Many G protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8 (H8), at the proximal region of the C-terminal tail. However, the significance of this domain is not fully understood. Here, we demonstrate the requirement of H8 for the proper folding of GPCRs for passage through the quality control in the endoplasmic reticulum (ER). In the human leukotriene B(4) type-2 receptor (hBLT2), lack of H8 led to an accumulation of the receptor (hBLT2/DeltaH8) in the ER. Similar results were obtained in two representative human GPCRs, dopamine type-1 and lysophosphatidic acid type-2 receptors, which were engineered to lack H8. Treatment with the several ligands, which act as pharmacological chaperones, facilitated the surface expression of hBLT2/DeltaH8. The surface-trafficked hBLT2/DeltaH8 exhibited an agonist-evoked increase in Ca(2+), demonstrating that H8 is not critical for ligand binding and activation of coupled G proteins. Thus, these results suggest that the H8 region of hBLT2 plays an important role in transport-competent receptor folding.
Abstract Cell competition is a biological process by which unfit cells are eliminated from “cell society.” We previously showed that cultured mammalian epithelial Madin‐Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by “normal” MDCK cells. However, the molecular mechanism underlying the elimination of active YAP‐expressing cells was unknown. Here, we used high‐throughput chemical compound screening to identify cyclooxygenase‐2 (COX‐2) as a key molecule triggering cell competition. Our work shows that COX‐2‐mediated PGE 2 secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase‐cyclic AMP‐PKA pathway that, in the presence of active YAP, induces E‐cadherin internalization leading to apical extrusion. Thus, COX‐2‐induced PGE 2 appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.
Recent evolution of silica-based nonlinear fibers and their applications are reviewed. Important design issues of the fiber in order to enhance the nonlinearity and tailor the dispersion performance are discussed. In addition, recent demonstration results such as ultra-broadband wavelength conversion and supercontinuum generation experiments are also introduced in this paper.
Dispersion-flattened and decreasing fiber is found highly effective for ultra-broadband supercontinuum generation through simulations, for the first time. Actually manufactured fiber produces 280 nm bandwidth supercontinuum with an input pulse peak power of only 12 W.
Abstract Although pneumococcal infection is a serious problem worldwide and has a high mortality rate, the molecular mechanisms underlying the lethality caused by pneumococcus remain elusive. Here, we show that BLT2, a G protein-coupled receptor for leukotriene B 4 and 12(S)-hydroxyheptadecatrienoic acid (12-HHT), protects mice from lung injury caused by a pneumococcal toxin, pneumolysin (PLY). Intratracheal injection of PLY caused lethal acute lung injury (ALI) in BLT2-deficient mice, with evident vascular leakage and bronchoconstriction. Large amounts of cysteinyl leukotrienes (cysLTs), classically known as a slow reactive substance of anaphylaxis, were detected in PLY-treated lungs. PLY-dependent vascular leakage, bronchoconstriction and death were markedly ameliorated by treatment with a CysLT1 receptor antagonist. Upon stimulation by PLY, mast cells produced cysLTs that activated CysLT1 expressed in vascular endothelial cells and bronchial smooth muscle cells, leading to lethal vascular leakage and bronchoconstriction. Treatment of mice with aspirin or loxoprofen inhibited the production of 12-HHT and increased the sensitivity toward PLY, which was also ameliorated by the CysLT1 antagonist. Thus, the present study identifies the molecular mechanism underlying PLY-dependent ALI and suggests the possible use of CysLT1 antagonists as a therapeutic tool to protect against ALI caused by pneumococcal infection.
Pneumonia is one of the major causes of death over the world, and the capital cause of infectious pneumonia is S. pneumoniae (pneumococcus). The key virulence factor of pneumococcus is a pore‐forming toxin pneumolysin (PLY). By its direct cytotoxic effect, PLY is known to cause acute lung injury, leading to pneumococcal invasion and subsequent severe bacteremia. Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G‐protein coupled receptor that was originally identified as a low‐affinity receptor for LTB4, and recently we elucidated that the high affinity endogenous ligand for BLT2 is 12‐hydroxyheptadecatrienoic acid (12‐HHT), an arachidonic acid metabolite by cyclooxygenase. Previously we reported that BLT2 is expressed in intestinal epithelial cells and skin keratinocytes, and that 12‐HHT/BLT2 signaling exhibits important roles in maintaining mucosal integrity and in accelerating skin wound healing. However, the biological roles of BLT2 in other organs are still uncertified. In this study, we investigated the protective role of BLT2 in mouse lung using PLY‐induced acute lung injury model. Interestingly, BLT2‐deficient mice were more susceptible to PLY administration, and also exhibited increased vascular permeability and potent bronchoconstriction by PLY administration when compared to wild‐type mice. We found BLT2 in mouse lung is expressed in alveolar type II cells and vascular endothelial cells. The molecular mechanisms of how BLT2 exhibits the protective role for PLY‐induced acute lung injury are now under investigation. Support or Funding Information This work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) Nos. 22116001, 22116002, 15H05901, 15H05904, and 15H04708.
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. Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins. FASEB J. 30, 933–947 (2016). www.fasebj.org
