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.
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.
Nam-Hyuk
NH
Cho
Seung-Yong
SY
Seong
Myung-Sook
MS
Huh
Na-Hyun
NH
Kim
Myung-sik
MS
Choi
Ik-sang
IS
Kim
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