ToxSci Advance Access published September 8, 2015

NcoA2-dependent inhibition of HIF-1α activation is regulated via AhR

Chi-Hao Tsai*, Po-Lin Liao*,†, Yu-Wen Cheng†, Cheng-Hui Lin†, Shih-Hsuan Huang†, Ching-Hao Li‡,§,¶, Jaw-Jao Kang*,¶

Institute of Toxicology, College of Medicine, National Taiwan University, Taipei,

Taiwan †

School of Pharmacy, College of Pharmacy, Taipei Medicine University, Taipei,

Taiwan ‡

Department of Physiology, School of Medicine, College of Medicine, Taipei Medical

University, Taipei, Taiwan §

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical

University, Taipei, Taiwan ¶

Corresponding author

Author email addresses: Chi-Hao Tsai: [email protected]; Po-Lin Liao: [email protected];

Yu-Wen Cheng: [email protected]; Cheng-Hui Lin: [email protected]; Shih-Hsuan Huang: [email protected]. 1

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*

Correspondence: Ching-Hao Li, PhD Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

E-mail: [email protected]

Jaw-Jao Kang, PhD Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan Tel: 886-2-23123456; Ext. 88603; Fax: 886-2-27374622 E-mail: [email protected]

Running title: NcoA2 regulate AhR and HIF-1α cross-talking

Conflict of Interest: The authors declare that they have no conflict of interest.

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Tel: 886-2-273761661; Ext. 6123; Fax: 886-2-27374622

ABSTRACT High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR ligand benzo[a]pyrene (B[a]P) and could alter cellular homeostasis and disease condition. Increasing evidence indicates that AhR might compete with aryl hydrocarbon receptor

factor-1α (HIF-1α) for transactivation, which could alter several physiological variables. Nuclear receptor coactivator 2 (NcoA2) is a transcription coactivator that regulates transcription factor activation and inhibition of basic helix-loop-helix Per (Period)-ARNT-SIM (single-minded) (bHLH-PAS) family proteins, such as HIF-1α, ARNT, and AhR, through protein-protein interactions. In the present study, we demonstrated that both hypoxia and hypoxia-mimic conditions decreased NcoA2 protein expression in HEK293T cells. Hypoxia-responsive element (HRE) and xenobiotic-responsive element (XRE) transactivation also were downregulated with NcoA2 knockdown under hypoxic conditions. In addition, B[a]P significantly decreased NcoA2 protein expression be accompanied with AhR degradation. We next evaluated

whether the absence of AhR could affect NcoA2 protein function under hypoxia-mimetic conditions. NcoA2 and HIF-1α nuclear localization decreased in both B[a]P-pretreated and AhR-knockdown HepG2 cells under hypoxia-mimic conditions. 3

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nuclear translocator (ARNT) for complex formation with hypoxia-inducible

Interestingly, NcoA2 overexpression downregulated HRE transactivation by competing

with HIF-1α and AhR to form protein complexes with ARNT. Both NcoA2 knockdown and overexpression inhibited endothelial cell tube formation in vitro. We also demonstrated using the in vivo plug assay that NcoA2-regulated vascularization decreased in mice. Taken together, these results revealed a biphasic role of NcoA2

the crosstalk between AhR and hypoxia that affects disease development and progression.

Key words: Aryl hydrocarbon receptor (AhR), Benzo[a]pyrene (B[a]P), Nuclear receptor coactivator 2 (NcoA2), Hypoxia-inducible factor-1α (HIF-1α), Hypoxia-responsive element (HRE)

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between AhR and hypoxic conditions, thus providing a novel mechanism underlying

INTRODUCTION Aryl hydrocarbon receptor (AhR) is a well-known mediator of the toxic effects of xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclicaromatic hydrocarbons (PAHs), benzene, and polychlorinated biphenyls (PCBs). Ligand-activated AhR can associate with aryl hydrocarbon receptor nuclear

and phase II xenobiotic metabolizing enzymes (e.g., cytochrome P450 (CYP1A)1, CYP1A2, CYP1B1, and glutathione S-transferases [GSTs]) (Denison et al., 2011; Nebert et al., 2004; Schmidt and Bradfield, 1996). AhR belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) superfamily of transcription factors (McIntosh et al., 2010). These proteins have been implicated in several physiological adaptation processes including mammalian development, cell transformation, stress responses, and tumorigenesis (Opitz et al., 2011). In addition to the formation of bHLH/PAS complexes, similar to the AhR/ARNT heterodimers, ARNT heterodimerizes with hypoxia-inducible factor-1α (HIF-1α), which is a hypoxia-stabilized protein, and this process is a prerequisite for transcriptional activation of hypoxia response element (HRE) sequences in hypoxia-inducible target genes (Majmundar et al., 2010; Semenza, 2003; Vorrink et al., 2014). It has been demonstrated that the AhR signaling pathway can be perturbed upon exposure to 5

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translocator (ARNT) to form AhR/ARNT heterodimers and transactivate both phase I

hypoxic conditions, which leads to inhibition of metabolizing enzymes (Vorrink et al., 2014). Because ARNT can form heterodimers with both AhR and HIF-1α, it seems likely that a crosstalk exists, which affects the regulation between AhR and hypoxia. Moreover, the novel function of AhR on normophysiology of hematopoiesis involving the HIF-1α signaling pathway has been discussed recently (Casado et al., 2010;

organs such as the liver and kidney. Owing to the direct influence of oxygen tension on HIF regulation, the liver has a decreased zonal distribution of oxygen tensions from the periportal fields toward the center of the liver lobules, in contrast to the kidney, which is very sensitive to hypoperfusion injury and hypoxic conditions (Maxwell, 2003; Wiesener et al., 2003). Although numerous studies have indicated that ARNT could be a mediator between AhR- and HIF-1α- regulation (Fleming et al., 2009; Nie et al., 2001), the underlying mechanism of crosstalk between AhR and hypoxia regulation is still unclear. Nuclear receptor coactivator 2 (NcoA2), also known as glucocorticoid receptor-interacting protein 1 (GRIP1), steroid receptor coactivator-2 (SRC-2), or transcriptional mediators/intermediary factor 2 (TIF2) (Duteil et al., 2010; Xu et al., 2009), is a member of the p160 family of transcriptional coactivators and has been demonstrated to regulate a number of physiological processes, especially metabolic 6

