Interaction of nitric oxide with human heme oxygenase-1

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.     

Nitric oxide priming protects nitric oxide-mediated apoptosis via heme oxygenase-1 induction

The role of nitric oxide (NO) as a cytotoxic effector molecule of the immune system is clearly established, but recent studies demonstrate cytoprotective functions of NO at low nontoxic concentrations. However, the mechanism of cytoprotection has not been defined completely. Thus, we investigate the involvement of heme oxygenase-1 (HO-1) in the cytoprotective effects of NO. Exposure of L929 cells to sodium nitroprusside (SNP) resulted in the induction of HO-1 protein expression and heme oxygenase activity. Pretreatment of the cells with a low dose of NO (200 microM SNP) significantly inhibited a high dose of (1000 microM SNP) NO-induced apoptosis in L929 cells. Cytoprotection by a low dose of NO was abrogated in the presence of the heme oxygenase inhibitor zinc protoporphyrin IX. A cytoprotective effect comparable to a low dose of SNP was observed when the cells were transfected with HO-1 gene or preincubated with another HO-1 inducer, hemin. Additional experiments revealed the involvement of carbon monoxide in the cytoprotective effect of SNP/HO-1 in L929 cells. Our results presented here provide evidence to support the essential role of HO-1 in the cytoprotective function of NO priming.     

Regulation of heme oxygenase-1 by nitric oxide during hepatopulmonary syndrome

During hepatopulmonary syndrome caused by liver cirrhosis, pulmonary endothelial nitric oxide (NO) synthase (NOS) expression and NO production are increased. Increased NO contributes to the blunted hypoxic pressor response (HPR) during cirrhosis and may induce heme oxygenase-1 (HO-1) expression and carbon monoxide (CO) production, exacerbating the blunted HPR. We hypothesized that NO regulates the expression of HO-1 during cirrhosis, contributing to hepatopulmonary syndrome. Cirrhosis was induced in rats by common bile duct ligation (CBDL). Rats were studied 2 and 5 wk after CBDL or sham surgery. Lung HO-1 expression was elevated 5 wk after CBDL. Liver HO-1 was increased at 2 wk and remained elevated at 5 wk. In catheterized rats, the blunted HPR was partially restored by HO inhibition. Rats treated with the NOS inhibitor N(G)-nitro-L-arginine methyl ester for the entire 2- or 5-wk duration had normalized HO-1 expression and HPR. These data provide in vivo evidence for the NO-mediated upregulation of HO-1 expression and support the concept that hepatopulmonary syndrome is multifactorial, involving not only NO, but also HO-1 and CO.     

Heme oxygenase-1 induction depletes heme and attenuates pulmonary artery relaxation and guanylate cyclase activation by nitric oxide

This study examines in endothelium-denuded bovine pulmonary arteries the effects of increasing heme oxygenase-1 (HO-1) activity on relaxation and soluble guanylate cyclase (sGC) activation by nitric oxide (NO). A 24-h organ culture with 0.1 mM cobalt chloride (CoCl2) or 30 microM Co-protoporphyrin IX was developed as a method of increasing HO-1 expression. These treatments increased HO-1 expression and HO activity by approximately two- to fourfold and lowered heme levels by 40-45%. Induction of HO-1 was associated with an attenuation of pulmonary arterial relaxation to the NO-donor spermine-NONOate. The presence of a HO-1 inhibitor 30 microM chromium mesoporphyrin during the 24-h organ culture (but not acute treatment with this agent) reversed the attenuation of relaxation to NO seen in arteries co-cultured with agents that increased HO-1. Relaxation to isoproterenol, which is thought to be mediated through cAMP, was not altered in arteries with increased HO-1. Inducers of HO-1 did not appear to alter basal sGC activity in arterial homogenates or expression of the beta(1)-subunit of sGC. However, the increase in activity seen in the presence of 1 microM spermine-NONOate was attenuated in homogenates obtained from arteries with increased HO-1. Since arteries with increased HO-1 had decreased levels of superoxide detected by the chemiluminescence of 5 microM lucigenin, superoxide did not appear to be mediating the attenuation of relaxation to NO. These data suggest that increasing HO-1 activity depletes heme, and this is associated with an attenuation of pulmonary artery relaxation and sGC activation responses to NO.     

Heme oxygenase-1 induction by endogenous nitric oxide: influence of intracellular glutathione

To investigate the influence of glutathione (GSH) on cellular effects of nitric oxide (NO) formation, human colon adenocarcinoma cells were transfected with a vector allowing controlled expression of inducible nitric oxide synthase (iNOS). Protein levels of oxidative stress-sensitive heme oxygenase-1 (HO-1) were analyzed in the presence or absence of GSH depletion using L-buthionine-[S,R]-sulfoximine and iNOS induction. While no effect was observed in the presence of iNOS activity alone, a synergistic effect on HO-1 expression was observed in the presence of iNOS expression and GSH depletion. This effect was prevented by addition of N-methyl-L-arginine. Therefore, targeting of endogenous NO may be modulated by intracellular GSH.     

Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential component of cellular defense against a vast variety of endogenous and exogenous insults, including oxidative stress. Nrf2 acts as a master switch in the circuits upregulating the expression of various stress-response proteins, especially heme oxygenase-1 (HO-1). Paradoxically, however, recent studies have demonstrated oncogenic functions of Nrf2 and its major target protein HO-1. Levels of Nrf2 and HO-1 are elevated in many different types of human malignancies, which may facilitate the remodeling of the tumor microenvironment making it advantageous for the autonomic growth of cancer cells, metastasis, angiogenesis, and tolerance to chemotherapeutic agents and radiation and photodynamic therapy. In this context, the cellular stress response or cytoprotective signaling mediated via the Nrf2–HO-1 axis is hijacked by cancer cells for their growth advantage and survival of anticancer treatment. Therefore, Nrf2 and HO-1 may represent potential therapeutic targets in the management of cancer. This review highlights the roles of Nrf2 and HO-1 in proliferation of cancer cells, their tolerance/resistance to anticancer treatments, and metastasis or angiogenesis in tumor progression.     

Heme oxygenase-1 induction improves ischemic renal failure: role of nitric oxide and peroxynitrite

The present study evaluated the effects of heme oxygenase-1 (HO-1) induction on the changes in renal outer medullary nitric oxide (NO) and peroxynitrite levels during 45-min renal ischemia and 30-min reperfusion in anesthetized rats. Glomerular filtration rate (GFR), outer medullary blood flow (OMBF), HO and nitric oxide synthase (NOS) isoform expression, and renal low-molecular-weight thiols (-SH) were also determined. During ischemia significant increases in NO levels and peroxynitrite signal were observed (from 832.1 +/- 129.3 to 2,928.6 +/- 502.0 nM and from 3.8 +/- 0.7 to 9.0 +/- 1.6 nA before and during ischemia, respectively) that dropped to preischemic levels during reperfusion. OMBF and -SH significantly decreased after 30 min of reperfusion. Twenty-four hours later, an acute renal failure was observed (GFR 923.0 +/- 66.0 and 253.6 +/- 55.3 microl.min(-1).g kidney wt(-1) in sham-operated and ischemic kidneys, respectively; P < 0.05). The induction of HO-1 (CoCl(2) 60 mg/kg sc, 24 h before ischemia) decreased basal NO concentration (99.7 +/- 41.0 nM), although endothelial and neuronal NOS expression were slightly increased. CoCl(2) administration also blunted the ischemic increase in NO and peroxynitrite (maximum values of 1,315.6 +/- 445.6 nM and 6.3 +/- 0.5 nA, respectively; P < 0.05), preserving postischemic OMBF and GFR (686.4 +/- 45.2 microl.min(-1).g kidney wt(-1)). These beneficial effects of CoCl(2) on ischemic acute renal failure seem to be due to HO-1 induction, because they were abolished by stannous mesoporphyrin, a HO inhibitor. In conclusion, HO-1 induction has a protective effect on ischemic renal failure that seems to be partially mediated by decreasing the excessive production of NO with the subsequent reduction in peroxynitrite formation observed during ischemia.     

Role of Pin1 in neointima formation: Down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr–Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.     

Homocysteine attenuates endothelial haem oxygenase-1 induction by nitric oxide (NO) and hypoxia.

The disrupted metabolism of homocysteine (Hcy) causes hyperhomocysteinemia, a condition associated with the impairment of nitric oxide (NO) bio-availability, tissue hypoxia and increased risk of vascular disease. Here, we examined how Hcy modulates the induction of the stress protein haem oxygenase-1 (HO-1) evoked by NO releasing agents and hypoxia in vascular endothelial cells. We found that Hcy (0.5 mM) markedly reduced the increase in haem oxygenase activity and HO-1 protein expression induced by sodium nitroprusside (SNP, 0.5 mM) but did not affect HO-1 activation mediated by S-nitroso-N-acetyl-penicillamine. Cells pre-treated with Hcy followed by addition of fresh medium containing SNP still exhibited an augmented haem oxygenase activity. Interestingly, high levels of Hcy were also able to abolish hypoxia-mediated HO-1 expression in a concentration-dependent manner. These novel findings indicate that hyperhomocysteinemia interferes with crucial signaling pathways required by cells to respond and adapt to stressful conditions.     

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO₂) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO₂ via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO₂-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO₂-FAs stimulated the phosphorylation of eNOS at Ser¹¹⁷⁹. These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO₂-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.