scholarly journals 12(S)-hydroxyheptadeca-5Z, 8E, 10E–trienoic acid is a natural ligand for leukotriene B4 receptor 2

2008 ◽  
Vol 205 (4) ◽  
pp. 759-766 ◽  
Author(s):  
Toshiaki Okuno ◽  
Yoshiko Iizuka ◽  
Hiroshi Okazaki ◽  
Takehiko Yokomizo ◽  
Ryo Taguchi ◽  
...  
Keyword(s):  
Mast Cells ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
High Performance ◽  
Blood Platelets ◽  
Leukotriene B4 ◽  
Trienoic Acid ◽  
Natural Ligand ◽  
Deficient Mice

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.

Mast cells in health and disease

2011 ◽  
Vol 120 (11) ◽  
pp. 473-484 ◽  
Author(s):  
Charlotte L. Weller ◽  
Sarah J. Collington ◽  
Tim Williams ◽  
Jonathan R. Lamb
Keyword(s):  
Wound Healing ◽  
Mast Cells ◽  
Allergic Disease ◽  
Effector Functions ◽  
Health And Disease ◽  
Developing Area ◽  
Deficient Mice ◽  
Made In

Although MCs (mast cells) were discovered over 100 years ago, for the majority of this time their function was linked almost exclusively to allergy and allergic disease with few other roles in health and disease. The engineering of MC-deficient mice and engraftment of these mice with MCs deficient in receptors or mediators has advanced our knowledge of the role of MCs in vivo. It is now known that MCs have very broad and varied roles in both physiology and disease which will be reviewed here with a focus on some of the most recent discoveries over the last year. MCs can aid in maintaining a healthy physiology by secreting mediators that promote wound healing and homoeostasis as well as interacting with neurons. Major developments have been made in understanding MC function in defence against pathogens, in recognition of pathogens as well as direct effector functions. Probably the most quickly developing area of understanding is the involvement and contribution MCs make in the progression of a variety of diseases from some of the most common diseases to the more obscure.

scholarly journals Early Postnatal Death and Motor Disorders in Mice Congenitally Deficient in Calnexin Expression

2002 ◽  
Vol 22 (21) ◽  
pp. 7398-7404 ◽  
Author(s):  
Angela Denzel ◽  
Maurizio Molinari ◽  
Cesar Trigueros ◽  
Joanne E. Martin ◽  
Shanti Velmurgan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Knockout ◽  
Nerve Fibers ◽  
Motor Disorders ◽  
Type I ◽  
Myelinated Nerve ◽  
Gene Knockout Mice ◽  
Deficient Mice ◽  
Insight Into

ABSTRACT Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific.

scholarly journals Phenotypic changes of bone marrow-derived mast cells after intraperitoneal transfer into W/Wv mice that are genetically deficient in mast cells.

1987 ◽  
Vol 165 (3) ◽  
pp. 615-627 ◽  
Author(s):  
K Otsu ◽  
T Nakano ◽  
Y Kanakura ◽  
H Asai ◽  
H R Katz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Adoptive Transfer ◽  
Gel Filtration ◽  
Chondroitinase Abc ◽  
Indirect Immunofluorescence Staining ◽  
Deficient Mice

The ability of mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC) to generate serosal mast cells (SMC) in vivo after adoptive transfer to mast cell-deficient mice has been defined by chemical and immunochemical criteria. BMMC differentiated and grown from WBB6F1-+/+ mouse progenitor cells in medium containing PWM/splenocyte-conditioned medium synthesized a approximately 350,000 Mr protease-resistant proteoglycan bearing approximately 55,000 Mr glycosaminoglycans, as defined by gel filtration of each. Approximately 85% of the glycosaminoglycans bound to the cell-associated BMMC proteoglycans were chondroitin sulfates based upon their susceptibility to chondroitinase ABC digestion; HPLC of the chondroitinase ABC-generated unsaturated disaccharides revealed these glycosaminoglycans to be chondroitin sulfate E. As determined by heparinase and nitrous acid degradations, approximately 10% of the glycosaminoglycans bound to BMMC proteoglycans were heparin. In contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient WBB6F1-W/Wv mice 15 wk after intraperitoneal injection of BMMC synthesized approximately 650,000 Mr protease-resistant proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 105,000 Mr. Thus, after adoptive transfer, the SMC of the previously mast cell-deficient mice were like those recovered from the normal WBB6F1-+/+ mice that were shown to synthesize approximately 600,000 Mr proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 115,000 Mr. As assessed by indirect immunofluorescence staining and flow cytometry using the B1.1 rat mAb (an antibody that recognizes an epitope located on the neutral glycosphingolipid globopentaosylceramide), approximately 5% of BMMC bound the antibody detectably, whereas approximately 72% of the SMC that were harvested from mast cell-deficient mice 15 wk after adoptive transfer of BMMC were B1.1-positive; approximately 82% of SMC from WBB6F1-+/+ mice bound the antibody. These biochemical and immunochemical data are consistent with the results of previous adoptive transfer studies that characterized mast cells primarily on the basis of morphologic and histochemical criteria. Thus, IL-3-dependent BMMC developed in vitro, cells that resemble mucosal mast cells, can give rise in vivo to SMC that express phenotypic characteristics of connective tissue mast cells.

scholarly journals Positive and Negative Regulation of FcεRI-Mediated Signaling by the Adaptor Protein LAB/NTAL

2004 ◽  
Vol 200 (8) ◽  
pp. 991-1000 ◽  
Author(s):  
Minghua Zhu ◽  
Yan Liu ◽  
Surapong Koonpaew ◽  
Olivia Granillo ◽  
Weiguo Zhang
Keyword(s):  
B Cells ◽  
Mast Cells ◽  
Cytokine Production ◽  
Cell Function ◽  
Adaptor Protein ◽  
Thymocyte Development ◽  
Positive Role ◽  
Erk Activation ◽  
Deficient Mice

Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcεRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcεRI-mediated signaling than LAT−/− mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcεRI-mediated signaling.

scholarly journals 12-hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor

2014 ◽  
Vol 211 (6) ◽  
pp. 1063-1078 ◽  
Author(s):  
Min Liu ◽  
Kazuko Saeki ◽  
Takehiko Matsunobu ◽  
Toshiaki Okuno ◽  
Tomoaki Koga ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Closure ◽  
Cultured Cells ◽  
Leukotriene B4 ◽  
Epidermal Keratinocytes ◽  
Skin Wound Healing ◽  
Wound Fluid ◽  
Deficient Mice

Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G protein–coupled receptor (GPCR) for 12(S)-hydroxyheptadeca-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. As mouse BLT2 is highly expressed in epidermal keratinocytes, we investigated the role of the 12-HHT/BLT2 axis in skin wound healing processes. 12-HHT accumulated in the wound fluid in mice, and BLT2-deficient mice exhibited impaired re-epithelialization and delayed wound closure after skin punching. Aspirin administration reduced 12-HHT production and resulted in delayed wound closure in wild-type mice, which was abrogated in BLT2-deficient mice. In vitro scratch assay using primary keratinocytes and a keratinocyte cell line also showed that the 12-HHT/BLT2 axis accelerated wound closure through the production of tumor necrosis factor α (TNF) and matrix metalloproteinases (MMPs). A synthetic BLT2 agonist accelerated wound closure in cultured cells as well as in C57BL/6J and diabetic mice. These results identify a novel mechanism underlying the action of the 12-HHT/BLT2 axis in epidermal keratinocytes and accordingly suggest the use of BLT2 agonists as therapeutic agents to accelerate wound healing, particularly for intractable wounds, such as diabetic ulcers.

scholarly journals A Targeting Mutation of Tyrosine 1062 in Ret Causes a Marked Decrease of Enteric Neurons and Renal Hypoplasia

2004 ◽  
Vol 24 (18) ◽  
pp. 8026-8036 ◽  
Author(s):  
Mayumi Jijiwa ◽  
Toshifumi Fukuda ◽  
Kumi Kawai ◽  
Akari Nakamura ◽  
Kei Kurokawa ◽  
...  
Keyword(s):  
Nervous System ◽  
Enteric Nervous System ◽  
Intracellular Signaling ◽  
Effector Proteins ◽  
Mutant Mice ◽  
Enteric Neurons ◽  
Kidney Size ◽  
Renal Hypoplasia ◽  
Deficient Mice

ABSTRACT The Ret receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in Ret represents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, phosphatidylinositol 3-kinase/AKT, and Jun-associated N-terminal kinase pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice in which tyrosine 1062 in Ret was replaced with phenylalanine were generated. Although homozygous knock-in mice were born normally, they died by day 27 after birth and showed growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract, as observed in Ret-deficient mice. The rest of the mutant mice developed enteric neurons in the intestine to various extents, although the size and number of ganglion cells were significantly reduced. Unlike Ret-deficient mice, a small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to a decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis.

p21-Activated Kinase 1 Regulates Hyperproliferation of Nf1 Haploinsufficient Mast Cells In Vitro and In Vivo Via Activation of the MAPK Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3063-3063
Author(s):  
Andrew S. McDaniel
Keyword(s):  
Mast Cells ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Type I ◽  
Wild Type ◽  
Kit Ligand