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Lindsey and Papoutsakis, 2012). Upon exposure to PAHs, this crosstalk may affect

homeostasis (Duteil et al., 2010). In addition to NcoA2, other members of p160 family of transcriptional coactivators, such as SRC1 and SRC3, also have been shown to regulate energy balance. These proteins could play critical roles in forming the bridge for the interactions between different transcription factors to affect gene transcription (Beischlag et al., 2002; Chen et al., 1997; Chopra et al., 2008).

signaling for stress adaption (Miyamoto et al., 2008). Recently, it was reported that the molecular mechanisms underlying the crosstalk between AhR and hypoxic conditions are important for physiological homeostasis upon exposure to both PAHs and hypoxia. PAHs can be ingested via the consumption of contaminated food and water or by inhalation of tobacco smoke and the exhausts from factories and motor vehicles (Li et al., 2010; Mastrangelo et al., 1996; Phillips, 1999). The liver is the principal site of bioactivation of environmental contaminants (Hussain et al., 2014). A few studies have shown that AhR and HIF-1α compete with ARNT; however, the critical mechanism of this competition remains unknown. In this study, we investigated the function of NcoA2 in the AhR and HIF-1α signaling pathways, especially the crosstalk, which affects HRE transactivation depending on the level of AhR.

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Homeostasis in different tissues could be regulated by negative feedback

MATERIALS AND METHODS Chemicals. PAHs, including B[a]P, and CoCl2 were all analytical grade and, unless otherwise indicated, were purchased from Sigma (St. Louis, MO). Cell culture and treatment.

(Manassas, VA). Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % heat- inactivated fetal bovine serum, 2 mM L-glutamine, 200 U/ mL penicillin, and 200 µg/mL streptomycin and incubated in a humidified atmosphere of 5 % CO2 at 37 °C. Before testing, cells were plated onto 48-well plates for the viability assay, 12-well plates for the luciferase assay, or 6-well plates for western blotting and mRNA analysis at a density of 2 × 105 cells/mL for overnight incubation. Cells then were pretreated with vehicle or B[a]P (>12 h) and then subjected to hypoxia or hypoxia-mimetic treatment. The cells were exposed to B[a]P for the entire duration of these experiments. For the tube formation assay, primary vascular endothelial cells (HUVECs) were isolated from human umbilical cords as described previously (Li et al., 2010). Confluent primary cultures obtained within 3–6 passages were used in the experiments. 8

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HepG2 and HEK293T cells were obtained from American Type Culture Collection

Lentivirus-mediated small hairpin RNA for AhR and NcoA2 gene silencing. The VSV-G expression vector pMDG, gag-pol expression vector pCMVD8.91, and pLKO.1 encoding the AhR small hairpin RNA (shRNA) (TRCN0000021254) were co-transfected into 293T cells by calcium phosphate precipitation. All plasmids were obtained from the National RNAi core facility (Academic Sinica, Taiwan). The

through a 0.45-µm syringe filter. The viral supernatants were added to the HUVEC culture in the presence of 10 µg/mL polybrene (hexadimethrine bromide; Sigma). Two days after infection, lentivirus-infected HUVECs were used for subsequent studies. Hypoxia treatment. A majority of healthy tissues are exposed to 2–9 % O2, while hypoxia is defined as less than 2 % O2 exposure (Mucaj et al., 2012). In this study, hypoxic conditions were achieved using the Anaerobic System ProOx model 110 (BioSpherix, Lacona, NY), with the parameter setting at less than 1 % O2. Cell viability assay. Cell viability was determined using the 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Li et al., 2010). Western blots. Whole-cell lysate, protein concentration determination, SDS-PAGE, and immunoblot 9

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virus-containing supernatants were collected after 24–48 h transfection and refined

were performed as described previously (Lin et al., 2013). For immunodetection, the polyvinylidene fluoride (PVDF) membrane was blocked in non-fat milk and incubated in bovine serum albumin (BSA) with antibodies specific to HIF-1α (BD Biosciences, San Jose, CA ), AhR, ARNT (Santa Cruz Biotechnology, Santa Cruz, CA ), NcoA2 (Genetex, Irvine, CA), LaminA/C, α-tubulin (Epitomic, Burlingame,

was performed according to the manufacturer's protocol (Millipore, Billerica, MA ). Confocal Immunofluorescence assay HEK293T cells were fixed with 4% paraformaldehyde with PBS for 15 min, and followed by treating 0.1% Triton –X100 for 5 min. 2% BSA was worked as blocking buffer and incubated with cells for 30 min. Immunostaining for ARNT (Santa Cruz Biotechnology, Santa Cruz, CA ) antibody and nuclei with Hoechst 33258 (Sigma, St. Louis, MO). Then the fluorescent were observed by Leica TCS SP5 confocal microscopy system. Immunoprecipitation analysis. Whole cell lysates were pre-cleared by short incubation with protein A magnetic beads (Millipore). Then, the lysates (1 mg) were subjected to immunoprecipitation by the addition of 1 µg/mL anti-ARNT antibodies , for overnight incubation with rotation at 4 °C. This was followed by shaking with 50 µL protein A magnetic beads for 10

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CA), and β-actin (Sigma, St. Louis, MO). Enhanced chemiluminescence detection

another 2 h. The captured immunocomplexes were precipitated and washed three times with radioimmunoprecipitation assay (RIPA) buffer prior to the addition of 100 µL of 2 × SDS sample buffer and heating at 95 °C for 6 min. Samples were electrophoresed on 7.5 % SDS–polyacrylamide gels and analyzed as described previously.

RNA samples were prepared using the TriPure reagent according to the manufacturer protocol (Roche, Rotkreuz, Switzerland). Vascular endothelial growth factor (VEGF) levels were analyzed by real time RT-PCR and compared against the housekeeping gene, β-actin, as an internal control. Briefly, first-strand complementary cDNA was synthesized from 6 µg of total RNA at 37 °C for 60 min using the MMLV high-performance reverse transcriptase (Epicentre, Madison, WI). The cDNA then was amplified by a DNA thermal cycler (Applied Biosystems, Waltham, MA) using the following program: denaturation for 5 min at 95 °C, followed by 30 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 55 °C, and extension for 40 s at 72 °C with a final extension for 10 min at 72 °C. PCR products were separated by 1.5 % agarose gel electrophoresis and visualized by ethidium bromide (EtBr) staining. Luciferase reporter assay. Cells were co-transfected using the Lipofectamine 2000 reagent (Invitrogen, Waltham, 11

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Real-time transcription-polymerase chain reaction (real time RT-PCR).

MA) with 2 µg of the HRE luciferase reporter construct (pGL2-HRE) and 0.5 µg of pRK5-LacZ. Both constructs were kindly provided by Dr. Tzeng Shu-Ling (Chung-Shan Medical University, Tai-chung, Taiwan). After 24 h transfection, cells were exposed to hypoxic conditions for 6–9 h, and then, cell extracts were harvested. Luciferase activity was measured using the reporter assay system (Promega, Madison,

Tube formation assay. The neovasculogenesis assay was performed in Matrigel-coated 48-well culture plates as described (Li et al., 2010). The Matrigel (130 µL/well, 8 mg/mL; BD Biosciences, Bedford, MA) was polymerized and allowed to gel for 60 min at 37 °C. NcoA2 overexpressed and silenced HUVECs were seeded onto coated plates at a density of 2 × 104 cells/well in M199 containing 5 % fetal bovine serum (FBS) and VEGF (50 ng/mL) as positive control at 37 °C under normoxia and hypoxia. Besides, the B[a]P treated group also been determined. Neovasculogenesis was optimal after 4–8 h. Based on this findings, images were taken at 6 h at 40X magnification using a digital output camera (Nikon, Tokyo, Japan) attached to an inverted phase-contrast microscope. In vivo Matrigel assay. In vivo angiogenesis was assayed using the Matrigel plug assay .500 µl of liquid 12

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WI).

growth factor reduced Matrigel (BD Biosciences) was injected subcutaneously into the flanks of male ICR mice(4 mouse per group). To study the effect of NcoA2 biphasic effect on HIF-1α -dependent vasculogenesis, 106 293T transfected (ctrl, pcDNA3.1-HIF-1α, pcDNA3.1-NcoA2, and sh-NcoA2) cells were placed in Matrigel for plug assays. After 10 days, the mice were sacrificed and Matrigel plugs were

Matrigel plugs were homogenized in hypotonic lysis buffer (250 µl of 0.1% Brij-35 per plug) and centrifuged for 5 min at 5,000 × g. The supernatant was incubated at 0.5 ml of Drabkin's solution for 15 min at room temperature, and the absorbance was measured at 540 nm with Drabkin's solution as a blank. Because absorbance is proportional to the total hemoglobin content, the relative hemoglobin content was calculated versus the negative and positive controls. Statistical analysis. All data are expressed as the mean ± standard deviation (SD) from at least three independent experiments (n ≥ 3). Statistically significant differences between the control and each experimental test condition were analyzed using the Student’s t test. Statistically significant differences among groups were determined using one-way analysis of variance (ANOVA). P values of < 0.05 were considered statistically significant. 13

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removed and hemoglobin content was determined using the Drabkin's reagent kit. The

RESULTS The protein level of NcoA2 was down-regulated in hypoxic conditions. CoCl2 is a hypoxia-mimetic agent, which could block HIF-1α degradation. At high treatment concentrations, CoCl2 causes cytotoxicity (Guo et al., 2009). In this study,

HEK293T cells after 6 h treatment (Supplementary Figure 1A), but CoCl2 treatment caused an increase in HIF-1α protein accumulation, either in a concentration- or time-dependent (which peaked in 3–6 h and returned to basal level at 24 h) manner. Conversely, the protein level of NcoA2 was decreased in CoCl2-treated HEK293T cells (Figure 1A, 1B), as well as hypoxia-conditioned HEK293T cells (Supplementary Figure 2A). However, there were no obvious changes in NcoA2 protein levels in HIF-1α overexpressing HEK293T cells (Supplementary Figure 2B), suggesting that the down-regulation of NcoA2 was unrelated to the transcriptional activity of HIF-1α. Moreover, we also found that the NcoA2 protein partition was not significantly changed in the cytosolic fraction of CoCl2-treated HEK293T cells, whereas NcoA2 was greatly decreased in the nuclear fraction. We also found the translocation of HIF-1α and ARNT to the nucleus after CoCl2 treatment in a concentration-dependent manner (Figure 1C). 14

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reduction in cell viability was not found in CoCl2-treated (0, 75, 150, and 300 µM)

NcoA2 knockdown decreased hypoxia- and CoCl2-induced HRE transactivation. The requirement of NcoA2 during pregnancy and embryonic development has been demonstrated, and both conditions were strongly correlated with hypoxia. The deletion of NcoA2 might have cause hypofertility or fetal death in mice (Gehin et al., 2002). In this study, we have screened five NcoA2 shRNA vector (Supplementary

5A). By transient transfection with NcoA2 shRNA (24–72 h), the expression of NcoA2 was silenced in cultured HEK293T cells (Figure 2A), and no obvious changes in cell viability were observed among the control (wild-type), vehicle (PolyJetTM), vector (pLKO.1), and sh-NcoA2 (NcoA2 shRNA) (Supplementary Figure 1B). Interestingly, knockdown of NcoA2 compared to the vector control showed dramatic changes in cell morphology to a cobblestone-like feature (Figure 2B). The transactivation activity of HRE was assayed using a luciferase reporter. In normoxia, HRE activity was inhibited in sh-NcoA2 transfected cells as compared to that in wt-HEK293T cells. Also, the induced HRE activities in response to CoCl2 treatment (150 µM) or hypoxia (0.5% O2) for 6 h (Figure 2C, 2D) were diminished in sh-NcoA2 transfected cells. The induction of vascular endothelial growth factor (VEGF) also was diminished in sh-NcoA2 transfected cells in response to CoCl2 treatment or hypoxic conditions (Figure 2E). These results suggest that the NcoA2 might function 15

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Table 1) and fifth clone was used for following experiments (Supplementary Figure

as a co-activator of HRE transactivation and that NocA2 down-regulation desensitized hypoxia-mediated gene transactivation. The protein level of NcoA2 was down-regulated while AhR activation. Recent reports suggest that the signaling cross-talks between both HIF-1α and AhR require ARNT (also called HIF-1β) to assist transcriptional regulation. AhR-activating

2001). B[a]P, a ligand of AhR, treatment (0.3–10 µM) decreased AhR and NcoA2 protein levels in both HepG2 (Figure 3A) and AhR-overexpressing HEK293T but not in wt-HEK293T cells (Figure 3B). We also investigated B[a]P-induced XRE level of the AhR-dependent transcription between HepG2 and wt-HEK293T cells. The data have shown that B[a]P up-regulated nearly 2 folds change of XRE transactivation in HepG2 significantly. In contrast, there were no obvious effects in wt-HEK293T cells (Figure 3C). B[a]P also resulted CYP1A1 protien increased in HepG2 but not HEK293T. This difference is due to the low basal expression of AhR in wt-HEK293T cells (Figure 3D). B[a]P-induced AhR nuclear translocation triggers proteolytic degradation of AhR via the ubiquitin-proteasome pathway as reported previously (Ma and Baldwin, 2000). Interestingly, AhR-overexpressing HEK293T cells had no changes in NcoA2 protein level (Supplementary Figure 3A), but TCDD treatment resulted both AhR and NcoA2 protein decreased in HepG2 and AhR-overexpressing 16

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ligands might perturb the HIF-1α-dependent pathway (Fritz et al., 2008; Nie et al.,

HEK293T cells (Supplementary Figure 3B).These results suggested that the down-regulation of NcoA2 was related to the ligand-activated AhR signaling. We also detected NcoA2 protein the cytosolic/nuclear partition in B[a]P-treated HepG2 cells. B[a]P dose we have used causing no cytotoxicity in HepG2 (Supplementary Figure 4). Western blots showed that NcoA2 was greatly decreased in

The translocation of AhR and ARNT to the nuclear fraction after B[a]P treatment also were demonstrated (Figure 4A). On the other hand, the transactivation of B[a]P-induced XRE expression also was down-regulated by NcoA2 knockdown in HepG2 cells (Figure 4B). These data suggest that NcoA2 was involved in the AhR signaling pathway. The decrease of NcoA2 was correlated to the AhR activation and accompanied by ligand-activated AhR regulation. Both B[a]P pretreatment and AhR knockdown in HepG2 cells decreased CoCl2– induced HIF-1α and NcoA2 nuclear localization. To determine whether the absence of AhR would affect NcoA2 protein function under hypoxia-mimetic conditions, we performed immunoblotting of the nuclear fraction of HepG2 cells, which have a higher AhR protein level than that of HEK293T cells. HepG2 cells were pretreated with B[a]P (10 µM) for 24 h followed by CoCl2 (150 µM) administration (Figure 5A). B[a]P or CoCl2 only treatments used as positive controls 17

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the nuclear fraction, whereas NcoA2 in the cytosolic fraction remained unaffected.

increased both AhR and HIF-1α nuclear localization but decreased NcoA2 nuclear localization independently. The nuclear localization of NcoA2 was clearly down-regulated by co-treatment of B[a]P with CoCl2. We also found that co-treatment of B[a]P with CoCl2 suppressed AhR and HIF-1α nuclear translocation, which previously have been reported to interfere with each other’s signaling pathways

suppressed NcoA2 nuclear localization and had an additive effect upon exposure to hypoxia-mimetic conditions. To confirm further whether the level of AhR affected NcoA2 regulation under hypoxic conditions, HepG2 cells were transfected with shAhR (Figure 5C), which have been chosen from two clone and second clone were used (Supplementary Figure 5B). And followed by CoCl2 (150 µM) treatment for 6 h to mimic hypoxia (Figure 5B). Similar to the results that we observed in B[a]P-treated HepG2 cells, both shAhR and CoCl2 treatment decreased NcoA2 nuclear localization. The significantly decreased level of NcoA2 in the nuclear fraction of AhR-silenced HepG2 cells was similar to that observed in cells exposed to hypoxia-mimetic conditions. As shown in Figures 1 and 3, decreased levels of NcoA2 protein are related to both B[a]P treatment and hypoxia. Taken together, these results indicate that the AhR protein level regulated NcoA2 nuclear localization, which could affect hypoxia-induced HRE transactivation. This suggests that NcoA2 is a key regulatory 18

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(Vorrink et al., 2014). These results suggest that B[a]P-induced AhR activation

crosslink between AhR and hypoxia. Collectively, our data suggest that NcoA2 is a regulator of negative feedback to maintain homeostasis of HRE transactivation. NcoA2 regulated HRE transactivation by competing with HIF-1α and AhR to form a protein complex with ARNT ARNT, a member of the bHLH/PAS protein family, is an important transcription

Immunofluorescent staining identified the nuclear localization of ARNT before and after B[a]P or hypoxia stimulation (Figure 6A). Subsequently, the protein complexes were captured by specific anti-ARNT antibodies, and then HIF-1α , AhR, and NcoA2 were detected by immunoblotting. Immunoprecipitation assay have been demonstrated under both normoxia and hypoxia. Under normoxia condition, HIF-1α /ARNT protein complexes were not detected and slightly interaction between AhR/ARNT, NcoA2/ARNT were found. In contrast, compared with the control group (wt-HEK293T under hypoxia for 6 h) that had obvious HIF-1α /ARNT protein complexes, we found that some ARNT had been replaced by AhR and NcoA2 in the AhR-overexpressing and NcoA2-overexpressing groups, respectively. We also found decreased amounts of HIF-1α /ARNT protein complexes in the sh-NcoA2 group. The vectors we used in our study also been determined as empty vector control for this experiments accuracy (Figure 6B). In essence, the HRE performance was strongly 19

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regulator, which forms a heterodimer with AhR and HIF-1α (Wolff et al., 2013).

induced in the control group (wt-HEK293T under hypoxia for 6 h), but the induction was diminished in AhR-overexpressing, NcoA2-overexpressing, and sh-NcoA2 groups (Figure 6C); these results provide strong evidence to confirm those shown in Figure 6B. These results suggest that decreased levels of HIF-1α /ARNT protein complexes could down-regulate HRE transactivation due to higher AhR levels or

HIF-1α /ARNT complex formation. Biphasic effects of NcoA2 protein on angiogenesis in vitro and in vivo As mentioned above, NcoA2 played an important role on HRE transactivation. Next, we determined the functional effect of NcoA2 on angiogenesis. Vasculogenesis (tube formation) was assayed using wt-HUVEC, sh-NcoA2 HUVEC, and NcoA2-overexpressing HUVEC. The vector we used in the tube formation were unaffected by empty vector (Supplementary Figure 6). B[a]P has been indicated that resulted angiogenesis inhibition (Li et al., 2010). We also investigated if B[a]P treatment affects tube formation with different NcoA2 expression level under normoxia and hypoxia. Both images and quantified data showed that the formation of an endothelial capillary-like network under hypoxia was enhanced by VEGF recombinant protein treatment compared with the control group. However, hypoxia-induced vasculogenesis was partially impaired in sh-NcoA2 HUVEC, but 20

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abnormal NcoA2 expression, which increases NcoA2/ARNT association and inhibits

fully suppressed in NcoA2-overexpressing HUVEC. B[a]P treatment also impaired VEGF-enhanced tube formation under normoxia and hypoxia condition. Besides, compared with non B[a]P-treated group that NcoA2-overexpressing and sh-NcoA2 HUVEC resulted inhibition of tube formation, we found B[a]P-inhibited tube formation in NcoA2-overexpressing and sh-NcoA2 HUVEC under normoxia and

To determine further whether NcoA2 could modulate angiogenesis in vivo, the murine plug assay was performed. Matrigel was impregnated with wt-HEK293T, HIF-1α -overexpressing HEK293T(positive control), HIF-1α /NcoA2-overexpressing HEK293T, and HIF-1α -overexpressing/sh-NcoA2 HEK293T cells. Then, the mixed Matrigels were injected into the subcutaneous tissue of ICR mice (n = 4) for 7 days. Both the imaged plugs and hemoglobin quantification data showed an increase in vascular structures in the HIF-1α-overexpressing group compared with the control group. However, HIF-1α-induced angiogenesis was reduced significantly in the sh-NcoA2 group and even more reduced in the NcoA2-overexpressing group (Figure 7B). Collectively, these results indicate that NcoA2-regulated hypoxia-induced angiogenesis in a biphasic manner. At physiological expression levels, NcoA2 functioned as a co-activator of HIF-1α and down-regulated HIF-1α activity by NcoA2 degradation in the nucleus. 21

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hypoxia, additively (Figure 7A).

DISCUSSION The crosstalk between the AhR-mediated and HIF-1α-mediated signaling pathways has been implicated in various cell responses, especially in cardiovascular disease, which could be owing to the hypoxic micro-environment caused

previous study, we found that neovascular formation failed after B[a]P exposure in HUVECs (Li et al., 2010); however, the molecular mechanism of AhR-affected neovascularization is still elusive. On the other hand, the pathology affected organs with different oxygen tensions and sensitivities such as the liver (Israel and Orrego, 1984; Jungermann and Kietzmann, 2000; Wiesener et al., 2003) and kidney (Maxwell, 2003) when exposed to PAHs. Exposure to PAHs might perturb HIF-α regulation because of AhR activation (Vorrink and Domann, 2014; Wolff et al., 2013). In the present study, we report how B[a]P-activated AhR affected HRE transactivation accompanied with NcoA2-dependent regulation. Ligand-activated AhR were accompanied with NcoA2 protein reduction, which could have down-regulated the HRE transactivation. On the other hand, AhR knockdown in HepG2 cells also decreased the nuclear NcoA2 protein expression and further HRE transactivation. These results indicated that the effect of HRE transactivation through the 22

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cardiovascular disease in contrast to heathy conditions (Semenza, 2014). In our

NcoA2-dependent pathway was based on the basal level of AhR with ligand-activated degradtion. These correlations suggested that NcoA2 might play an important role.between HRE transactivation and endogenous AhR by B[a]P exposure. However, we also found biphasic effects of NcoA2 protein on HRE transactivation. This was supported further by the inhibitory effect on HIF-1α-ARNT interaction and

We studied the molecular mechanism of NcoA2-dependent effects on the AhR-ARNT-HIF-1α signaling node and found that NcoA2 protein expression was decreased in dose- and time-dependent manners under hypoxia-mimetic conditions, whereas NcoA2 protein expression was unaffected in HIF-1α overexpressing cells. NcoA2 is a member of the SRC family, which contains a bHLH-PAS domain. Some studies have indicated that phosphorylation of SRC family members could regulate gene transcriptional activity. NcoA2 phosphorylation by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) pathway at Ser-736 also enhanced p300/CREB-binding protein (CBP) activity and increased the interaction with bHLH-PAS transcription factors such as estrogen receptor (ER) (Frigo et al., 2006; Lopez et al., 2001) and androgen receptor (AR) (Gregory et al., 2004). Moreover, the phosphorylation of NcoA2 increased the activity of transcription factors and accompanied by rapid proteasome degradation (Borud et al., 2002; Hoang 23

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angiogenesis effects in HEK293T cells overexpressing NcoA2.

et al., 2004). On the other hand, arrest defect-1 protein (ARD1) enhanced the Von Hippel–Lindau tumor suppressor (pVHL)-HIF-1α interaction through acetylation, which caused HIF-1α degradation under hypoxic conditions (Chang et al., 2006; Jeong et al., 2002). Interestingly, it also has been reported that acetylation and methylation of SRC family members could disassociate SRC-associated transcription

might have a key role in feedback loop regulation of HRE transactivation. In contrast, our findings (Supplementary Figures 2B and Figures 6B) showed that HIF-1α overexpression did not affect the NcoA2 protein level, but NcoA2 overexpression could decrease the HIF-1α protein level under hypoxia. The previous study indicated that the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is a crucial regulator of VEGF activation and angiogenesis through HRE transactivation by HIF-1α-ARNT complex nuclear translocation. However, it has been shown that HIF-1α regulation by the PI3K/Akt pathway is non-specific in different cell types, such as HepG2 and HEK293T (Alvarez-Tejado et al., 2002). The activity of the PI3K/Akt is not sufficient for the activation of HIF nor is it essential for its induction by hypoxia (Epstein et al., 2001). However, HIF-1α overexpression did not alter the protein level of NcoA2 but enhanced neovasculation in vivo in the Matrigel plug assay (Figure 7B). The previous 24

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factors accelerate SRC degradation (Naeem et al., 2007). This suggests that NcoA2

study also indicated that HIF-1α overexpression could enhance HRE transactivation (Brahimi-Horn and Pouyssegur, 2009). This suggested that the regulation of the NcoA2 feedback loop could not directly be activated by the HIF-1α protein level. It has been reported that ARNT is related to bHLH-PAS transcription factors, such as SRC1 and SRC3 (Wang et al., 2010). In our present data, we found that

and hypoxia treatments (Figure 2); however, the HRE transactivation was weakened, rather than enhanced, by NcoA2 overexpression, which is in contrast to that of the control under hypoxia (Figure 6). To clarify this confounding result,we demonstrated the HIF-1α-ARNT-NcoA2 interaction by immunoprecipitation. Moreover, the data further showed that the NcoA2-ARNT interaction was much greater than the HIF-1α-ARNT interaction when NcoA2 was overexpressed under hypoxia. In addition, NcoA2 overexpression also caused a decrease in HIF-1α protein level. Earlier studies have indicated that the acetylation of the HIF-1α oxygen-dependent degradation (ODD) domain could reduce HIF protein stability, which affects HIF-1α and heat-shock protein 90 (HSP90) binding (Jeong et al., 2002; Wei and Yu, 2007). On the other hand, SRC1 indeed triggered maximal HRE transactivation, but knockdown of SRC1 did not affect HRE downstream gene expression, though SRC3 knockdown did (Wang et al., 2010). Obviously, the regulation between these 25

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silencing of NcoA2 decreased the HRE transactivation under both hypoxia-mimetic

co-activators is complicated, and the co-activators exhibit different specificities for different HRE target genes and can behave differently in the transcriptional regulation of different target genes mediated by the same transcription factor. Together with previous studies, we revealed that NcoA2 could affect HIF-ARNT complex formation and trigger HRE transactivation, but we think that the affinity of NcoA2-ARNT is

Interestingly, we found AhR protein level was up-regulated in nuclear fraction even after treated with B[a]P for 24h (Figure 6A). As the past studies have indicated, the regulation of AhR is ligand-activated, and degraded by the proteasome (Lees et al., 2003; Pollenz and Dougherty, 2005). But most results were investegated in whole cell lysate. In our study, we found B[a]P decreased AhR protein level exactly in whole cell lyaste extraction. The reasonable explanation might be B[a]P-induced AhR nuclear translocation. On the otherhand, half-life of AhR play a significant role in regulation of AhR level. Cytosolic AhR concentrations are modulated by AhR ligands exposure, but not subsequent AhR-ligand nuclear translocation in Hepa 1 cells (Swanson et al., 1993). The competitive inhibition on AhR and HIF-1α signaling was because ARNT is a bridge that crosslinks these two signaling pathways. In our study, we firstly found NcoA2-dependent inhibition on HRE transactivation because the AhR protein level could regulate NcoA2 function. Pretreatment with B[a]P in HepG2 cells, which have 26

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higher than HIF-1α-ARNT and also is affected by AhR signaling.

a high endogenous AhR protein level and be accompany with proteasome degradation upon B[a]P-treatment, decreased NcoA2 protein in the nucleus (Figures 5 and 6). On the other hand, it has been reported that ARNT overexpression in the ZR-75 breast cancer cell line could not reverse hypoxia–induced inhibition of AhR downstream genes such as ethoxyresorufin-O-deethylase (EROD) and CYP1A1 (Khan et al.,

HIF-1α signaling. Moreover, our data provide a novel observation different from previous observations and suggest that NcoA2 has a key role for AhR signaling and HIF-1α signaling node. In the present studies, we found that NcoA2 function was not only to assist HRE transactivation but also to inhibit HRE over-activation. Both the tube formation assay in HUVECs and the Matrigel plug assay in vivo revealed that NcoA2 has a biphasic function (Figure 7). Moreover, this is the first finding of NcoA2-regulation on the AhR-ARNT-HIF-1α interaction, which might depend not only on ARNT but also NcoA2. In conclusion, although the regulatory crosstalk and interference between the xenobiotic- and hypoxia-sensing pathways at the AhR-ARNT-HIF1α signaling node is complicated, our studies highlight the role of NcoA2, elucidate the mechanism of the crosstalk, and discuss the physiological implications for exposure to AhR-inducing compounds in the context of hypoxia. 27

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2007). This suggests that ARNT might not be a limiting step between AhR and

Funding. This study was supported in part by a grant (NSC 100-2320-B-002 -087 -MY3) from the Ministry of Science and Technology, Taiwan.

Alvarez-Tejado, M., Alfranca, A., Aragones, J., Vara, A., Landazuri, M. O., and del Peso, L. (2002). Lack of evidence for the involvement of the phosphoinositide 3-kinase/Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. JBC. 277, 13508-13517. Beischlag, T. V., Wang, S., Rose, D. W., Torchia, J., Reisz-Porszasz, S., Muhammad, K., Nelson, W. E., Probst, M. R., Rosenfeld, M. G., and Hankinson, O. (2002). Recruitment of the NCoA/SRC-1/p160 family of transcriptional coactivators by the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator complex. Mol. Cell. Biol. 22, 4319-4333. Borud, B., Hoang, T., Bakke, M., Jacob, A. L., Lund, J., and Mellgren, G. (2002). The nuclear receptor coactivators p300/CBP/cointegrator-associated protein (p/CIP) and transcription intermediary factor 2 (TIF2) differentially regulate PKA-stimulated transcriptional activity of steroidogenic factor 1. Mol. Endocrinol. 16, 757-773. 28

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FIGURE LEGENDS Figure 1. The expression of NcoA2 protein decreased in CoCl2-treated HEK293T cells. Western blots showing the levels of NcoA2 protein expression following CoCl2 treatment in a (A) dose-dependent manner (75, 150, and 300, µM) and (B) time-dependent manner (1, 3, 6, 12, and 24 h). (C) HEK293T cells were treated with

nuclear localization levels were quantified. Lamin A/C and α-tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control. Figure 2. Both hypoxia- and CoCl2-induced HRE transactivation reduced in NcoA2-silenced HEK293T cells. (A) Western blots showing levels of NcoA2 protein expression following NcoA2-knockdown in HEK293T cells. The cells were pre-transfected with a firefly luciferase reporter gene driven by HREs (pGL2-HRE) for 24 h. (B) Morphology of HEK293T with NcoA2 knockdown. (C) Fold change of HRE reporter vector activity in HEK293T wild type and NcoA2-knockdown cells under CoCl2 treatment or nomoxia conditions for 6 h. The data show the HRE activity increased in the CoCl2 treatment group; the fold change of HRE reduced in HEK293T cells transfected with shNcoA2. (D) Fold change of HRE reporter vector activity in HEK293T wild-type and NcoA2-knockdown cells under hypoxia (0.5% O2) or 37

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75, 150, and 300, µM CoCl2 for 6 h. The nuclear extract was obtained, and the NcoA2

nomoxia conditions for 6 h. (E) Fold change of vegf mRNA expression in HEK293T wild-type and NcoA2-knockdown cells under hypoxia (0.5% O2) or nomoxia conditions quantified by qPCR. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control. #p < 0.05, ##p < 0.01, and ###p < 0.001 indicate a statistical difference from the hypoxia or hypoxia-mimic treated only groups.

AhR-overexpressed, but not wild-type, HEK293T cells. (A) NcoA2 and AhR protein levels reduced in B[a]P-treated HepG2 cells, which have a higher endogenous AhR basal level than HEK293T cells, in dose-dependent manner. HEK293T cells were transfected with pcDNA 3.1-AhR overexpression vector for 24 h and then treated with B[a]P. (B) The NcoA2 and AhR content of HEK293T cells, which have a low endogenous AhR basal level, did not change after B[a]P treatment. However, both NcoA2 and AhR protein levels reduced in B[a]P-treated AhR-overexpressing HEK293T cells in a dose-dependent manner. (C) Fold change of XRE reporter vector activity in HepG2 and HEK293T treated with B[a]P for 6 h. (D) Due to the AhR endogenous level, B[a]P-treated induced CYP1A1 in HepG2 but not HEK293T cells. *

p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control.

Figure 4. B[a]P decreased NcoA2 protein nuclear localization levels, and XRE transactivation reduced in NcoA2-silenced HepG2 cells. Representative western 38

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Figure 3. The AhR ligand B[a]P decreased NcoA2 protein levels in

blot of nuclear extracts and quantitation of the NcoA2 nuclear translocation levels. (A) HepG2 cells treated with the AhR ligand B[a]P (10 µM) for 24 h, which decreased NcoA2 nuclear translocation. (B) Fold change of XRE reporter vector activity in HepG2 wild-type and NcoA2-knockdown cells treated with B[a]P for 6 h. ***p < 0.001 indicate a statistical difference from the control. ##p < 0.01, and ###p < 0.001

Figure 5. Both HIF-1α α and NcoA2 protein nuclear localization induced by CoCl2 treatment reduced in B[a]P-pretreated HepG2 cells transfected with AhR shRNA. Representative western blot of the nuclear extract and quantitation of the NcoA2 nuclear translocation levels. (A) HepG2 cells pretreated with the AhR ligand B[a]P (10 µM) for 24 h and then incubated under normoxia or CoCl2 treatment for 6 h. CoCl2 inhibited HIF-1α and NcoA2 nuclear translocation with B[a]P treatment. (B) Wild-type and AhR-knockdown HepG2 cells were incubated under normoxia or CoCl2 treatment for 6 h. AhR shRNA inhibited NcoA2 nuclear translocation with CoCl2 treatment. Western blots were normalized with the loading control, and data are presented as mean ± S.E. as compared with the control group for three independent experiments *p < 0.05 indicate a statistical difference from the control. #p < 0.05 as compared with B[a]P-untreated HepG2 cells under CoCl2 treatment. α-tubulin and lamin A/C were used as the internal controls for the cytosolic and nuclear fractions, 39

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indicate a statistical difference from the B[a]P only treatment group.

respectively. (C) The AhR shRNA was transfected to knockdown AhR expression in HepG2 cells. Figure 6. NcoA2 protein acts as a regulatory factor in HRE transactivation through changes in protein-protein interactions with ARNT, AhR, and HIF-1α.. (A) Immunofluorescence microscopy of ARNT (green) in HEK293T cells following

visualized with hoechst 33258 (blue). (B) HEK293T cells were transfected with with NcoA2 shRNA to silence NcoA2 expression, pcDNA3.1-AhR vector to overexpress AhR, or pcDNA3.1-NcoA2 vector to overexpress NcoA2, and then were incubated under hypoxic conditions (0.5% O2) or normoxia for 6 h. Empty vector control pcDNA3.1, pLKO.1 were tested. ARNT was immunoprecipitated with anti-ARNT, anti-HIF-1α, anti-NcoA2 antibody, and protein A/G beads, and the precipitated ARNT/HIF-1α, ARNT /AhR, and ARNT/NcoA2 were identified by western blotting. (C) Fold change of HRE reporter vector activity in HEK293T cells and HEK293T cells overexpressing NcoA2 or AhR under hypoxia (0.5% O2) or nomoxic conditions for 6 h. The cells were pre-transfected with a firefly luciferase reporter gene driven by HREs (pGL2-HRE) for 24 h. ***p < 0.001 indicate a statistical difference from the control. ##p < 0.01 and ###p < 0.001 indicate a statistical difference from the hypoxia-only treatment group. 40

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exposure to hypoxia (0.5% O2) for 6 h and 10 µM B[a]P for 6 h. Nuclei were

Figure 7. The effects of NcoA2 protein expression on the regulation of angiogenesis in vivo. (A) VEGF recombinant protein were added as a positive control for tube formation. NcoA2 shRNA and pcDNA3.1-NcoA2 vector to overexpress NcoA2 were transfected in HUVECs. Representative images show that tube formation was suppressed in both the NcoA2 knockdown and NcoA2 overexpressed

knockdown and NcoA2 overexpressed HUVECs under normoxia and hypoxia. (B) The Matrigel plug assay for neovasculogenesis in vivo was performed as described in the ‘‘Materials and Methods’’ section. Plugs were harvested for hemoglobin content measurement 6 d after implanting. Images and quantitative results show that both NcoA2 knockdown and overexpression suppressed the HIF-1α -induced neovasculogenesis. **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control. ##p < 0.01 indicate a statistical difference from the HIF-1α.

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HUVECs, and B[a]P treatment also inhibited tube formation additivity both in NcoA2

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Figure 1. The expression of NcoA2 protein decreased in CoCl2-treated HEK293T cells. Western blots showing the levels of NcoA2 protein expression following CoCl2 treatment in a (A) dose-dependent manner (75, 150, and 300, µM) and (B) time-dependent manner (1, 3, 6, 12, and 24 h). (C) HEK293T cells were treated with 75, 150, and 300, µM CoCl2 for 6 h. The nuclear extract was obtained, and the NcoA2 nuclear localization levels were quantified. Lamin A/C and α-tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control. 190x275mm (300 x 300 DPI)

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Figure 2. Both hypoxia- and CoCl2-induced HRE transactivation reduced in NcoA2-silenced HEK293T cells. (A) Western blots showing levels of NcoA2 protein expression following NcoA2-knockdown in HEK293T cells. The cells were pre-transfected with a firefly luciferase reporter gene driven by HREs (pGL2-HRE) for 24 h. (B) Morphology of HEK293T with NcoA2 knockdown. (C) Fold change of HRE reporter vector activity in HEK293T wild type and NcoA2-knockdown cells under CoCl2 treatment or nomoxia conditions for 6 h. The data show the HRE activity increased in the CoCl2 treatment group; the fold change of HRE reduced in HEK293T cells transfected with shNcoA2. (D) Fold change of HRE reporter vector activity in HEK293T wildtype and NcoA2-knockdown cells under hypoxia (0.5% O2) or nomoxia conditions for 6 h. (E) Fold change of vegf mRNA expression in HEK293T wild-type and NcoA2-knockdown cells under hypoxia (0.5% O2) or nomoxia conditions quantified by qPCR. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control. #p < 0.05, ##p < 0.01, and ###p < 0.001 indicate a statistical difference from the hypoxia or hypoxia-mimic treated only groups. 190x275mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at University of Sussex on September 25, 2015

Downloaded from http://toxsci.oxfordjournals.org/ at University of Sussex on September 25, 2015

Figure 3. The AhR ligand B[a]P decreased NcoA2 protein levels in AhR-overexpressed, but not wild-type, HEK293T cells. (A) NcoA2 and AhR protein levels reduced in B[a]P-treated HepG2 cells, which have a higher endogenous AhR basal level than HEK293T cells, in dose-dependent manner. HEK293T cells were transfected with pcDNA 3.1-AhR overexpression vector for 24 h and then treated with B[a]P. (B) The NcoA2 and AhR content of HEK293T cells, which have a low endogenous AhR basal level, did not change after B[a]P treatment. However, both NcoA2 and AhR protein levels reduced in B[a]P-treated AhR-overexpressing HEK293T cells in a dose-dependent manner. (C) Fold change of XRE reporter vector activity in HepG2 and HEK293T treated with B[a]P for 6 h. (D) Due to the AhR endogenous level, B[a]P-treated induced CYP1A1 in HepG2 but not HEK293T cells. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control. 190x275mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at University of Sussex on September 25, 2015

Figure 4. B[a]P decreased NcoA2 protein nuclear localization levels, and XRE transactivation reduced in NcoA2-silenced HepG2 cells. Representative western blot of nuclear extracts and quantitation of the NcoA2 nuclear translocation levels. (A) HepG2 cells treated with the AhR ligand B[a]P (10 µM) for 24 h, which decreased NcoA2 nuclear translocation. (B) Fold change of XRE reporter vector activity in HepG2 wild-type and NcoA2-knockdown cells treated with B[a]P for 6 h. ***p < 0.001 indicate a statistical difference from the control. ##p < 0.01, and ###p < 0.001 indicate a statistical difference from the B[a]P only treatment group. 190x275mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at University of Sussex on September 25, 2015

Figure 5. Both HIF-1α and NcoA2 protein nuclear localization induced by CoCl2 treatment reduced in B[a]Ppretreated HepG2 cells transfected with AhR shRNA. Representative western blot of the nuclear extract and quantitation of the NcoA2 nuclear translocation levels. (A) HepG2 cells pretreated with the AhR ligand B[a]P (10 µM) for 24 h and then incubated under normoxia or CoCl2 treatment for 6 h. CoCl2 inhibited HIF-1α and NcoA2 nuclear translocation with B[a]P treatment. (B) Wild-type and AhR-knockdown HepG2 cells were incubated under normoxia or CoCl2 treatment for 6 h. AhR shRNA inhibited NcoA2 nuclear translocation with CoCl2 treatment. Western blots were normalized with the loading control, and data are presented as mean ± S.E. as compared with the control group for three independent experiments *p < 0.05 indicate a statistical difference from the control. #p < 0.05 as compared with B[a]P-untreated HepG2 cells under CoCl2 treatment. α-tubulin and lamin A/C were used as the internal controls for the cytosolic and nuclear fractions, respectively. (C) The AhR shRNA was transfected to knockdown AhR expression in HepG2 cells. 190x275mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at University of Sussex on September 25, 2015

Figure 6. NcoA2 protein acts as a regulatory factor in HRE transactivation through changes in protein-protein interactions with ARNT, AhR, and HIF-1α (A) Immunofluorescence microscopy of ARNT (green) in HEK293T cells following exposure to hypoxia (0.5% O2) for 6 h and 10 µM B[a]P for 6 h. Nuclei were visualized with hoechst 33258 (blue). (B) HEK293T cells were transfected with with NcoA2 shRNA to silence NcoA2 expression, pcDNA3.1-AhR vector to overexpress AhR, or pcDNA3.1-NcoA2 vector to overexpress NcoA2, and then were incubated under hypoxic conditions (0.5% O2) or normoxia for 6 h. Empty vector control pcDNA3.1, pLKO.1 were tested. ARNT was immunoprecipitated with anti-ARNT, anti-HIF-1α, anti-NcoA2 antibody, and protein A/G beads, and the precipitated ARNT/HIF-1α, ARNT /AhR, and ARNT/NcoA2 were identified by western blotting. (C) Fold change of HRE reporter vector activity in HEK293T cells and HEK293T cells overexpressing NcoA2 or AhR under hypoxia (0.5% O2) or nomoxic conditions for 6 h. The cells were pre-transfected with a firefly luciferase reporter gene driven by HREs (pGL2-HRE) for 24 h. ***p < 0.001 indicate a statistical difference from the control. ##p < 0.01 and ###p < 0.001 indicate a statistical difference from the hypoxia-only

treatment group.

190x275mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at University of Sussex on September 25, 2015

Downloaded from http://toxsci.oxfordjournals.org/ at University of Sussex on September 25, 2015

Figure 7. The effects of NcoA2 protein expression on the regulation of angiogenesis in vivo. (A) VEGF recombinant protein were added as a positive control for tube formation. NcoA2 shRNA and pcDNA3.1NcoA2 vector to overexpress NcoA2 were transfected in HUVECs. Representative images show that tube formation was suppressed in both the NcoA2 knockdown and NcoA2 overexpressed HUVECs, and B[a]P treatment also inhibited tube formation additivity both in NcoA2 knockdown and NcoA2 overexpressed HUVECs under normoxia and hypoxia. (B) The Matrigel plug assay for neovasculogenesis in vivo was performed as described in the ‘‘Materials and Methods’’ section. Plugs were harvested for hemoglobin content measurement 6 d after implanting. Images and quantitative results show that both NcoA2 knockdown and overexpression suppressed the HIF-1α -induced neovasculogenesis. **p < 0.01, and ***p < 0.001 indicate a statistical difference from the control. ##p < 0.01 indicate a statistical difference from the HIF-1α. 190x275mm (300 x 300 DPI)

NcoA2-Dependent Inhibition of HIF-1α Activation Is Regulated via AhR.

High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR liga...
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