Hypoxic Regulation of Inducible Nitric Oxide Synthase via Hypoxia Inducible Factor-1 in Cardiac Myocytes

Abstract

The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.

Expression of constitutively stable hybrid hypoxia-inducible factor-1α protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury

“…It is known that hypoxic activation of iNOS transcription is mediated by HIF in cardiomyocytes (19,25). The increase of iNOS mRNA levels by the infection of cultured neonatal cardiomyocytes with Ad2/HIF-1␣/VP16 or Ad2/HIF-1␣/ NF-B implies that the upregulation of this protein is an important protective mechanism by expression of a constitutively stable hybrid HIF-1␣ in this particular experimental setting.…”

“…It has been suggested that AP-1 and NF-B may mediate the transcriptional activation of several mediators that are involved in ischemic preconditioning (22,36). It is also known that transcription of iNOS, an important mediator/effector of myocardial preconditioning (8), is directly activated by HIF-1 in response to hypoxia (9,12,19,32). Protection of rat brain and retinal tissues against subsequent lethal ischemia by prior exposure to sublethal hypoxia is associated with elevated levels of the HIF-1␣ protein (6,7,14).…”

“…Significant elevation of HSP70 mRNA levels persisted up to 24 h after preconditioning. It is conceivable that upregulation of Glut-1, Glut-4, and iNOS is mediated by HIF because they are known HIF-1 target genes (19,25,32). Furthermore, expression of a constitutively stable hybrid HIF-1␣ protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury and induced a similar set of protective genes.…”

Overexpression of hypoxia-inducible-factor 1α(HIF-1α) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage

“…Hypoxiainducible-factor-1 is an important component of a widely operative transcriptional response, activated by hypoxia, cobaltous ions, and iron chelation. Hypoxia-inducible-factor 1 activates transcription of hypoxia-inducible genes, including those encoding erythropoietin (Wang and Semenza 1993), vascular endothelial growth factor (VEGF) (Forsythe et al, 1996), heme oxygenase-1 (Hoetzel et al, 2001), inducible nitric oxide synthase (Jung et al, 2000), and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A , phosphofructokinase I (Minchenko et al, 2002), and phosphoglycerate kinase I (Li et al, 1996). The C-terminal of HIF-1a binds to p300, and p300/CBP-HIF complexes participate in the induction of hypoxia-responsive genes, including VEGF (Arany et al, 1996).…”

Toll-like receptor 7/8 agonist resiquimod induces late preconditioning in neonatal cardiac myocytes

“…Expression of HIF1α mRNA and protein is induced in the brains of neonatal rats following hypoxia, intraperitoneal injection of cobalt chloride or desferrioxamine, and this up-regulation is associated with protection against cerebral infarction following hypoxia and ischemia [28] . HIF1α expression is also likely to be involved in cardiac preconditioning because NOS2 expression is essential for late preconditioning, and an intact HIF1 DNA binding site in the NOS2 promoter is required for its transcriptional induction in hypoxic myocardial cells [29] . Previous reports demonstrated that HIF1 interacts with the iNOS promoter directly in myocardial extracts after intermittent hypoxic preconditioning by using chromatin immunoprecipitation assay (CHIP) analysis [30] .…”