Abstract p21-activated kinases (Paks) are downstream mediators of Rho GTPase proteins and have been implicated in yeast and immortalized cells as positive regulators of MAPK pathway members in modulating cell growth and cytoskeletal functions. However, their role in primary mammalian cells has not been described. NF1 encodes neurofibromin, which negatively regulates p21Ras activity by stimulating its intrinsic GTPase activity, and accelerating hydrolysis of Ras from the GTP to the GDP confirmation. Disruption of the NF1 locus results in neurofibromatosis type I (NF1), an inherited disorder characterized by the development of neurofibromas that contain large numbers of degranulating mast cells that have been implicated in tumor progression. Utilizing a genetic intercross of Pak 1−/− mice with mice haploinsufficient at the Nf1 locus, we studied the role of Pak1 in the context of normal and hyperactivated Ras-MAPK signaling in primary inflammatory mast cells. Pak1 was found to directly contribute to Ras-dependent signaling by modulating both Raf-1, Mek-1 and ERK1/2 activation. Loss of Pak1 fully corrects the hyperphosphorylation of ERK1/2 found in Nf1+/− mast cells to that of wild type controls. Deletion of Pak1 in Nf1+/− mast cells is associated with a correction of Kit ligand mediated proliferation to wild type levels in vitro. Further, after subcutaneous administration of Kit ligand via micro osmotic pumps, which is an established model that stimulates local proliferation of mast cells in vivo (Ingram, JEM 2001), we confirmed that genetic disruption of Pak1 corrects the proliferation of Nf1+/− mast cells in vivo to that of wild type controls. These data provide direct genetic evidence that Pak1 modulates the Ras-Raf-Mek-Erk pathway and identifies a specific molecular target within the inflammatory tumor microenvironment for the treatment or prevention of neurofibromas.

scholarly journals Kiss1/Gpr54 Prevents Bone Loss through Src Dephosphorylation by Dusp18 in Osteoclasts

2021 ◽  
Author(s):  
Zhenxi Li ◽  
Xinghai Yang ◽  
Jian Jiao ◽  
Zhipeng Wu ◽  
Zhixiang Wu ◽  
...  
Keyword(s):  
Bone Loss ◽  
Therapeutic Strategy ◽  
Natural Ligand ◽  
C Terminus ◽  
Functional Defect ◽  
Approved Drugs ◽  
G Protein Coupled ◽  
Deficient Mice ◽  
Arginine Rich Motif

Abstract Osteoclasts were over-activated as we age, which leads to bone loss. Src-deficient mice lead to only one phenotype -severe osteopetrosis due to functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCR) have been targets for ∼35% of approved drugs. However, how Src kinase activity is negatively regulated by GPCRs remains largely elusive. Herein we report that Src is dephosphorylated at Tyr 416 by Dusp18 upon GPR54 activation by its natural ligand Kp-10. Mechanically, both active Src and the Dusp18 phosphatase are recruited by GPR54 through the proline/arginine-rich motif (PR motif) in the C terminus, which is dependent on the Gαq signal pathway. As such, Kiss1, Gpr54, Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss. Accordingly, Kp-10 abrogated bone loss by suppressing osteoclasts activity in vivo. Therefore, Kiss1/Gpr54 is a promising therapeutic strategy governing bone resorption through Src dephosphorylation by Dusp18.

scholarly journals ORMDL2 Deficiency Potentiates the ORMDL3-Dependent Changes in Mast Cell Signaling

2021 ◽  
Vol 11 ◽  
Author(s):  
Viktor Bugajev ◽  
Ivana Halova ◽  
Livia Demkova ◽  
Sara Cernohouzova ◽  
Petra Vavrova ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Signaling ◽  
Mammalian Cells ◽  
Cell Activation ◽  
De Novo ◽  
Mast Cell Activation ◽  
Whole Body

The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3). However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO exhibited increased intracellular levels of sphingosine-1-phosphate (S1P). Furthermore, we found that concurrent ORMDL2 and ORMDL3 deficiency increased IκB-α phosphorylation, degranulation, and production of IL-4, IL-6, and TNF-α cytokines in antigen-activated mast cells. Interestingly, the chemotaxis towards antigen was increased in all mutant cell types analyzed. Experiments in vivo showed that passive cutaneous anaphylaxis (PCA), which is initiated by mast cell activation, was increased only in ORMDL2,3 double KO mice, supporting our in vitro observations with mast cells. On the other hand, ORMDL3 KO and ORMDL2,3 double KO mice showed faster recovery from passive systemic anaphylaxis, which could be mediated by increased levels of blood S1P presented in such mice. Our findings demonstrate that Ormdl2 deficiency potentiates the ORMDL3-dependent changes in mast cell signaling.

scholarly journals Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

2021 ◽  
Author(s):  
Kiryl D Piatkevich ◽  
Siranush Babakhanova ◽  
Erica Jung ◽  
Kazuhiko Namikawa ◽  
Hanbin ZHANG ◽  
...  
Keyword(s):  
Molecular Evolution ◽  
Mammalian Cells ◽  
High Performance ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Model Organisms ◽  
Multicolor Imaging ◽  
Directed Molecular Evolution

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2x107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

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