Crosstalk between nitric oxide and hypoxia-inducible factor signaling pathways: an update

Figures & data

Figure 1 A comparison of the HIF family members shows varying degrees of conservation.

Notes: This comparison shows that all HIF family members contain a bHLH domain, which allows the dimerized proteins to bind to DNA, and two PAS domains, PAS A and PAS B, which facilitate dimerization. HIF-1α and HIF-2α also contain CTADs and the HIF-βs have a TAD, both of which allow p300 to bind the dimerized factor and increase transcriptional activity.⁸
Abbreviations: HIF-1, hypoxia-inducible factor 1; PAS, Per-ARNT-Sim; CTAD, C-terminal activation domain; TAD, transactivation domain; ODDD, oxygen-dependent degradation domain.

Figure 2 Post-translational modifications of HIF-1α.

Notes: HIF-1α is capable of being modified in many ways, and most modifications fall in the C-terminal half of the protein. HIF-1α is hydroxylated at Pro402 and Pro564 by PHD family members and at Asn803 by FIH.^19,21 HIF-1α can be acetylated at Lys532 by Arrest-defective-1 protein (ARD1), Lys674 by p300/CBP associated factor (PCAF), and Lys709 by p300.^82,85,165 HIF-1α is also phosphorylated at Ser641/3 by p42/44 mitogen-activated protein kinase (MAPK).⁸⁷ S-nitrosylation of HIF-1α has been observed at Cys533 and Cys800.^70–72 This figure does not include all known modifications of HIF-1α, but those most relevant to this review.
Abbreviations: HIF-1 α, hypoxia-inducible factor 1α; PHD, prolyl hydroxylase domain; ARD1, arrest-defective-1 protein; CBP, CREB-binding protein; PCAF, p300/CBP associated factor; MAPK, mitogen activated protein kinase; PAS, Per-ARNT-Sim.

Table 1 A representative list of proteins whose expression is regulated by HIF-1

Pathway	Proteins involved
Angiogenesis	VEGF-A,^Citation161 PDGFA,^Citation162 VEGFR-I^Citation163
Cell survival	MET,^Citation160 IGF-2,^Citation163 IGFBP-I^Citation163
Apoptosis	BNIP3,^Citation163 NOXA^Citation162
Metabolism	PDK,^Citation83 PGKI,^Citation161 GAPDH,^Citation161 HK2,^Citation161
	GLUT1,^Citation164 HO-I^Citation164
Secreted factors	TGFα,^Citation161 TGFβ,^Citation161 EPO^Citation161
HIF regulation	PHD3^Citation161
NO production	iNOS,^Citation161 COX4-2^Citation88
Autophagy	BNIP3^Citation30

Notes: Many of these processes are influenced by factors outside of HIF-1 regulation. For example, although apoptotic and prosurvival proteins are induced by HIF-1, the environment in which they are expressed will ultimately determine whether HIF-1 promotes apoptosis or survival.

Abbreviations: HIF-1, hypoxia-inducible factor 1; VEGF-A, vascular endothelial growth factor A; VEGFR-1, VEGF receptor-1; IGF-2, insulin-like growth factor 2; PDGFA, platelet-derived growth factor subunit A; IGFBP-1, insulin-like growth factor binding protein-1; iNOS, inducible nitric oxide synthase; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; NOXA, phorbol-12-myristate-13-acetate-induced protein 1; PHD3, prolyl hydroxylase domain 3; PDK, pyruvate dehydrogenase kinase; PGK1, phosphglycerate kinase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HK2, hexokinase 2; GLUT1, glucose transporter 1; HO-1, heme oxygenase 1; TGFα, transforming growth factor α; TGFβ, transforming growth factor b; EPO, erythropoietin.

Figure 3 A model for the effects of oxygen on arginine and $O_2^{-}$-dependent NO synthesis.

Notes: Nitrite-dependent NO synthesis is favored at low O₂ levels while the arginine-dependent pathway for NO synthesis is favored at high O₂ levels.
Abbreviation: NO, nitric oxide.

Table 2 Positive and negative regulators of NO production

Regulators of activity
NOS		-dependent
Positive	Negative	Positive	Negative
O2	NO		O2
L-Arginine	ADMA	H^+	ATP^a
Ca^2+a	L-NMA	ADP^a	COX5a^a
H^+		COX5b^a
NADPH

Notes: Arginine and -dependent NO production each have various activators and inhibitors. In addition to positive and negative feedback by substrates and products, NOS is inhibited by the arginine analogs ADMA and l-NMA, and nNOS and eNOS are induced by increases in Ca²⁺. -dependent reactions are also subject to feedback regulation. Cco/NO activity is dependent on which subunit 5 isoform is present – 5a or 5b (yeast) – and ATP/ADP levels. High ADP increases Cco/NO activity, while high ATP inhibits it. ^aDenotes Cco/NO specific regulators of activity.

