Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1β

Abstract

Background: The main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer.

Methods: This project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed.

Results: BaP can enhance the binding ability of HIF-1α protein to HIF-1β/ARNT in a dose-dependent manner without CoCl₂. However, the binding ability of AhR protein to HIF-1β/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl₂.

Conclusion: It is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway.

Keywords: Aryl hydrocarbon receptor; Aryl hydrocarbon receptor nuclear translocator; Benzopyrene; CoCl2; Hypoxia-inducible factor-1α.

Fig. 1

Establishment of cell spent oxygen microenvironment and selection of BaP concentration. A Expression level of HIF-1α mRNA after 24 h of treatment with different concentrations of CoCl₂. B Effects of 300 μM CoCl₂ on expression level of HIF-1α mRNA at different times in A549 cells. C Effects of different concentrations BaP on cell viability at different times in A549 cells. D A549 cell culture map, *compared to control group P < 0.05, **compared to control group P < 0.01, n = 6

Fig. 2

Schematic diagram of eukaryotic expression vector. A Bioassay based on FRET. B pAcGFP1-AhR and pDsRed-Monomer-ARNT enzyme-digested results of agarose gel electrophoresis. C Recombinant plasmid transfection

Fig. 3

BaP in A549 cells enhanced the FRET fluorescence signal of pAcGFP1-N1-AhR and pDsRed-Monomer-ARNT. A pAcGFP1-N1-AhR and pDsRed-Monomer-ARNT recombinant plasmids were transfected into A5F49 cells. After 24 h, the cells were treated with 0, 2, 4, and 8 μM BaP, and then the following channels were used for high content detection: nucleus, donor: AhR, receptor: ARNT and FRET. B Bar chart showing changes in the ratios of FRET signal strength,* P < 0.05, ** P < 0.01, n = 3

Fig. 4

CoCl₂ in A549 cells reduced the FRET fluorescence signal of pAcGFP1-AhR and pDsRed-Monomer-ARNT. A pAcGFP1-N1-AhR and pDsRed-Monomer-ARNT recombinant plasmids were transfected into A549 cells; 24 h later, the cells were treated with 8 μM BaP for 24 h. B pAcGFP1-AhR and pDsRed-Monomer-ARNT recombinant plasmids were transfected into A549 cells; 24 h later, the cells were treated for 24 h with 8 μM BaP under hypoxic conditions. In A549 cells, BaP enhanced the pAcGFP1-AhR and pDsRed-Monomer-ARNT FRET fluorescent signal in dose-dependent manner,* P < 0.05, ** P < 0.01, n = 3

Fig. 5

Effects of BaP on CAIX (A), VEGF (B), CYP1A1 (C), and CYP1B1 (D) expression at the mRNA level without (□) or with (■) CoCl₂ and with 0–8 μM BaP. Data are presented as the fold-increase compared to without B(α) P and CoCl₂, * P < 0.05, ** P < 0.01. Without (□) or with (■) CoCl₂ and with 0–8 μM BaP. Data are presented as the fold-increase compared to without BaP and CoCl₂, * P < 0.05, ** P < 0.01, n = 3

Fig. 6

Effects of BaP on CAIX, VEGF, CYP1A1 and CYP1B1 expression at the protein level without (□) or with (■) CoCl₂ and with 0–8 μM BaP. Data are presented as the fold-increase compared to without B(α) P and CoCl₂, * P < 0.05, ** P < 0.01. Without (□) or with (■) CoCl₂ and with 0–8 μM BaP. Data are presented as the fold-increase compared to without B(α) P and CoCl₂, * P < 0.05, ** P < 0.01, n = 3

Fig. 7

Effects of BaP and CoCl₂ exposure on HIF-1α, AhR, and ARNT protein-protein interaction. A549 cells were incubated without or with 300 μM CoCl₂ and with 0–8 μM BaP. Representative western blots show the amount of HIF-1α, AhR bound to ARNT and ARNT bound to AhR. Data are presented as the fold-increase compared to without CoCl₂ and BaP corrected for the amount of protein loaded, n = 3