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. 2015 Jul 15;286(2):92-101.
doi: 10.1016/j.taap.2015.03.023. Epub 2015 Mar 29.

Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

Shaojie Zhang  1 , Ananddeep Patel  1 , Chun Chu  1 , Weiwu Jiang  1 , Lihua Wang  1 , Stephen E Welty  1 , Bhagavatula Moorthy  1 , Binoy Shivanna  2
Affiliations

Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

Shaojie Zhang et al. Toxicol Appl Pharmacol. .

Abstract

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells.

Keywords: Antioxidant enzymes; Aryl hydrocarbon Receptor; Fetal human pulmonary microvascular endothelial cells; Hyperoxic injury; Oxidant stress; RelB.

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Figures

Figure 1
Figure 1. Hyperoxia functionally activates AhR in HPMEC
HPMEC were exposed to air or hyperoxia for up to 48 h. The cell lysates were separated into cytoplasmic and nuclear fractions at the indicated time points, and western blotting was performed using anti-AhR, anti-NQO1, anti-β-actin, or anti-lamin B antibodies. (A) Representative western blot showing nuclear AhR apoprotein expression. (B) Densitometric analysis of the nuclear AhR apoprotein and lamin B. RNA, isolated at 6, 12, 24 and 48 h of exposure, was subjected to real-time RT-PCR analysis of CYP1A1 (C) and NQO1 (D) mRNA. (E) Representative western blot showing cytoplasmic NQO1 protein expression. (F) Densitometric analysis of the cytoplasmic NQO1 protein and β- actin. Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). Two-way ANOVA showed an effect of hyperoxia and time of exposure and an interaction between them for NQO1 expression, and an effect of hyperoxia without any interaction for CYP1A1 mRNA expression in this figure. *, p < 0.05 vs. air exposed cells.
Figure 2
Figure 2. Validation of siRNA mediated knockdown of AhR in HPMEC
HPMEC were transfected with either 50 nM control (SiC) or AhR (SiAhR) siRNA. Twenty-four hours after transfection, cells were exposed to air for up to 48 h. RNA was extracted at the indicated time points for real-time RT-PCR analyses of AhR mRNA (A) expression. The whole-cell protein extract was used for western blot analysis with the anti-AhR or anti-β-actin antibodies (B). AhR band intensities were quantified and normalized to β-actin (C). Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). *, p < 0.05 vs. control siRNA.
Figure 3
Figure 3. AhR deficiency potentiates hyperoxia-induced cytotoxicity in HPMEC
Control (SiC) or AhR (SiAhR) siRNA transfected HPMEC were exposed to air or hyperoxia for up to 48 h, following which: (A) cell viability was assessed by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide) reduction activities; (B) cell proliferation was determined based on the measurement of cellular DNA content via fluorescent dye binding using the CyQUANT NF cell proliferation assay; (C) cell necrosis and apoptosis was determined by annexin V and propidium iodide staining of the cells as measured by flow cytometry. Cell apoptosis was also determined by western blotting with anti-cleaved poly (ADP-ribose) polymerase (CPARP) and anti- β-actin antibodies (D), and normalizing CPARP to β-actin band intensities (E). Values are presented as means ± SEM (n=4). Data are representative of at least three independent experiments. Two-way ANOVA showed an effect of hyperoxia and AhR gene and an interaction between them for all the dependent variables, except for CPARP in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p < 0.05. Significant differences between hyperoxia-exposed AhR-sufficient and –deficient cells are indicated by †, p < 0.05.
Figure 4
Figure 4. AhR deficiency potentiates hyperoxia-induced ROS generation in HPMEC
Control (SiC) or AhR (SiAhR) siRNA transfected HPMEC were exposed to air or hyperoxia for 24 h, following which the oxidation of the fluorescent dye, CM-H2DCF-DA, was measured by flow cytometry. The graph represents the mean CM-H2DCF fluorescence intensity for at least three independent experiments. Values are presented as means ± SEM (n=4). Two-way ANOVA showed an effect of hyperoxia and AhR gene and an interaction between them for the dependent variable in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p < 0.05. Significant differences between hyperoxia-exposed AhR-sufficient and –deficient cells are indicated by †, p < 0.05.
Figure 5
Figure 5. AhR deficiency potentiates hyperoxia-induced MIP-1α and MIP-1β concentrations in HPMEC
Control (SiC) or AhR (SiAhR) siRNA transfected HPMEC were exposed to air or hyperoxia for 48 h, following which the cell free supernatants were analyzed for cytokines/chemokines by multiplex luminex assay. The graph represents the mean MIP-1α (A) and MIP-1β (B) concentrations for at least three independent experiments. Values are presented as means ± SEM (n=4). Two-way ANOVA showed an effect of hyperoxia and AhR gene and an interaction between them for all the dependent variables in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p < 0.05. Significant differences between hyperoxia-exposed AhR-sufficient and –deficient cells are indicated by †, p < 0.05. Significant differences between air-exposed AhR-sufficient and –deficient cells are indicated by π, p < 0.05.
Figure 6
Figure 6. AhR deficiency decreases hyperoxia-induced CYP1A1 and NQO1 mRNA expression
Control (SiC) or AhR (SiAhR) siRNA transfected HPMEC were exposed to air or hyperoxia for up to 48 h, following which RNA was extracted for real-time RT-PCR analyses of CYP1A1 (A), NQO1 (B), HO1 (C), and SOD1 (D) mRNA expression. Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). Two-way ANOVA showed an effect of hyperoxia and AhR gene and an interaction between them for the dependent variables, CYP1A1 and NQO1, in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p < 0.05. Significant differences between hyperoxia- exposed AhR-sufficient and –deficient cells are indicated by †, p < 0.05.
Figure 7
Figure 7. AhR deficiency decreases hyperoxia-induced NQO1 and SOD1 protein expression
Control (SiC) or AhR (SiAhR) siRNA transfected HPMEC were exposed to air or hyperoxia for up to 48 h, following which cytoplasmic protein was extracted and western blotting was performed using anti-NQO1, HO1, SOD1, or β-actin antibodies. Representative western blots showing cytoplasmic NQO1 (A), HO1 (C), and SOD1 (E) protein expression. Densitometric analyses wherein NQO1 (B), HO1 (D), and SOD1 (F) band intensities were quantified and normalized to β-actin. Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). Two-way ANOVA showed an effect of hyperoxia and AhR gene and an interaction between them for the dependent variables, NQO1 and SOD1, in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p < 0.05. Significant differences between hyperoxia-exposed AhR-sufficient and –deficient cells are indicated by †, p < 0.05.
Figure 8
Figure 8. AhR deficiency decreases hyperoxia-induced nuclear RelB protein expression
Control (SiC) or AhR (SiAhR) siRNA transfected HPMEC were exposed to air or hyperoxia for up to 6 h, following which nuclear protein was extracted and western blotting was performed using anti-AhR, RelB, or lamin B antibodies. (A) Representative western blot showing nuclear RelB protein expression. (B) Densitometric analyses wherein RelB band intensities were quantified and normalized to lamin B. Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). Two-way ANOVA showed an effect of hyperoxia and AhR gene and an interaction between them for the dependent variable in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p < 0.05. Significant differences between hyperoxia-exposed AhR-sufficient and –deficient cells are indicated by †, p < 0.05.
Figure 9
Figure 9. Proposed mechanism(s) by which AhR deficiency potentiates hyperoxic lung injury
Deficient AhR signaling increases hyperoxia-induced ROS generation and inflammation, by decreasing the expression of antioxidant enzymes (AOE) and by modulating NF-κB activation via RelB. CYP1A1 - cytochrome P450 1A1; NQO1 – NADP(H) quinone oxidoreductase 1; SOD1 – superoxide dismutase 1.
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