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The enzymatic machinery of leukotriene biosynthesis: Studies on ontogenic expression, interactions and function
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Leukotrienes (LTs) are biologically active arachidonic acid (AA) derivatives generated by the 5-lipoxygenase (5-LO) pathway. They are produced by myeloid cells. 5-LO converts AA to LTA4 in cooperation with 5-LO activating protein (FLAP). LTA4 is converted to LTB4, by LTA4-hydrolase (LTA4H) or to LTC4 by LTC4-synthase (LTC4S). LTs act on cells through plasma membrane bound G-protein coupled receptors found on leukocytes, smooth muscle and endothelial cells. We report here protein-protein interactions of proteins involved in LTC4 synthesis. 5-LO interacts with cytosolic domains of the integral membrane proteins FLAP and LTC4S at the nuclear envelope, in addition LTC4S interacts with FLAP through its hydrophobic membrane spanning regions. We constructed an LTC4S promoter controlled GFP reporter vector, displaying cell specific expression and sensitivity to agents known to affect LTC4S expression. The vector was used to create transgenic mice expressing GFP as a reporter for LTC4S. Ontogenic mouse expression studies revealed that the complete LT biosynthesis machinery was present at e11.5 primarily in the hematopoietic cells colonizing the liver. Although mature myeloid cells were the main contributors, a substantial amount of FLAP message was also detected in hematopoietic stem and progenitor cells, indicating possible functions for FLAP in hematopoietic regulation. Functional analyses using FLAP knockout mice suggested fine-tuning roles for LTs during differentiation, primarily along the B-lymphocyte differentiation path.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 103 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1287
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-74785ISBN: 978-91-7519-987-0 (print)OAI: oai:DiVA.org:liu-74785DiVA: diva2:492952
Public defence
2012-03-09, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2017-07-07Bibliographically approved
List of papers
1. Leukotriene C4 synthase promoter driven expression of GFP reveals cell specificity
Open this publication in new window or tab >>Leukotriene C4 synthase promoter driven expression of GFP reveals cell specificity
2008 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 366, no 1, 80-85 p.Article in journal (Refereed) Published
Abstract [en]

Leukotriene C4 synthase is a key enzyme in leukotriene biosynthesis. Its gene has been cloned and mapped to mouse chromosome 11. Expression occurs in cells of myeloid origin and also in the choroid plexus, the hypothalamus and the medial eminence of mouse brain. In this study a vector that expresses enhanced green fluorescent protein (eGFP) under the control of the mouse leukotriene C4 synthase promoter was constructed and used to study promoter activity in different cell lines. Specific eGFP expression was observed in human monocytic leukemia (THP-1) and rat basophilic leukemia (RBL-1) myeloid cells which both express leukotriene C4 synthase, but not in human embryonic kidney (HEK293/T) epithelial cells which do not express this enzyme. In the myeloid cells, but not in the epithelial cells, we observed that the leukotriene C4 synthase promoter activity was stimulated by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid. In contrast dimethyl sulfoxide did not affect promoter activity. © 2007 Elsevier Inc. All rights reserved.

Keyword
Leukotriene; Leukotriene C4 synthase; Expression; GFP; TPA; Promoter; Retinoic acid; DMSO
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-42196 (URN)10.1016/j.bbrc.2007.11.097 (DOI)000252392400013 ()61334 (Local ID)61334 (Archive number)61334 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
2. Distinct parts of leukotriene C-4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein
Open this publication in new window or tab >>Distinct parts of leukotriene C-4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein
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2009 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 381, no 4, 518-522 p.Article in journal (Refereed) Published
Abstract [en]

Leukotriene C-4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C-4 synthase (LTC4S) participate in its biosynthesis. We report evidence that LTC4S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC4S. FLAP bound to the N-terminal part/first hydrophobic region of LTC4S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC4S. Fluorescent FLAP- and LTC4S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC4S co-localized with a fluorescent ER marker. In testing HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC4S. Direct interaction of 5-LO and LTC4S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC4S.

