Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes.
Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12-LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.
Medizinische Klinik, Klinikum Innenstadt, Ludwig-Maximilians Universität, Munich, Germany.
W E
WE
Kaminski
E
E
Jendraschak
K
K
Baumann
R
R
Kiefl
S
S
Fischer
A J
AJ
Marcus
M J
MJ
Broekman
C
C
von Schacky
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D058927:5-Lipoxygenase-Activating Proteins; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D006098:Granulocytes / Q000201:enzymology*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D018076:DNA, Complementary; D006893:Hydroxyeicosatetraenoic Acids; D008565:Membrane Proteins; D012333:RNA, Messenger; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase