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IFN- Induces Cysteinyl Leukotriene Receptor 2 Expression and Enhances the Responsiveness of Human Endothelial Cells to Cysteinyl Leukotrienes¹

Grzegorz Woszczek^*, Li-Yuan Chen^*, Sahrudaya Nagineni^*, Sara Alsaaty^*, Anya Harry^*, Carolea Logun^*, Rafal Pawliczak^*, and James H. Shelhamer^2,*

^* Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892; and Department of Immunopathology, Medical University of Lodz, Lodz, Poland

Cysteinyl leukotrienes (cysLTs) are important mediators of cell trafficking and innate immune responses, involved in the pathogenesis of inflammatory processes, i.e., atherosclerosis, pulmonary fibrosis, and bronchial asthma. The aim of this study was to examine the regulation of cysLT signaling by IFN- in human primary endothelial cells. IFN- increased cysLT receptor 2 (CysLTR2) mRNA expression and CysLTR2-specific calcium signaling in endothelial cells. IFN- signaled through Jak/STAT1, as both AG490, a Jak2 inhibitor, and expression of a STAT1 dominant-negative construct, significantly inhibited CysLTR2 mRNA expression in response to IFN-. To determine mechanisms of IFN--induced CysLTR2 expression, the human CysLTR2 gene structure was characterized. The CysLTR2 gene has a TATA-less promoter, with multiple transcription start sites. It consists of six variably spliced exons. Eight different CysLTR2 transcripts were identified in endothelial and monocytic cells. Gene reporter assay showed potent basal promoter activity of a putative CysLTR2 promoter region. However, there were no significant changes in gene reporter and mRNA t_1/2 assays in response to IFN-, suggesting transcriptional control of CysLTR2 mRNA up-regulation by IFN- response motifs localized outside of the cloned CysLTR2 promoter region. Stimulation of endothelial cells by cysLTs induced mRNA and protein expression of early growth response genes 1, 2, and 3 and cycloxygenase-2. This response was mediated by CysLTR2 coupled to G_q/11, activation of phospholipase C, and inositol-1,4,5-triphosphate, and was enhanced further 2- to 5-fold by IFN- stimulation. Thus, IFN- induces CysLTR2 expression and enhances cysLT-induced inflammatory responses.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The nucleotide sequences presented in this article have been submitted to GenBank with the following accession numbers: EF141523, EF141524, EF141525, EF141526, EF141527, EF141528, EF141529, and EF141530.

² Address correspondence and reprint requests to Dr. James H. Shelhamer, Critical Care Medicine Department, Clinical Center, National Institutes of Health, Building 10, Room 2C145, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: jshelhamer{at}cc.nih.gov

³ Abbreviations used in this paper: cysLT, cysteinyl leukotriene; 5-LO, 5-lipoxygenase; 5'UTR, 5' untranslated region; Cox, cyclooxygenase; CysLTR, cysLT receptor; Egr, early growth response; EST, expressed sequenced tag; GPCR, G protein-coupled receptor; HMVEC-L, human microvascular endothelial cells from the lung; IP3, inositol-1,4,5-triphosphate; LTC₄, leukotriene C₄; LTD₄, leukotriene D₄; PLC, phospholipase C; TSS, transcription start site.