti‐inflammatory role of BLT2 that is distinct from the proinflammatory roles of BLT1.—Iizuka, Y., Okuno, T., Saeki, K., Uozaki, H., Okada, S., Misaka, T., Sato, T., Toh, H., Fukayama, M., Takeda, N., Kita, Y., Shimizu, T., Nakamura, M., Yokomizo, T. Protective role of the leukotriene B4 receptor BLT2 in murine inflammatory colitis. FASEB J. 24, 4678–4690 (2010). http://www.fasebj.org

Aims Interferon-γ (IFN-γ) exhibits hepatotoxicity through signal transducer and activator of transcription 1 (STAT1) activation. On the contrary, interleukin-11 (IL-11) shows tissue-protective effects on various organs including the liver through STAT3 activation. Here, we found that IL-11 pretreatment protects hepatocytes from IFN-γ-induced death and investigated the molecular mechanisms, particularly focusing on signal crosstalk. Methods and results Primary culture mouse hepatocytes were treated with IL-11 prior to IFN-γ, and cell death was evaluated by lactate dehydrogenase release into media. As a result, IL-11 pretreatment effectively suppressed IFN-γ-induced hepatocyte death. Since IFN-γ-induced hepatocyte death requires STAT1 signaling, the activity of STAT1 was analyzed. IFN-γ robustly activated STAT1 with its peak at 1 hr after stimulation, which was significantly attenuated by IL-11 pretreatment. Consistently, IL-11 pretreatment impeded mRNA increase of STAT1-downstream molecules promoting cell death, i.e., IRF-1, caspase 1, bak, and bax. IL-11-mediated suppression of STAT1 signaling was presumably due to upregulation of the suppressor of cytokine signaling (SOCS) genes, which are well-known negative feedback regulators of the JAK/STAT pathway. Interestingly, however, IFN-γ pretreatment failed to affect the following IL-11-induced STAT3 activation, although IFN-γ also upregulated SOCSs. Finally, we demonstrated that IL-11 pretreatment mitigated oxidative stress through increasing expression of ROS scavengers. Conclusion IL-11 protects hepatocytes from IFN-γ-induced death via STAT1 signal suppression and ROS scavenging. Further investigation into the mechanisms underlying selective negative feedback regulation of IFN-γ/STAT1 signaling compared to IL-11/STAT3 signaling may shed new light on the molecular biology of hepatocytes.

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BACKGROUND AND AIMS Proteinuria is a major risk factor for the progression of chronic kidney disease. Protein overload in the proximal tubular epithelial cells causes oxidative stress, lysosomal dysfunction, inflammation and apoptosis, resulting in proximal tubule dysfunction. Recent studies have focussed on the association between proximal tubule injury and cellular senescence and the development of drugs targeting and removing senescent proximal tubular cells. Senescent cells show resistance to apoptosis and persistently secrete inflammatory cytokines, resulting in chronic inflammation. It has been suggested that excess proliferation induced by fatty acids causes senescence of proximal tubular cells. However, the association between protein overload, proliferation and cellular senescence in proximal tubular cells remains unclear. The present study aimed to clarify the effect of protein overload on cell proliferation and senescence in a proteinuric mouse model and in an immortalized proximal tubular epithelial cell line. Moreover, it was evaluated whether the endocytic receptors for protein uptake, megalin and cubilin, affect protein overload-induced proliferation and senescence using knockout (KO) of megalin and cubilin in the proteinuric mouse model. METHOD Experiments were performed using podocin-KO (proteinuric mouse model) and megalin-cubilin-podocin triple-KO mice. Kidneys from these mice were analysed using immunohistochemical and immunofluorescent staining. Immortalized proximal tubular cells (RPTEC/TERT1, ATCC) were used for in vitro experiments, wherein RPTEC/TERT1 cells were incubated with 0.1–10 mg/mL of human serum albumin (HSA; Sigma), fatty acid-free HSA (Sigma), and transferrin (Sigma). Western blotting, quantitative rt-PCR, senescence-associated beta-galactosidase (SA-β-gal) staining and immunofluorescence were performed to analyse cellular senescence. The effect of a PKC activator (phorbol 12-myristate 13-acetate) and an inhibitor (Go6983) on proliferation and senescence was also evaluated. RESULTS The proliferation markers EdU incorporation, PCNA (Figure 1) and Ki-67 were detected in proximal tubular cells of podocin-KO mice, but not in wild-type mice. In triple-KO mice, a decrease in PCNA-positive tubules was observed. This suggests that protein reabsorption via megalin and cubilin provoked cell proliferation. Light microscopy analysis and a proliferation assay with BrdU incorporation revealed that HSA overload-induced the proliferation of RPTEC/TERT1 cells, whereas fatty acid-free HSA or transferrin had no effect on the proliferation of RPTEC/TERT1 cells. Western blot analysis showed that HSA treatment, but not fatty acid-free HSA treatment, induced alterations in proliferation (PCNA and Ki-67), cell cycle (cyclin A, D, and Rb), cellular senescence (p21 and p16), and DNA injury (γ-H2AX). Using immunofluorescence, an increase in p21 and p16 expression was also observed in HSA-treated cells. Moreover, the HSA-treated cells showed positive staining for SA-β-gal. These results suggest that fatty acids bound to HSA induce cell proliferation and senescence. Furthermore, results showed that a PKC inhibitor suppressed HSA-induced proliferation and senescence, while a PKC activator accelerated these alterations without HSA treatment. CONCLUSION The present study showed that fatty acid-associated albumin induced proliferation and senescence of the proximal tubule cells, which were dependent on megalin/cubilin endocytosis of filtered protein. PKC activation is at least in part related to cell proliferation and senescence. It is unknown which molecular switch determine the cell cycle fate.