Cytokine-stimulated GTP cyclohydrolase I expression in endothelial cells requires coordinated activation of nuclear factor-kappaB and Stat1/Stat3.
Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH4), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH4 levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH4 biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear. We sought to investigate the signaling pathways whereby cytokines induce GTPCH I expression in human umbilical vein endothelial cells (HUVECs). Interferon-gamma (IFN-gamma) induced endothelial cell GTPCH I protein and BH4 modestly, whereas high-level induction required combinations of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha). In the presence of IFN-gamma, TNF-alpha increased GTPCH I mRNA in a manner dependent on nuclear factor-kappaB (NF-kappaB), as this effect was abrogated by overexpression of a dominant-negative IkappaB construct. HUVEC IFN-gamma treatment resulted in signal transducer and activator of transcription 1 (Stat1) activation and DNA binding in a Jak2-dependent manner, as this was inhibited by AG490. Conversely, overexpression of Jak2 effectively substituted for IFN-gamma in supporting TNF-alpha-mediated GTPCH I induction. The role of IFN-gamma was also Stat1-dependent as Stat1-null cells exhibited no GTPCH I induction in response to cytokines. However, Stat1 activation with oncostatin M failed to support TNF-alpha-mediated GTPCH I induction because of concomitant Stat3 activation. Consistent with this notion, siRNA-mediated Stat3 gene silencing allowed oncostatin M to substitute for IFN-gamma in this system. These data implicate both NF-kappaB and Stat1 in endothelial cell cytokine-stimulated GTPCH I induction and highlight the role of Stat3 in modulating Stat1-supported gene transcription. Thus, IFN-gamma and TNF-alpha exert distinct but cooperative roles for BH4 biosynthesis in endothelium that may have important implications for vascular function during vascular inflammation.
Huang
Annong
A
Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass 02118, USA.
Zhang
Ying-Yi
YY
Chen
Kai
K
Hatakeyama
Kazuyuki
K
Keaney
John F
JF
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology / Q000502:physiology; D007375:Interleukin-1 / Q000494:pharmacology; D053614:Janus Kinase 2; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D053683:Oncostatin M; D010455:Peptides / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D020032:Tyrphostins / Q000494:pharmacology; D014471:Umbilical Veins
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C508351:OSM protein, human; C508352:Osm protein, mouse; D010455:Peptides; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494080:Stat1 protein, mouse; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D053683:Oncostatin M; D000072000:NF-KappaB Inhibitor alpha; D001708:Biopterins; D009569:Nitric Oxide; D007371:Interferon-gamma; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D006136:GTP Cyclohydrolase; C003402:sapropterin