J Biol Chem. 2001 Mar 16;276(11):8445-52. Epub 2000 Dec 08. Mitogen-activated protein kinases mediate activator protein-1-dependent human inducible nitric-oxide synthase promoter activation.
Kristof AS, Marks-Konczalik J, Moss J.
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434, USA. kristofih.gov
Inducible nitric-oxide synthase (iNOS) is an important signaling protein involved in the regulation of biological processes (e.g. vasodilation, inflammation) and is subject to transcriptional regulation by cytokines and lipopolysaccharide (LPS). Full activation of the human iNOS (hiNOS) promoter by cytokines (i.e., tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma (IFN-gamma)) required downstream and upstream nuclear factor-kappaB (-115, -8283) and activator protein-1 (AP-1) (-5115, -5301) transcription factor binding sites. Human lung epithelial (A549) cells were transiently transfected with luciferase reporter plasmids containing an 8.3-kilobase human iNOS promoter to examine the molecular signaling events necessary for hiNOS transcriptional activation. The combination of LPS and IFN-gamma, but neither alone, increased hiNOS promoter activity 28-fold, in a reaction requiring two critical AP-1 (JunD-Fra-2) promoter binding sites. Mitogen-activated protein kinases (MAPKs) were assessed as potential activators of AP-1 and the hiNOS promoter. Both pharmacological and molecular inhibitors of the extracellular signal-related kinase (ERK) and p38 pathways reduced cytokine mixture (CM)- and LPS/IFN-gamma-induced promoter activation. By gel retardation analysis, the addition of MAP/ERK kinase-1 and p38 inhibitors significantly diminished AP-1 binding in both CM- and LPS/IFN-gamma-stimulated cells. Thus, p38- and ERK-dependent pathways, through effects on the AP-1 complex, activate the hiNOS promoter in cells stimulated with CM or LPS/IFN-gamma.
PMID:_11112784
J Diabetes Complications. 2000 Jul-Aug;14(4):207-14. Correction of glycosylated oxyhemoglobin-induced impairment of endothelium-dependent vasodilatation by gliclazide.
Vallejo S, Angulo J, Peiro C, Sanchez-Ferrer A, Cercas E, Nevado J, Sanchez-Ferrer CF, Rodriguez-Manas L.
Unidad de Investigacion y Servicio de Geriatria, Hospital Universitario de Getafe, Getafe, Spain.
We have investigated whether gliclazide, a second-generation sulfonylurea hypoglycemic agent, interferes with the impairment of endothelium-dependent nitric-oxide-mediated relaxation produced by 14%-glycosylated human oxyhemoglobin (GHHb). For comparative purposes, other agents, like glibenclamide, aminoguanidine, ascorbic acid or superoxide dismutase (SOD), were also tested. GHHb (10 nM) caused a reduction in endothelium-dependent relaxation induced by acetylcholine (1 nM to 10 microM) in both isolated aortic segments and mesenteric microvessels from normoglycemic nondiabetic rats. Preincubation of the vessels with gliclazide (100 nM to 10 microM) prevented the impairment of endothelial relaxation, the threshold concentration of gliclazide being 300 nM. In addition, 10 microM gliclazide also prevented the reduction by 10 nM GHHb of the relaxation induced by exogenous nitric oxide (NO, 10 nM to 100 microM). Determination of superoxide anion release measured by the reduction in ferricytochrome c indicated that GHHb produced significant amounts of these free radicals that were concentration-dependently inhibited by gliclazide. The impairment of endothelium-mediated responses was also prevented by 100 U/ml SOD or 10 microM ascorbic acid, but not by 10 microM glibenclamide or 100 microM aminoguanidine. We conclude that gliclazide can reduce the impairment of nitric-oxide-mediated endothelium-dependent relaxation produced by GHHb. This reduction is likely related to the antioxidant properties of the drug, a mechanism suggested by these studies which demonstrate the inactivation of superoxide anions produced by the glycosylated protein by gliclazide.
PMID:_11004430
J Biol Chem. 2000 Dec 29;275(52):40732-41. Characterization of an upstream enhancer region in the promoter of the human endothelial nitric-oxide synthase gene.
Laumonnier Y, Nadaud S, Agrapart M, Soubrier F.
INSERM U525, Hopital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
The endothelial nitric-oxide synthase gene is constitutively expressed in endothelial cells. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including a GATA-2 and an Sp-1 binding site. Because they cannot account for the constitutive expression of endothelial nitric-oxide synthase gene in a restricted number of cells, we have searched for other cell-specific regulatory elements. By DNase I hypersensitivity mapping and deletion studies we have identified a 269-base pair activator element located 4.9 kilobases upstream from the transcription start site that acts as an enhancer. DNase I footprinting and linker-scanning experiments showed that several regions within the 269-base pair enhancer are important for transcription factor binding and for full enhancer activity. The endothelial specificity of this activation seems partly due to interaction between this enhancer in its native configuration and the promoter in endothelial cells. EMSA experiments suggested the implication of MZF-like, AP-2, Sp-1-related, and Ets-related factors. Among Ets factors, Erg was the only one able to bind to cognate sites in the enhancer, as found by EMSA and supershift experiments, and to activate the transcriptional activity of the enhancer in cotransfection experiments. Therefore, multiple protein complexes involving Erg, other Ets-related factors, AP-2, Sp-1-related factor, and MZF-like factors are important for the function of this enhancer in endothelial cells.
PMID:_11013235
J Biol Chem. 2000 Dec 29;275(52):40974-80. Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins.
Pleschke JM, Kleczkowska HE, Strohm M, Althaus FR.
Institute of Pharmacology and Toxicology, University of Zurich, Tierspital, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland.
Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21(CIP1/WAF1), xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase epsilon, DNA-PK(CS), Ku70, NF-kappaB, inducible nitric-oxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.
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