Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells

doi:10.1016/j.bbrc.2009.04.007

Biochemical and Biophysical Research Communications

Volume 383, Issue 3, 5 June 2009, Pages 336-339

https://doi.org/10.1016/j.bbrc.2009.04.007 Get rights and content

Abstract

Leukotriene C₄ is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C₄ synthase (LTC₄S) participate in its biosynthesis. We report evidence from insitu 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.

Section snippets

Materials and methods

Materials. Antibodies for cell surface staining were from Biolegend, San Diego, CA [PECY5-CD19 (RA3–6B2), GR-1 (RB6–8C5)]; BD-Biosciences, San Jose, CA [CD11b/MAC1 (M1/70), CD3 (17A2), CD32/16]; or eBioscience, San Diego, CA [TER-119 (LY-76), CD45-PECY7(30-F11)]. [α-³⁵S] UTPαS was from PerkinElmer, Boston, MA; MAXI script in vitro transcription kit for probe preparation was from Ambion, Austin, TX and quick spin columns for probe purification were from Roche Diagnostics, Indianapolis, IN.

Liver is a major site of FLAP mRNA expression during mouse embryogenesis

In order to investigate the expression pattern of FLAP message during mouse embryogenesis whole body sections of embryos at different stages of development were examined by insitu hybridization using [³⁵S]-labeled FLAP antisense probe. Autoradiography showed abundant FLAP mRNA expression in the fetal liver already at e11.5 (Fig. 1A). From this stage until e17.5 liver was the only site where FLAP expression was observed. At e17.5 and e18.5 expression was also detected in some other organs,

Discussion

We report here that FLAP mRNA is expressed in the liver from mouse embryonic day 11.5. Liver expression continues throughout gestation but vanishes in the adult mouse. The cellular source of FLAP expression was identified by sorting total e15.5 liver cells by FACS followed by expression analyses by qPCR with HPRT1 as a reference gene. The results demonstrated that only CD45⁺ hematopoietic cells transcribed the FLAP gene. Moreover, mature hematopoietic cells formed higher levels of FLAP mRNA

Acknowledgments

This work was supported by grants from the Swedish Research Council (31X-05914), the Swedish Cancer Foundation (to Mikael Sigvardsson) and Östergötland County Research Council.

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Leukotriene (LT) C4 is a potent inflammatory mediator formed from arachidonic acid, and glutathione,5-lipoxygenase, 5-lipoxygenase activating protein, and leukotriene C4 synthase (LTC4S) participate in its biosynthesis [1].
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We randomly divided adult male Sprague-Dawley rats into sham (control), I/R, and IP groups (n = 6). We subjected rat liver to 60 min partial hepatic ischemia followed by 5 h reperfusion with saline administered intravenously. We detected protein expression of LTC4 synthase (LTC4S) with Western blot, and measured LTC4 synthesis enzymes' activities and content by reverse-phase high-performance liquid chromatography. We assessed tissue injury using serum aspartate aminotransferase and aspartate aminotransferase activities and histologic changes. We examined liver tissue glutathione levels by a biochemical method.
Ischemic preconditioning markedly decreased LTC4 content, reduced LTC4S protein expressions, and inhibited LTC4 synthesis enzymes' activities in rat liver compared with the I/R group (P < 0.05). We also observed a decline in serum alanine aminotransferase and aspartate aminotransferase activities (P < 0.05), together with hepatic tissue glutathione elevation (P < 0.05) in the IP groups. Positive expression of LTC4S on hepatocytes and sinusoidal endothelial cells in the IP group was significantly lower than that in the I/R group.
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Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells

Abstract

Section snippets

Materials and methods

Liver is a major site of FLAP mRNA expression during mouse embryogenesis

Discussion

Acknowledgments

Vitam. Horm.

Prostaglandins Leukot. Essent. Fatty Acids

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Cell. Differ.

Bioinformatic and enzymatic characterization of the MAPEG superfamily

FEBS J.

Leukotrienes and lipoxins: structures, biosynthesis, and biological effects

Science

5-Lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase

Eur. J. Biochem.

Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis

Nature

Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases

Biochem. J.