Keyword
BRET, Confocal fluorescence microscopy, Eicosanoids, Fusion proteins, GFP, GST pull-down assay, Nuclear envelope
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17904 (URN)10.1016/j.bbrc.2009.02.074 (DOI)000264929400013 ()
Note

Original Publication: Tobias Strid, Jesper Svartz, Niclas Franck, Elisabeth Hallin, Björn Ingelsson, Mats Söderström and Sven Hammarström, Distinct parts of leukotriene C-4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein, 2009, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, (381), 4, 518-522. http://dx.doi.org/10.1016/j.bbrc.2009.02.074 Copyright: Elsevier Science B.V., Amsterdam http://www.elsevier.com/

Available from: 2009-04-30 Created: 2009-04-24 Last updated: 2017-12-13Bibliographically approved
3. Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells
Open this publication in new window or tab >>Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells
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2009 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 383, no 3, 336-339 p.Article in journal (Refereed) Published
Abstract [en]

Leukotriene C-4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (540), 5-lipoxygenase activating protein (FLAP) and leukotriene C-4 synthase (LTC4S) participate in its biosynthesis. We report evidence from in situ hybridization experiments that FLAP mRNA is abundantly expressed in fetal mouse liver from e11.5 until delivery. In contrast very little or no FLAP mRNA was detected in adult liver. The fetal expression in liver was not uniform but occurred in patches. Cells from e15.5 livers were fractionated by fluorescence activated cell sorting into hepatocytes and other CD45(-) cells and CD45(+) hematopoietic cells. The latter were further separated into immature (Lin(-)) and mature (Lin(+)) cells and analyzed for FLAP mRNA content by quantitative RT-PCR. FLAP mRNA expression was confined to CD45(+) cells and the mature cells had approximately 4-fold higher FLAP mRNA levels compared to the immature cells.

Keyword
Embryonic development, Fluorescence-activated cell sorting, In situ hybridization, Leukotrienes, 5-Lipoxygenase activating protein, Liver, Hematopoietic cells
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18568 (URN)10.1016/j.bbrc.2009.04.007 (DOI)000265966800012 ()
Available from: 2009-06-01 Created: 2009-06-01 Last updated: 2017-12-13Bibliographically approved
4. Expression of leukotriene biosynthesis proteins in fetal and adult hematopoietic cells and its functional effects on hematopoiesis
Open this publication in new window or tab >>Expression of leukotriene biosynthesis proteins in fetal and adult hematopoietic cells and its functional effects on hematopoiesis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reactions catalyzed by 5-lipoxygenase and either leukotriene A4 hydrolase or leukotriene C4 synthase. 5-lipoxygenase activating protein (FLAP) is also required. We have previously reported expression of FLAP in the hematopoietic compartment of the fetal liver raising questions regarding the role of leukotrienes in hematopoietic regulation. Here we report evidence from in situ hybridization, immunohistochemistry and qRT-PCR experiments that the complete LT biosynthesis machinery is abundantly expressed in hematopoietic cells of the fetal mouse liver from e11.5 until birth. FACS sorting of hematopoietic cells from e15.5 liver and adult bone marrow into different subpopulations followed by quantitative RT-PCR analysis showed that expression was confined mainly to myeloid cells but also detected in hematopoietic stem and progenitor cells. Analysis of FLAP knockout mice showed that a lack of this gene abolished LT and reduced 5(S)- hydroxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (HETE) production. Furthermore,  decreased relative numbers of B-lymphocytes and increased numbers of T-lymphocytes were observed in peripheral blood and increased numbers of common lymphoid progenitor cells were observed in BM. Taken together these findings suggest that production of LTs can occur in cells of the fetal and adult hematopoietic compartments and that deficiency of the FLAP gene (and leukotrienes) may affect lymphocyte maturation.

Keyword
Leukotriene, hematopoiesis, adult, fetal liver, bone marrow
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-74784 (URN)
Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2013-10-23Bibliographically approved

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