Induction of the gene encoding macrophage chemoattractant protein 1 by Orientia tsutsugamushi in human endothelial cells involves activation of transcription factor activator protein 1.
Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-kappaB and AP-1. Orientia tsutsugamushi causes vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated the cis-acting and trans-acting elements involved in O. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-kappaB activation was observed in HUVEC infected with O. tsutsugamushi, inhibition of NF-kappaB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position -69 to -63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position -69 to -63-96 to -90 or deletion of NF-kappaB-binding site at position -69 to -63-88 to -79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-kappaB binding sites at positions -2640 to -2632, -2612 to -2603 in the enhancer region, or the AP-1 biding site at position -2276 to -2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position -69 to -63 in proximal promoter and TRE at position -2276 to -2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected with O. tsutsugamushi. Although NF-kappaB activation is not essential per se, the kappaB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the involvement of the NF-kappaB may be due to the function of chromatin structures and other transcription cofactors in the regulation of MCP-1 gene expression in response to O. tsutsugamushi infectioin.
Cho
Nam-Hyuk
NH
Department of Microbiology and Immunology, Seoul National University College of Medicine, and Institute of Endemic Disease, Seoul National University Medical Research Center, Chongno-gu, Seoul 110-799, Republic of Korea.
Seong
Seung-Yong
SY
Huh
Myung-Sook
MS
Kim
Na-Hyun
NH
Choi
Myung-sik
MS
Kim
Ik-sang
IS
D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D016328:NF-kappa B / Q000378:metabolism; D012285:Orientia tsutsugamushi / Q000472:pathogenicity*; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012612:Scrub Typhus / Q000209:etiology; D013859:Thiocarbamates / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014162:Transfection; D014657:Vasculitis / Q000209:etiology
D000975:Antioxidants; D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D012333:RNA, Messenger; D013859:Thiocarbamates; D018808:Transcription Factor AP-1; C066229:prolinedithiocarbamate; D014108:Tosylphenylalanyl Chloromethyl Ketone; D004247:DNA; D011392:Proline; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases