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Pulmonary expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) is critically important for normal functional maturation of alveolar macrophages. We found previously that lung GMCSF is dramatically suppressed in mice exposed to hyperoxia. Alveolar epithelial cells (AEC) are a major source of GM-CSF in the peripheral lung, and in vivo hyperoxia resulted in greatly reduced expression of GM-CSF protein by AEC ex vivo. We now explore the mechanisms responsible for this effect, using primary cultures of murine AEC exposed to hyperoxia in vitro. Exposure of AEC to 80% oxygen/5% C[O.sub.2] for 48 h did not induce overt toxicity, but resulted in significantly decreased GM-CSF protein and mRNA expression compared with cells in normoxia. Similar effects were seen when AEC were stressed with serum deprivation, an alternative inducer of oxidative stress. The effects in AEC were opposite those in a murine lung epithelial cell line (MLE-12 cells), in which hyperoxia induced GM-CSF expression. Both hyperoxia and serum deprivation resulted in increased intracellular reactive oxygen species (ROS) in AEC. Hyperoxia and serum deprivation induced significantly accelerated turnover of GM-CSF mRNA. Treatment of AEC with catalase during oxidative stress preserved GM-CSF protein and mRNA and was associated with stabilization of GM-CSF mRNA. We conclude that hyperoxia-induced suppression of AEC GM-CSF expression is a function of ROS-induced destabilization of GM-CSF mRNA. We speculate that AEC oxidative stress results in significantly impaired pulmonary innate immune defense due to effects on local GM-CSF expression in the lung. lung; innate immunity; growth factors; oxidative stress doi:10.1152/ajplung.00161.2009.

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-[beta]1 (TGF-[beta]1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-[beta]1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-[beta]1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-[beta]1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-[beta]1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

The receptor for advanced glycation end-products (RAGE) is a member of the immunoglobin superfamily of multiligand receptors. Following ligand binding, mechanisms associated with host defense, tissue remodeling, and inflammation are activated. RAGE is highly expressed in pulmonary epithelium transitioning from alveolar type (AT) II to ATI cells and is upregulated in the presence of ligand; however, the regulation and function of RAGE during development are less clear. Herein, immunohistochemistry demonstrated a temporal-spatial pattern of RAGE expression in pulmonary epithelial cells from embryonic clay 17.5 to postnatal day 10. Cotransfection experiments revealed that the mouse RAGE promoter was activated by early growth response gene 1 (Egr-1) and inhibited by thyroid transcription factor-1 (TTF-1) via interaction with specific regulatory elements. A rat ATI cell line (R3/1) with endogenous RAGE expression also differentially regulated RAGE when transfected with TTF-1 or Egr-1. Because Egr-1 is markedly induced in pulmonary epithelial cells exposed to cigarette smoke extract (CSE; Reynolds PR, Hoidal JR. Am J Respir Cell Mol Biol 35: 314-319, 2006.), we sought to investigate RAGE induction by CSE. Employing RT-PCR and Western blotting, RAGE and common ligands (amphoterin and S 100A12) were upregulated in epithelial (R3/1 and A549) and macrophage (RAW) cell lines following exposure to CSE. Immunostaining for RAGE in cells similarly exposed and in lungs from mice exposed to cigarette smoke for 6 mo revealed elevated RAGE expression in pulmonary epithelium. After the addition of glyoxylated BSA, an advanced glycation end-product that binds RAGE, real-time RT-PCR detected a 200-fold increase in Egr-1. These results indicate that Egr-1 regulates RAGE expression during development and the likelihood of positive feedback involving Egr-1 and RAGE in cigarette smoke-related disease. development; disease; lung; receptor for advanced glycation endproducts; early growth response gene 1

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component ([p47.sup.phox]) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in [p47.sup.phox-/-] than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in [p47.sup.phox-/-] compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 [+ or -] 2.4 and 9.2 [+ or -] 2.3% in null and wild-type mice (P < 0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the [p47.sup.phox] subunit of NAD(P)H oxidase plays a role in ASM relaxation. airways; airway responsiveness; contractility; reactive oxygen species