Abbreviations: NO, nitric oxide; Ca²⁺, calcium; NADPH, nicotinamide adenine dinucleotide phosphate; ADMA, N^G, N^G-dimethyl-l-arginine; l-NMA, N^G-methyl-l-arginine; NOS, nitric oxide synthase; O^-₂, nitrite; COX5a, yeast cytochrome c oxidase subunit 5 isoform a; COX5b, yeast cytochrome c oxidase subunit 5 isoform b; O₂, oxygen; ATP, adenosine triphosphate; ADP, adenosine diphosphate.

Figure 4 Impact of O₂ levels on ROS/RNS signaling.

Notes: O₂ levels have a large impact on ROS/RNS signaling. High $O_2^{-}$ and H₂O₂ can stabilize HIF-1α by oxidizing and inhibiting PHD2 and FIH though FIH appears to be far more sensitive to these oxidants.^23,166,167 SIRT1 activity is also negatively affected by ROS.⁸⁴ A mix of high ROS production and high NO production results in the formation of highly toxic molecules such as ONOO^–. ONOO^– is capable of nitrating a large number of proteins, including dimethylarginine dimethylaminohydrolase 1 (DDAH1), catalase, and α-ketoglutarate (or 2-OG) dehydrogenase complex (KGDHC), which results in decreased activity of all three enzymes. DDAH1 is fundamental to NOS functioning by facilitating removal of inhibitors ADMA and l-NMA, and catalase removes H₂O₂ from cells.¹⁶⁸ KGDHC is a tricarboxylic acid cycle enzyme, and its inhibition will decrease 2-OG levels and affect PHD/FIH function.¹⁶⁹ High NO production coupled with low ROS allows for thiol nitrosylation, which can affect HIF-1α, PHD2, and SIRT1.^70,73,84
Abbreviations: RNS, reactive nitrogen species; $O_2^{-}$, superoxide; H₂O₂, hydrogen peroxide; ONOO^–, peroxynitrite; HIF-1 α, hypoxia-inducible factor 1α; NO, nitric oxide; PHD2, prolyl hydroxylase domain 2; FIH, factor inhibiting HIF-1; SIRT1, sirtuin 1; DDAH1, dimethylarginine dimethylaminohydrolase 1; KGDHC, α-ketoglutarate (or 2-OG) dehydrogenase complex; ADMA, N^G, N^G-dimethyl-l-arginine; l-NMA, N^G-methyl-l-arginine; 2-OG, 2-oxoglutarate.

Figure 5 NO is both upstream and downstream of PI3K/Akt and HIF-1 signaling and can affect many proteins and pathways within the cell, including Ras and HIF-1α.

Notes: The specific modifications and functional effects NO exerts may be a result of differing sources and concentrations of NO, or whether the added NO is the result of transnitrosylation via GADPH or NO release from GSNO.
Abbreviations: NO, nitric oxide; PI3K, phosphatidylinositide 3-Kinase; Akt, protein kinase B; HIF-1, hypoxia-inducible factor 1; ER, estrogen receptors; mAchR, muscarinic acetylcholine receptor; COX4-2, gene encoding cytochrome c oxidase subunit 4 isoform 2; NOS2, gene encoding inducible nitric oxide synthase; GAPDH, glyceraldehyde phosphate dehydrogenase; GSNO, S-nitrosylate glutathione.

Figure 6 HIF-NO signaling supports several processes in the body, including development, angiogenesis, immunity, apoptosis, survival, and aging.

Abbreviations: HIF, hypoxia-inducible factor; NO, nitric oxide.

Figure 7 Oncogenic and tumor suppressive activities of HIF-1.

Notes: HIF-1 has both oncogenic and tumor suppressive activities. While HIF-1 does increase cell migration, angiogenesis, glucose uptake, and lipid availability, all of which promote tumor growth and metastasis, HIF-1 also increases autophagy, apoptosis, and p53 accumulation and decreases Myc activity, all of which slow tumor growth and progression. Certain activities indicated with an * can be either oncogenic or tumor suppressive, depending on the specific tumor and its environment.
Abbreviation: HIF-1, hypoxia-inducible factor 1.

Kewley R, Whitelaw M, Chapman-Smith A. The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int J Biochem Cell Biol. 2004;36:189–204.

 PubMed Web of Science ®Google Scholar

Berra E, Benizri E, Ginouvès A, Volmat V, Roux D, Pouysségur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1α in normoxia. EMBO J. 2003;22:4082–4090.

 PubMed Web of Science ®Google Scholar

Lando D, Peet D, Whelan D, Gorman J, Whitelaw M. Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch. Science. 2002;295(5556):858–861.

