Negative regulation of human heme oxygenase in microvessel endothelial cells by dexamethasone.
Heme oxygenase-1 (HO-1) is a stress protein, and its induction has been suggested to participate in defense mechanisms against agents that promote oxidative injury such as endotoxins and heme. We have shown that the inflammatory cytokines, interleukin-6 (IL-6) and heme-induced HO-1 gene expression, were suppressed by dexamethasone (Dex) in a sustained manner. We examined the mechanism by which the anti-inflammatory agent, Dex, inhibits IL-6 and heme-induced HO-1 expression in rabbit coronary endothelial cells. Endothelial cells treated with heme (10 microM) and IL-6 (25 ng/ml), increased HO-1 mRNA 15- and 60-fold, respectively. The activity of HO was increased 3-fold after such treatment. Although Dex failed to inhibit heme-mediated HO-1 mRNA and HO activity, it was able to reverse IL-6-stimulated HO activity. Several human HO-1 promoter-drive chloramphenicol acetyltransferase (CAT) constructs were examined to analyze IL-6 and Dex-mediated modulation of the HO-1 gene in endothelial cells. CAT assays revealed that the HO-1 promoter region between -180 and -1500 might contain a Dex-mediated negative regulator. Gel mobility shift assays using nuclear extracts from IL-6-treated endothelial cells showed a binding to the synthetic 21 base pairs of the HO-1 sequence that contains the putative STAT3 sequence. STAT3-specific probe inhibited nuclear binding protein to the putative HO-1-STAT3 sequence. This suggests that IL-6 induction of human HO-1 is mediated via the JAK-STAT pathway and that Dex inhibition of gene expression is carried out by activation of a transcriptional protein in competition with the STAT3 binding site.
Deramaudt
T B
TB
Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
da Silva
J L
JL
Remy
P
P
Kappas
A
A
Abraham
N G
NG
D000209:Acute-Phase Proteins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003326:Coronary Circulation; D004268:DNA-Binding Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006418:Heme / Q000494:pharmacology*; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D051547:Heme Oxygenase-1; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins; D008833:Microcirculation; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D000209:Acute-Phase Proteins; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D006418:Heme; D003907:Dexamethasone; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D015500:Chloramphenicol O-Acetyltransferase