Transforming growth factor-[beta]1 (TGF-[beta]1) plays a pivotal role in increasing airway smooth muscle mass in severe asthma by inducing proliferation and hypertrophy of human airway smooth muscle. The mechanism(s) for these effects of TGF-[beta]1 have not been fully elucidated. In this study, we demonstrate that TGF-[beta]1 is a potent inducer of expression of the nonphagocyte NAD(P)H oxidase catalytic homolog Nox4, diphenylene iodonium-inhibitable reactive oxygen species production, proliferation, and hypertrophy in cultured human airway smooth muscle cells. By confocal microscopy, TGF-[beta]1-induced Nox4 was localized with the endoplasmic reticulum and the nucleus, implying a role for Nox4 in regulation of both the cell cycle and protein synthesis. Consistent with this hypothesis, TGF-[beta]1 increased retinoblastoma protein phosphorylation at both Ser807/811 and Ser780. Silencing Nox4 prevented TGF-[beta]1-mediated retinoblastoma protein phosphorylation, proliferation, and cell hypertrophy. TGF-[beta]1 also increased phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 at Thr37/46, and this was likewise blocked by silencing Nox4. This is the first report to suggest a functional role for Nox4 in cell cycle transition and to demonstrate that Nox4 influences the pathobiochemistry of asthma by generating reactive oxygen species that promote TGF-[beta]1-induced proliferation and hypertrophy of human airway smooth muscle. Myc; cdc2; eukaryotic translation initiation factor 4E binding protein- 1; nicotinamide adenine dinucleotide phosphate oxidase; reactive oxygen species; translation doi: 10.1152/ajplung.00430.2006

Transforming growth factor-[beta]1 (TGF-[beta]1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-[beta]1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-[beta]1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-[beta]1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-[beta]1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-[beta]1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-[beta]1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-[beta]1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-[beta]1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-[beta]1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling. Smad 2/3; pulmonary arterial hypertension; hypertrophy; superoxide anion; rac 1; ERK 1/2; phosphatases

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, [p22.sup.phox] and [gp91.sup.phox] as well as the [Ca.sup.2+] concentration-responsive [gp.syup.91phox] homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for [p22.sup.phox] was not detectable, both [gp91.sup.phox] and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to [p22.sup.phox] or [gp91.sup.phox] did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death. superoxide anion; diphenylene iodonium; [p22.sup.phox]; [gp91.sup.phox]; adenosine 3',5'-cyclic monophosphate response element; caspases

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion ([O.sup.-.sub.2]) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating ([O.sup.-.sub.2]) as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for [p22.sup.phox]. Transfection with p22phox antisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-[kappa]B (NF-[kappa]B), and overexpression of a superrepressor form of the NF-[kappa]B inhibitor I[kappa]B[alpha] significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing [p22.sup.phox] regulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-[kappa]B. superoxide anion; diphenylene iodonium; [p22.sup.phox]

Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-[kappa]B (NF-[kappa]B). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-[kappa]B activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The [p22.sup.phox], [gp91.sup.phox], and [p67.sup.phox] components of the human phagocyte NAD(P)H oxidase and the [gp91.sup.phox] homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both [p22.sup.phox] and [gp91.sup.phox] was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only [p22.sup.phox] and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for [p22.sup.phox] and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-[kappa]B and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD(P)H oxidase may play a role in signaling malignant melanoma growth. superoxide anion; diphenylene iodonium; [p22.sup.phox]; [gp91.sup.phox]; [p67.sup.phox]; NOX1; NOX4; nuclear factor-[kappa]B; cAMP response element; dicumarol

Brar, Sukhdev S., Thomas P. Kennedy, A. Richard Whorton, Anne B. Sturrock, Thomas P. Huecksteadt, Andrew J. Ghio, and John R. Hoidal. Reactive oxygen species from NAD(P)H:quinone oxidoreductase constitutively activate NF-[Kappa]B in malignant melanoma cells. Am J Physiol Cell Physiol 280: C659-C676, 2001.--The transcription factor nuclear factor-[Kappa]B (NF-[Kappa]B) is constitutively activated in malignancies from enhanced activity of inhibitor of NF-[Kappa]B (I[Kappa]B) kinase, with accelerated I[Kappa]B[Alpha] degradation. We studied whether redox signaling might stimulate these events. Cultured melanoma cells generated superoxide anions [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] without serum stimulation. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] generation was reduced by the NAD(P)H:quinone oxidoreductase (NQO) inhibitor dicumarol and the quinone analog capsaicin, suggesting that electron transfer from NQO through a quinone-mediated pathway may be an important source of endogenous reactive oxygen species (ROS) in tumor cells. Treatment of malignant melanoma cells with the [H.sub.2][O.sub.2] scavenger catalase, the sulfhydryl donor N-acetylcysteine, the glutathione peroxidase mimetic ebselen, or dicumarol decreased NF-[Kappa]B activation. Catalase, N-acetylcysteine, ebselen, dicumarol, and capsaicin also inhibited growth of melanoma and other malignant cell lines. These results raise the possibility that ROS produced endogenously by mechanisms involving NQO can constitutively activate NF-[Kappa]B in an autocrine fashion and suggest the potential for new antioxidant strategies for interruption of oxidant signaling of melanoma cell growth. nuclear factor-[Kappa]B; superoxide anion; hydrogen peroxide; tumor; neoplasm; dicumarol