 PubMed Web of Science ®Google Scholar

Arif M, Vedamurthy B, Choudhari R, et al. Nitric oxide-mediated histone hyperacetylation in oral cancer: target for a water-soluble HAT inhibitor, CTK7A. Chem Biol. 2010;17:903–913.

 PubMedGoogle Scholar

Laemmle A, Lechleiter A, Roh V, et al. Inhibition of SIRT1 impairs the accumulation and transcriptional activity of HIF-1α protein under hypoxic conditions. PLoS One. 2012;7:e33433.

 PubMed Web of Science ®Google Scholar

Jeong J, Bae M, Ahn M, et al. Regulation and destabilization of HIF-1α by ARD1-mediated acetylation. Cell. 2002;111:709–720.

 PubMed Web of Science ®Google Scholar

Callsen D, Pfeilschifter J, Brüne B. Rapid and delayed p42/p44 mitogen-activated protein kinase activation by nitric oxide: the role of cyclic GMP and tyrosine phosphatase inhibition. J Immunol. 1998;161:4852–4858.

 Web of Science ®Google Scholar

Yasinska I, Sumbayev V. S-nitrosation of Cys-800 of HIF-1α protein activates its interaction with p300 and stimulates its transcriptional activity. FEBS Lett. 2003;549:105–109.

 PubMed Web of Science ®Google Scholar

Chowdhury R, Flashman E, Mecinovic J, et al. Studies on the reaction of nitric oxide with the hypoxia-inducible factor prolyl hydroxylase domain 2 (EGLN1). J Mol Biol. 2011;410:268–279.

 PubMed Web of Science ®Google Scholar

Gerber H, Condorelli F, Park J, Ferrara N. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes: Flt-1, but not Flk-1/KDR, is up-regulated by hypoxia. J Biol Chem. 1997;272:23659–23667.

 PubMed Web of Science ®Google Scholar

Semenza G. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003;3:721–732.

 PubMed Web of Science ®Google Scholar

Bergeron M, Yu A, Solway K, Semenza G, Sharp F. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. European J Neurosci. 1999;11:4159–4170.

 PubMed Web of Science ®Google Scholar

Benita Y, Kikuchi H, Smith A, Zhang M, Chung D, Xavier R. An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res. 2009;37:4587–4602.

 PubMed Web of Science ®Google Scholar

Kornberg M, Sen N, Hara M, et al. GAPDH mediates nitrosylation of nuclear proteins. Nat Cell Biol. 2010;12:1094–1100.

 PubMed Web of Science ®Google Scholar

Yang Z, Zou A. Transcriptional regulation of heme oxygenases by HIF-1α in renal medullary interstitial cells. Am J Physiol Renal Physiol. 2001;281:F900–F908.

 PubMed Web of Science ®Google Scholar

Fukuda R, Zhang H, Kim J, Shimoda L, Dang C, Semenza G. HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell. 2007;129:111–122.

 PubMed Web of Science ®Google Scholar

Zhang H, Bosch-Marce M, Shimoda L, et al. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem. 2008;283:10892–10903.

 PubMed Web of Science ®Google Scholar

Masson N, Singleton R, Sekirnik R, et al. The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity. EMBO Rep. 2012;13:251–257.

 PubMed Web of Science ®Google Scholar

Pan Y, Mansfield K, Bertozzi C, et al. Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro. Mol Cell Biol. 2007;27(3):912–925.

 PubMed Web of Science ®Google Scholar

Caito S, Rajendrasozhan S, Cook S, et al. SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress. FASEB J. 2010;24(9):3145–3159.

 PubMed Web of Science ®Google Scholar

Lim J, Lee Y, Chun Y, Chen J, Kim J, Park J. Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1α. Mol Cell. 2010;38:864–878.

 PubMed Web of Science ®Google Scholar

Tyther R, McDonagh B, Sheehan D. Proteomics in investigation of protein nitration in kidney disease: Technical challenges and perspective from the sponaneously hypertensive rat. Mass Spectrom Rev. 2011;30(1):121–141.

 PubMed Web of Science ®Google Scholar

Shi Q, Xu H, Yu H, et al. Inactivation and reactivation of the mitochondrial -ketoglutarate dehydrogenase complex. J Biol Chem. 2011;286(20):17640–17648.

 PubMed Web of Science ®Google Scholar

Park Y, Ahn D, Oh M, et al. Nitric oxide donor, (±)-S-nitroso-N-acetylpenicillamine, stabilizes transactive hypoxia-inducible factor-1 by inhibiting von Hippel-Lindau recruitment and asparagine hydroxylation. Mol Pharmacol. 2008;74:236–245.

 Web of Science ®Google Scholar