Hepatic Levels of DHA-Containing Phospholipids Instruct SREBP1-Mediated Synthesis and Systemic Delivery of Polyunsaturated Fatty Acids.

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.

IL-11 prevents IFN-γ-induced hepatocyte death through selective downregulation of IFN-γ/STAT1 signaling and ROS scavenging.

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.

Protective role of the leukotriene B4 receptor BLT2 in murine inflammatory colitis.

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)-1ß, 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.

12(S)-Hydroxyheptadeca-5Z, 8E, 10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2.

Activated blood platelets and macrophages metabolize prostaglandin H(2) into thromboxane A(2) 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 B(4) (LTB(4)) receptor-2 (BLT2), a G protein-coupled receptor that was originally identified as a low-affinity receptor for LTB(4). 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 [(3)H]LTB(4) showed that 12-HHT binds to BLT2 with a higher affinity than LTB(4). 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.

Characterization of a mouse second leukotriene B4 receptor, mBLT2: BLT2-dependent ERK activation and cell migration of primary mouse keratinocytes.

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.