Thourani, Vinod H., Sukhdev S. Brar, Thomas P. Kennedy, Lisa R. Thornton, John A. Watts, Russell S. Ronson, Zhi-Qing Zhao, Anne L. Sturrock, John R. Hoidal, and Jakob Vinten-Johansen. Nonanticoagulant heparin inhibits NF-[Kappa]B activation and attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol 48: H2084-H2093, 2000.--Heparin reduces ischemia-reperfusion injury to myocardium. This effect has been attributed to complement inhibition, but heparin also has other activities that might diminish ischemia-reperfusion. To further probe these mechanisms, we compared heparin or an o-desulfated nonanticoagulant heparin with greatly reduced anticomplement activity. When given at the time of coronary artery reperfusion in a canine model of myocardial infarction, heparin or o-desulfated heparin equally reduced neutrophil adherence to ischemic-reperfused coronary artery endothelium, influx of neutrophils into ischemic-reperfused myocardium, myocardial necrosis, and release of creatine kinase into plasma. Heparin or o-desulfated heparin also prevented dysfunction of endothelial-dependent coronary relaxation following ischemic injury. In addition, heparin and o-desulfated heparin inhibited translocation of the transcription nuclear factor-[Kappa]B (NF-[Kappa]B) from the cytoplasm to the nucleus in human endothelial cells and decreased NF-[Kappa]B DNA binding in human endothelium and ischemic-reperfused rat myocardium. Thus heparin and nonanticoagulant heparin decrease ischemia-reperfusion injury by disrupting multiple levels of the inflammatory cascade, including the novel observation that heparins inhibit activation of the proinflammatory transcription factor NF-[Kappa]B. ischemia-reperfusion; myocardial infarction; endothelium neutrophils; endothelial dysfunction

The physicochemical properties of combustion particles that promote lung toxicity are not fully understood, hindered by the fact that combustion particles vary based on the fuel and combustion conditions. Real-world combustion-particle properties also continually change as new fuels are implemented, engines age, and engine technologies evolve. This work used laboratory-generated particles produced under controlled combustion conditions in an effort to understand the relationship between different particle properties and the activation of established toxicological outcomes in human lung cells (H441 and THP-1). Particles were generated from controlled combustion of two simple biofuel/diesel surrogates (methyl decanoate and dodecane/biofuel-blended diesel (BD), and butanol and dodecane/alcohol-blended diesel (AD)) and compared to a widely studied reference diesel (RD) particle (NIST SRM2975/RD). BD, AD, and RD particles exhibited differences in size, surface area, extractable chemical mass, and the content of individual polycyclic aromatic hydrocarbons (PAHs). Some of these differences were directly associated with different effects on biological responses. BD particles had the greatest surface area, amount of extractable material, and oxidizing potential. These particles and extracts induced cytochrome P450 1A1 and 1B1 enzyme mRNA in lung cells. AD particles and extracts had the greatest total PAH content and also caused CYP1A1 and 1B1 mRNA induction. The RD extract contained the highest relative concentration of 2-ring PAHs and stimulated the greatest level of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFα) cytokine secretion. Finally, AD and RD were more potent activators of TRPA1 than BD, and while neither the TRPA1 antagonist HC-030031 nor the antioxidant N-acetylcysteine (NAC) affected CYP1A1 or 1B1 mRNA induction, both inhibitors reduced IL-8 secretion and mRNA induction. These results highlight that differences in fuel and combustion conditions affect the physicochemical properties of particles, and these differences, in turn, affect commonly studied biological/toxicological responses. [ABSTRACT FROM PUBLISHER]

NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species and has a central role in airway smooth muscle (ASM) cell proliferation. Since reactive oxygen species also affect ASM contractile response, we hypothesized a regulatory role of NAD(P)H oxidase in ASM contractility. We therefore studied ASM function in wild-type mice (C57BL/6J) and mice deficient in a component (p47phox) of NAD(P)H oxidase. In histological sections of the trachea, we found that the area occupied by ASM was 17% more in p47phox than in wild-type mice. After correcting for the difference in ASM content, we found that force generation did not vary between the two genotypes. Similarly, their ASM shortening velocity, maximal power, and sensitivity to acetylcholine, as well as airway responsiveness to methacholine in vivo, were not significantly different. The main finding of this study was a significantly reduced ASM relaxation in p47phox compared with wild-type mice both during the stimulus and after the end of stimulation. The tension relaxation attained at the 20th second of electric field stimulation was, respectively, 17.6 ± 2.4 and 9.2 ± 2.3% in null and wild-type mice (P <0.01 by t-test). Similar significant differences were found in the rate of tension relaxation and the time required to reduce tension by one-half. Our data suggest that NAD(P)H oxidase may have a role in the structural arrangement and mechanical properties of the airway tissue. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a role in ASM relaxation. [ABSTRACT FROM AUTHOR]

Determines whether reactive oxygen species might mediate constitutive nuclear factor-kappa B activation in malignant melanoma cell lines. Effect of antioxidants on the constitutive activation of nuclear factor-kappa B in malignant human cell lines; Effect of antioxidants the growth of malignant cell lines.