Characterization of heme oxygenase 1 (heat shock protein 32) induction by atrial natriuretic peptide in human endothelial cells.
BACKGROUND
Atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory and cytoprotective potential. The aim of this study was to characterize induction of heme oxygenase (HO)-1 by ANP in human umbilical vein endothelial cells (HUVEC).


METHODS
HUVEC were treated with ANP, 8-bromo-cyclic GMP (cGMP), or cANF in the presence or absence of various inhibitors. HO-1 was determined by Western blot and RT-PCR, c-jun N-terminal kinase (JNK) and ERK by the use of phospho-specific antibodies. Activator protein (AP)-1 activation was assessed by gelshift assay. Reporter gene assays were performed using native or mutated AP-1 binding sites of the HO-1 promoter. TNF-alpha-induced cell death was investigated by Hoechst staining, fluorescence-activated cell sorting analysis, caspase-3-measurement, and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide test.


RESULTS
ANP (10(-9)-10(-6) mol/liter) induced the expression of HO-1 protein and mRNA. Induction was mediated via the guanylate-cyclase-coupled receptor because 8-Br-cGMP mimicked the effect of ANP, whereas the clearance receptor agonist cANF did not induce HO-1. Endogenously produced cGMP also induced HO-1 because phosphodiesterase inhibition markedly elevated HO-1. The lack of effect of the cGMP-dependent protein kinase inhibitor 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-pCT-cGMPS) suggested no involvement for this cGMP effector pathway in the signal transduction. ANP lead to activation of the transcription factor AP-1, and subsequently of JNK, as well as of ERK. Cotreatment of the cells with U0126 or SP600125, as well as reporter gene assays revealed the involvement of AP-1/JNK activation in HO-1 induction. Abrogation of HO-1 induction by PD-98059 showed also a role for ERK. Treatment of HUVEC with ANP did not protect from TNF-alpha-induced apoptosis.


CONCLUSIONS
This work characterizes the induction of HO-1 by ANP in HUVEC, which is shown to be mediated via JNK/AP-1 and ERK pathways. ANP-induced HO-1 does not confer protection against TNF-alpha-induced apoptosis.
Kiemer
Alexandra K
AK
Department of Pharmacy, Center of Drug Research, University of Munich, 81377 Munich, Germany. Alexandra.Kiemer@cup.uni-muenchen.de
Bildner
Nicole
N
Weber
Nina C
NC
Vollmar
Angelika M
AM
D000818:Animals; D017209:Apoptosis; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis* / Q000235:genetics; D051547:Heme Oxygenase-1; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D008565:Membrane Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins
D008565:Membrane Proteins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; C016276:8-bromocyclic GMP; D009320:Atrial Natriuretic Factor; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D006152:Cyclic GMP