12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) induces cell growth and improves barrier function through BLT2 interaction in intestinal epithelial Caco-2 cell cultures.

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is an unusual product of the cyclooxygenase pathway that is an endogenous ligand of the low-affinity receptor for leukotriene 4 (LTB), BLT2. Recent findings suggested that BLT2 possibly plays an important role in the healing of intestinal lesions and the regulation of barrier function. Here, we studied the role of 12-HHT on intestinal epithelial cell growth and the paracellular permeability of intestinal epithelium using Caco-2 cell cultures as experimental model. Our results demonstrated that 12-HHT stimulates intestinal epithelial Caco-2 cell growth through 12-HHT-BLT2-p38-PKC axis and improves paracellular permeability in differentiated Caco-2 cell cultures through the regulation of tight junction elements such as myosin light chain phosphorylation through 12-HHT-BLT2-p38-PKC-MYPT1 axis. Thus, 12-HHT-BLT2 interaction can be involved in intestinal epithelial cell growth and consequently in the epithelium regeneration/repair processes, together with an interesting improvement on the paracellular permeability. These effects appoint that 12-HHT/BLT2 axis may be a suitable strategy for treating wound healing epithelium and barrier-disrupted intestinal processes.

Storniolo CE ，Pequera M ，Company F ，Moreno JJ ... -  《-》

Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins.

GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2-overexpressing Madin-Darby canine kidney (MDCK) II cells and in the small intestine of BLT2-transgenic mice. BLT2-deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK-BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK-Mock cells, and 12-HHT stimulation accelerated TER recovery only in MDCK-BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12-HHT/BLT2 axis up-regulated claudin-4 (CLDN4) expression in MDCK-BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12-HHT-dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12-HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12-HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein-p38 MAPK pathway.

Ishii Y ，Saeki K ，Liu M ，Sasaki F ，Koga T ，Kitajima K ，Meno C ，Okuno T ，Yokomizo T ... -  《-》

Metabolism and biological functions of 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid.

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is a 17-carbon hydroxy fatty acid that is biosynthesized either by enzymatic pathways, like thromboxane synthase (TXAS) and cytochrome P450 or a non-enzymatic pathway. TXAS catalyzes the isomerization reaction from PGH to 12-HHT, malondialdehyde, and TXA at a ratio of 1:1:1. Furthermore, 12-HHT has been considered as a mere byproduct of TXA biosynthesis, and its biological function has long been uncertain. BLT2 was initially identified as a low-affinity leukotriene B (LTB) receptor, which is also activated by various hydroxy-eicosatetraenoic acids (HETEs), suggesting that BLT2 may be activated by other endogenous ligands apart from LTB and HETEs. By unbiased ligand screening using crude lipids from rat organs, 12-HHT has been identified as an endogenous agonist for BLT2. The 12-HHT-BLT2 axis induces mast cell migration and contributes to allergic inflammation. BLT2 is also expressed in epithelial cells of the small intestine and skin in mice and contributes to in vivo epithelial barrier functions.

Okuno T ，Yokomizo T 《PROSTAGLANDINS & OTHER LIPID MEDIATORS》

The leukotriene B4 receptor BLT2 protects barrier function via actin polymerization with phosphorylation of myosin phosphatase target subunit 1 in human keratinocytes.

Leukotriene B4 (LTB4 ) receptor type 2 (BLT2) is a novel G-protein-coupled receptor, which selectively binds to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) with stronger affinity than to LTB4 . Recently, 12-HHT has been shown to have a protective effect on the epidermal barrier in human keratinocytes or transfectant cells overexpressing BLT2. Because the protective activity of BLT2 in high-calcium conditions, which occurs in well-differentiated cells, is exerted through increasing the integrity of tight junctions, we investigated the effects of 12-HHT on the barrier function of human keratinocytes in low-calcium conditions that mimic the basal layer; to our knowledge, this has not been reported previously. After stimulation with or without 12-HHT, barrier function was measured using transepithelial electrical resistance (TER) and dextran permeability assay. Expression levels of adhesion molecules and actin polymerization were also evaluated. Treatment with 12-HHT increased TER, along with decreased epidermal permeability of dextran in human keratinocytes. Furthermore, 12-HHT induced actin polymerization with phosphorylation of myosin phosphatase target subunit 1. These results suggest that the ligation of BLT2 protects permeability barrier function by enhancing cell-cell contact, even under low-calcium conditions, and indicate that a BLT2 agonist could be a novel therapeutic target for barrier-disrupted skin diseases.

Chiba T ，Nakahara T ，Hashimoto-Hachiya A ，Yokomizo T ，Uchi H ，Furue M ... -  《-》

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.

Okuno T ，Iizuka Y ，Okazaki H ，Yokomizo T ，Taguchi R ，Shimizu T ... -  《-》