Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells - PubMed (original) (raw)
Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells
Srikanth Pendyala et al. Antioxid Redox Signal. 2009 Apr.
Abstract
In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22(phox) compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22(phox), but not Nox2 or p47(phox), are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2(-/-) ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs.
Figures
FIG. 1.
Effect of hyperoxia on mRNA expression of Nox and phox family NADPH oxidases in HPAECs. HPAECs grown to ∼90% confluence were exposed to normoxia (room air) or hyperoxia (95% O2) for 12, 24, 48, and 72 h. Total RNA was extracted and expressions of Nox and phox homologues were quantified by real-time RT-PCR and normalized with 18S rRNA. The values are mean ± S.E.M for three independent experiments. *significantly different from normoxia (p < 0.05); **significantly different from normoxia (p < 0.01).
FIG. 2.
Hyperoxia increases protein expression of Nox4 and Nox2 in HPAECs. HPAECs (∼90% confluence) were subjected to normoxia or hyperoxia for 3 or 24 h. Cells were rinsed twice in ice-cold PBS , total cell lysates (20 μg of protein) were subjected to SDS-PAGE, as described in “Materials and Methods” and analyzed by Western blot analysis with anti-Nox4 (Santa Cruz, CA), -Nox2 (gp91_phox_), and -p22_phox_ antibodies. Shown are representative Western blots from three independent experiments. Blots were scanned and quantified by automated digitized system UN-SCAN-IT-GEL. *significantly different from cells exposed to normoxia (p < 0.05).
FIG. 3.
Localization of Nox4 and p22 phox under normoxia and hyperoxia in HPAECs. (A) HPAECs grown on glass coverslips were exposed to normoxia (N) or hyperoxia (HO) for 3 h, washed, fixed, permeabilized and probed with anti-Nox4 (from Dr. Lambeth) or anti-p22_phox_ antibodies. Hyperoxia induced association of Nox4 with p22_phox_ protein (yellow in merged image) shown is a representative confocal immunofluorescence image from three independent experiments. (B) HPAECs in 100-mm dishes grown to ∼90% confluence were exposed to normoxia or hyperoxia for 3 or 24 h. Nuclear and cytoplasmic fractions were prepared using Active Motif according to manufacturer's protocol as described in “Materials and Methods.” Lysates from nuclear and cytoplasmic fractions (20 μg protein) were subjected to SDS-PAGE and Western blot analysis with antibodies against Nox4 (Santa Cruz, CA), p22_phox_, Lamin B1, lactate dehydrogenase (LDH), and calreticulin. Representative blots from three independent experiments are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at
).
FIG. 4.
Nox4 siRNA attenuates hyperoxia induced expression of Nox4 in nucleus. HPAECs grown on 35-mm dishes (A and C) or glass coverslips (B) to ∼50% confluence were transfected with scrambled siRNA (Sc) or Nox4 siRNA (50 n_M_) for 48 h. (A) Total RNA was extracted and Nox4 mRNA expression was quantified and normalized to 18S rRNA by real-time RT-PCR. Values are average of three independent determinations. (B) Sc or Nox4 siRNA transfected cells were exposed to normoxia or hyperoxia (3 h), washed, fixed, permeabilized, and probed with anti-Nox4 antibody (from Dr. Lambeth) or DAPI for nuclear staining and examined by immunofluorescence microscopy using a 60X oil objective. The Nox4 (red) and DAPI (blue) images show matched cell fields for each condition. A representative image from several independent experiments is shown. (C) Isolated nuclear or cytoplasmic fraction (20 μg protein) were subjected to SDS-PAGE and probed with anti-Nox4 (Santa Cruz, CA), or anti-β actin or -lamin B1 antibodies. Representative blots from three independent experiments are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at
).
FIG. 5.
Role of Nox4 in hyperoxia-induced ROS generation. (A and B) HPAECs grown to ∼50% confluence in 35-mm dishes were transfected with scrambled siRNA or Nox4 siRNA (50 n_M_) for 48 h as described in “Materials and Methods.” After 48 h, cells were pretreated with medium alone or medium containing PEG-conjugated SOD at 400 Units/mL for 1 h prior to addition of 10 μ_M_ DCFDA (A) or hydroethidine (B) for 30 min and washed once in basal medium. Cells in A or B were exposed to normoxia or hyperoxia (3 h) and at the end of the exposure, formation of total ROS (A) or superoxide (B) was measured by spectrofluorimetry. Values are mean ± S.E.M from three independent experiments done in triplicate and normalized to total protein. *significantly different from normoxia (p < 0.05); **significantly different from scrambled siRNA transfected cells exposed to hyperoxia (p < 0.01). (C) HPAECs grown to ∼70% confluence on 35-mm dishes were transfected with 1 μg of plasmid DNA/ml of Nox4 wild type. 72 h later, cells were loaded with 10 μM DCFDA for 30 min, washed once in basal medium and exposed to normoxia or hyperoxia (3 h). Total ROS production was visualized under immunofluorescence microscopy and quantified as described in “Materials and Methods.” Values are mean ± S.E.M of three independent experiments. *significantly different from normoxia (p < 0.05); **significantly different from vector control transfected cells under normoxia (p < 0.01).
FIG. 6.
Role of ]Nuclear Nox4 in hyperoxia-induced ROS generation. (A) HPAECs grown to ∼50% confluence in 100-mm dishes were transfected with scrambled siRNA or Nox4 siRNA (50nM) or p22_phox_ siRNA (50 n_M_) for 48 h. Nuclear fractions were prepared as described in “Materials and Methods” and exposed to normoxia or hyperoxia for 3 h. Accumulation of H2O2 was measured using an Amplex Red assay. Values are mean ± S.E.M from three independent experiments and expressed in % control. *significantly different from scrambled siRNA normoxic nuclear fraction (p < 0.05), **, significantly different from scrambled siRNA normoxic nuclear fraction (p < 0.01). (B and C) HPAECs grown to ∼50% confluence in glass-bottom dishes were transfected with scrambled siRNA or Nox4 siRNA (50 n_M_) for 48 h. Cells were loaded with10 μ_M_ hydroethidine for 30 min and washed once with basal medium, and further exposed to normoxia or hyperoxia for 3 h. Formation of superoxide and enhanced intercalation of oxidized hydroethidine with nuclear DNA was visualized under fluorescence microscopy and quantified. Values are mean ± S.E.M from three independent experiments in triplicates and expressed as % control. *significantly different from scrambled siRNA transfected cells under normoxia (p < 0.05), **significantly different from scrambled siRNA transfected cells under normoxia (p < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at
).
FIG. 7.
Silencing of Nox2 and Nox4 by siRNA upregulates expression of Nox4 and Nox2, respectively. HPAECs grown on 35-mm dishes to ∼50% confluence were transfected with 50 n_M_ scrambled siRNA or Nox2 siRNA or Nox4 siRNA or p22_phox_ siRNA for 48 h. (A and B) Total RNA was isolated and mRNA levels of Nox2 and Nox4 were quantified by real-time RT-PCR and normalized to 18S rRNA. In parallel experiments, total cell lysates (20 μg protein) from scrambled siRNA or Nox2 siRNA or Nox4 siRNA transfected cells were subjected to SDS-PAGE and Western blotted anti-Nox4 (Santa Cruz, CA) (A) or anti-Nox2 (B) antibodies. Values are average of three independent experiments. Shown are representative of blots from three separate experiments. (C) Total RNA was isolated and mRNA levels of p22_phox_ were determined by real-time RT-PCR and values are average of three independent experiments.
FIG. 8.
Effect of hyperoxia on lung injury in C57BL/6J and Nox2−/− mice. (A) C57BL/6J mice or Nox2−/− mice were exposed to normoxia (room air-21% oxygen) or hyperoxia (95% oxygen) for 24, 48, and 72 h. Mice were anesthetized by intraperitoneal injection of 30 μl of ketamine (100 mg/kg)/xylazine (10 mg/kg). BAL (bronchoalveolar lavage) fluid was aspirated from the lungs and stored on ice before measuring total protein. Each group had five mice and values are mean ± S.E.M of three independent experiments. (B) The levels of extravasated Evans blue dye (μg/ml/g body weight) in mouse lung homogenate of wild type and Nox2−/− mice are shown. Values are mean ± S.E.M of three independent experiments. (C) BAL fluids were centrifuged at 200 g for 10 min, and the pellet was immediately resuspended in physiological saline for total cell counts. Differential cell counts were carried out in the centrifuged cells after Wright– Giemsa staining. (D) shows the histological staining of the lung tissue from mice exposed to normoxia or hyperoxia. Lung tissues were stored in formalin for 24 h before processing to cut and mount the section for staining with H&E. Shown is a representative staining of the lung tissue from three independent determinations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at
).
FIG. 9.
