2.2. Mechanisms of hypoxia-mediated regulation of CYP
2.2.1. Nuclear receptors
2.2.1.1. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR)
PXR and CAR are members of the orphan receptor subfamily. The targets and regulatory functions of PXR and CAR are similar in many ways. Activation of PXR and CAR also induces CYP expression (Bautista-Olivier et al., 2022; Smutny et al., 2021). Hypoxia affected PXR expression Yuan et al. found that the expression of PXR mRNA in the rat liver substantially decreased by 63%, 96%, 86%, and 85% after 12, 24, 36, and 48 h of exposure to acute hypoxia, respectively. PXR protein expression increased substantially by 93% and 99% after 36 and 48 h of exposure to hypoxia, respectively (Yuan et al., 2019). Such changes in PXR or CAR under hypoxic conditions may be the potential mechanisms mediating CYP changes during hypoxia. Legendre et al. found that the expression of CAR and PXR is downregulated, whereas the activities of CYP3A4, CYP1A2, CYP2E1, and CYP2C9 are reduced during hypoxia (Legendre et al., 2009). Our study also confirmed that hypoxia inhibited the expression of CYP isoforms through the PXR and CAR regulatory pathways. Hypoxia is effective in inhibiting the expression of CYP isoforms, PXR, and CAR proteins and mRNAs in both rat and HepG2 cells. We conducted additional research in vitro using substances that either inhibit or activate PXR and CAR. Our findings indicate that hypoxia and inhibitors suppress the expression of CYP450 isoforms, while activators increase the expression of CYP isoforms, PXR, and CAR. Furthermore, hypoxia counteracts the activating effect of activators, suggesting that the decrease in expression of CYP1A2, CYP2C9, CYP2E1, and CYP3A4 under hypoxia is primarily due to the inhibition of the regulatory effects of PXR and CAR (Duan et al., 2020).
2.2.1.2. Aryl hydrocarbon receptor (AHR)
AHR promotes the transcription of its target genes, including CYP1A1 and CYP1A2, by binding to the aryl hydrocarbon receptor nuclear translocator (ARNT) to form the AHR/ARNT complex (Dolly et al., 2023), which also occurs under hypoxic conditions. Zhang et al. measured CYP1A1 mRNA in human lung microvascular endothelial cells exposed to hypoxia and 25 nM AHR siRNA, and found that hypoxia substantially reducedCYP1A1 and AHR mRNA, and AHR siRNA also substantially reduced CYP1A1 mRNA. Hypoxia, when paired with AHR siRNA, resulted in a greater reduction of CYP1A1 mRNA compared to hypoxia alone with AHR siRNA. This suggests that hypoxia and AHR inhibition may have a shared route for lowering CYP1A1 expression (Zhang et al., 2007). This regulatory role was further confirmed in a later study using the AHR activator-like dioxin PCB (PCB126), where Vorrink et al. found that exposure to hypoxia (1% O2) substantially inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 promoter-luciferase reporter activity after PCB126 stimulation was also substantially reduced by hypoxic stimulation, and hypoxic preconditioning reduced PCB126-induced AHR binding to the promoters of CYP1 target genes (Coelho et al., 2020; Jacob et al., 2015; Vorrink et al., 2014).
2.2.1.3. Peroxisome proliferator-activated receptor (PPAR)
PPARα is a member of the peroxisome proliferator-activated receptor family, which is involved in the regulation of a variety of biological processes. PPARα activates members of CYP1A1, CYP2E1, CYP2J2, CYP4A, and CYP7A1 (Cizkova et al., 2012), and further studies on the role of PPARα ligands will provide insight into the link between inhibition and CYP expression. Treatment with PPARα ligands, such as Wy-14643 or fibrate downregulated CYP2C9 and CYP2C44 expression and reduced the biosynthesis of the CYP epoxide metabolite, EET, in humans and mice, respectively.PPAR is a key gene in hypoxia adaptation, is highly susceptible to hypoxia regulation, and promotes adaptation to hypoxia (Kudo et al., 2023; S. Wang et al., 2022). This change in PPAR under hypoxic conditions also regulates CYP expression. Bonazzi et al. found that in rabbit corneal epithelial cells, hypoxia increased PPAR-inducedCYP4B1 mRNA (Bonazzi et al., 2000). van Malenstein et al. found that in human HepG2 hepatocytes chronically hypoxic for 72 h at 2% O2, a PPARα/RXRα expression signal was affected and the hepatic CYP450-specific detoxification pathway was downregulated (van Malenstein et al., 2012).
2.2.1.4. Hepatocyte nuclear factor (HNF)
HNF1α and HNF4α are direct trans-activators of CYP genes involved in regulating the constitutive expression of most CYP genes in the human liver. Knockdown of HNF4α and HNF1α alters mRNA levels of CYP2B6 ,CYP2C8 , CYP2C9 , CYP2C19 , CYP2E1 ,CYP1A2 and CYP3A4 (Chen et al., 2018; Chiang et al., 2014). Hypoxia inhibits HNF expression, and Mazure et al. initially found that hypoxia downregulates HNF4 protein and gene expression in HepG2 cells (Mazure et al., 2001). Sato et al. found that hypoxia decreased HNF4α protein expression and stability in β-cells (Sato et al., 2017). Changes in the expression of HNF1α and HNF4α in hypoxia may be a potential regulatory mechanism for the changes in CYP expression under hypoxia. Yang showed that HNF1α and HNF4α synergistically downregulate CYP1A2 through mutual inhibition under hypoxia; the regulatory mechanism of CYP under hypoxia may be different from that under normoxia, where HNF1α and HNF4α inhibit each other and synergistically downregulate CYP1A2; and there is a protein-protein interaction between HNF1α and HNF4α. HNF1A binds to the HNF1A binding site (-2892 bp to -2886 bp) in the CYP1A2 promoter, thereby activating the CYP1A2 promoter and the effect of HNF4A on CYP1A2 is not in the promoter region (Yang et al., 2022).