Effect of hyperoxia on Nox4 mRNA expression and immunohistochemical staining of control and Nox2−/− mice. (A) Lung tissues were harvested from wild-type-C57BL/6J mice exposed to normoxia or hyperoxia (24 h). Total RNA was isolated, Nox2 and Nox4 mRNA were quantified by real-time RT-PCR and normalized to 18S rRNA. *significantly different from normoxic lung tissue (p < 0.05). (B) Nox2−/− mice were exposed to either normoxia or hyperoxia (95% O2 for 24 h). Total RNA was isolated and mRNA levels of Nox4 were quantified by real-time RT-PCR and normalized to 18S rRNA. (C) Lung tissues from wild-type and Nox2−/− mice were stained with Nox2 and Nox4 antibodies using standard immunohistochemistry (immuno-peroxidase) techniques. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at
).
FIG. 10.
N -acetylcysteine attenuates hyperoxia-induced ROS generation and wound healing. HPAECs grown on 35-mm dishes (A) or glass coverslips to ∼90% confluence were pretreated with vehicle or vehicle plus N_-acetylcysteine (NAC) 1 m_M for 60 min. (A) Cells were wounded by scratching across the monolayer with a 10 μl sterile pipette tip. The scratched monolayers were rinsed twice in serum-free medium and exposed to either normoxia (N) or hyperoxia (HO) for 16 h. The effect of NAC on hyperoxia-mediated cell migration was quantified by calculating the % of free area not occupied by cells adjacent to an area of the initial wound that was defined as closure of wounded area as described in “Materials and Methods.” Values are mean ± S.E.M of three independent experiments in triplicates. *significantly different from normoxia and without NAC (p < 0.05); **significantly different from cells exposed to hyperoxia without NAC (p < 0.01). (B) After NAC pretreatment for 1 h, cells were loaded with 10 μ_M_ DCFDA for 30 min, washed once in basal medium, and total ROS formation was quantified using fluorescence microscopy as described in “Materials and Methods.” Values are mean ± S.E.M of three independent experiments and ROS generation expressed as % of control. *significantly different from cells exposed to normoxia without NAC (p < 0.05); **significantly different from cells exposed to hyperoxia without NAC (p < 0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at
).
FIG. 11.
Nox4 and Nox2siRNA attenuates hyperoxia-mediated cell migration. (A and B) HPAECs grown on 35-mm dishes to ∼50% confluence were transfected with scrambled siRNA or Nox4 siRNA or Nox2 siRNA for 48 h. Cells were wounded as described in “Materials and Methods” and Fig. 10, and exposed to either normoxia or hyperoxia for 16 h. Values are mean ± S.E.M of three independent experiments in triplicates. *significantly different from scrambled siRNA transfected cells under normoxia (p < 0.05); *significantly different from scrambled siRNA transfected cells under normoxia (p < 0.01); ***significantly different from scrambled siRNA transfected cells under hyperoxia (p < 0.001).
FIG. 12.
Nox4 and Nox2 siRNA attenuates hyperoxia-mediated capillary tube formation. (A and B) HPAECs grown in 100-mm dishes to ∼50% confluence were transfected with scrambled siRNA or Nox4 siRNA or Nox2 siRNA for 48 h. After 48 h, cells were trypsinized and seeded onto matrigel-coated 35-mm dishes, as described in “Materials and Methods.” After 24 h of seeding, cells were either exposed to normoxia or hyperoxia (24 h) and formation of capillary tubes were visualized under phase-contrast microscope and quantified by counting different areas. Values are mean ± S.E.M of three independent experiments. *significantly different from scrambled siRNA transfected cells under normoxia (p < 0.05); **significantly different from scrambled siRNA transfected cells under normoxia (p < 0.01); ***significantly different from scrambled siRNA transfected cells under hyperoxia (p < 0.00).
References
- Babior BM. The leukocyte NADPH oxidase. Isr Med Assoc J. 2002;11:1023–1024. - PubMed
- Babior BM. NADPH oxidase: An update. Blood. 1999;93:1464–1476. - PubMed
- Bokoch GM. Knaus UG. NADPH oxidases: Not just for leukocytes anymore. Trends Biochem Sci. 2003;28:502–508. - PubMed
- Brueckl C. Kaestle S. Kerem A. Habazettl H. Krombach F. Kuppe H. Kuebler WM. Hyperoxia-induced reactive oxygen species formation in pulmonary capillary endothelial cells in situ. Am J Respir Cell Mol Biol. 2006;34:453–463. - PubMed
- Cai H. Hydrogen peroxide regulation of endothelial function: Origins, mechanisms, and consequences. Cardiosvasc Res. 2005;68:26–36. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous