Functions of the aryl hydrocarbon receptor (AHR) beyond the canonical AHR/ARNT signaling pathway

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor regulating adaptive and maladaptive responses toward exogenous and endogenous signals. Research from various biomedical disciplines has provided compelling evidence that the AHR is critically involved in the pathogenesis of a variety of diseases and disorders, including autoimmunity, inflammatory diseases, endocrine disruption, premature aging and cancer. Accordingly, AHR is considered an attractive target for the development of novel preventive and therapeutic measures. However, the ligand-based targeting of AHR is considerably complicated by the fact that the receptor does not always follow the beaten track, i.e. the canonical AHR/ARNT signaling pathway. Instead, AHR might team up with other transcription factors and signaling molecules to shape gene expression patterns and associated physiological or pathophysiological functions in a ligand-, cell- and micromilieu-dependent manner. Herein, we provide an overview about some of the most important non-canonical functions of AHR, including crosstalk with major signaling pathways involved in controlling cell fate and function, immune responses, adaptation to low oxygen levels and oxidative stress, ubiquitination and proteasomal degradation. Further research on these diverse and exciting yet often ambivalent facets of AHR biology is urgently needed in order to exploit the full potential of AHR modulation for disease prevention and treatment.

The EGFR is a receptor tyrosine kinase (RTK) of the ErbB family and plays a key role in embryonic development and physiology [ 41 ]. As EGFR has an intra- and extracellular domain to bind ligands, activation can occur via both ways. This activation includes receptor trans -autophosphorylation, the hetero- or homodimerization with a member of the ErbB family or another EGFR molecule and the recruitment of signaling proteins or adaptors [ 41 ]. Its downstream signaling includes the mitogen-activated protein kinase (MAPK) RAS- RAF- MEK1/2-ERK1/2 and AKT-PI3K-mTOR pathways as well as protein kinase C (PKC), STAT, SRC and NF-κB. Moreover, EGFR is a well-known oncogenic protein and involved in the pathogenesis of several cancers, commonly carrying a mutation that leads to a constitutive activation of the receptor [ 42 , 43 ]. Thus, EGFR plays an important role in several cellular processes such as proliferation, differentiation and apoptosis. The seemingly first AHR-related interaction with the EGFR was described in 1982 suggesting that benzo[ a ]pyrene (BaP) and other PAHs may not only activate AHR but also inhibit binding of EGFR by its ligand, the epidermal growth factor (EGF) [ 44 ]. Since then, it has been repeatedly reported that an exposure to AHR agonists interferes with the binding of radiolabeled EGF to the plasma membrane [ 45 – 47 ]. The underlying molecular mechanism may involve an AHR ligand-mediated enforcement of EGFR internalization either by stimulating a phosphorylation of the RTK via c-Src or by inducing the release of growth factors that bind to EGFR extracellular domain (ECD). However, in contrast to PAHs that, presumably due to recycling of EGFR, only cause a transient decline [ 46 , 47 ], TCDD reduces the EGF-binding capacity of the plasma membrane for up to 4 days in human keratinocytes [ 47 ] and 40 days in rat liver [ 45 ]. In 2022, we published a mechanistic study which may serve to better understand the interaction between different AHR ligands and EGFR internalization [ 48 ]. Briefly, we found that treatment of human keratinocytes with PAHs, i.e. BaP and benzo[ k ]fluoranthene, causes a biphasic stimulation of EGFR phosphorylation and downstream MEK/ERK signal transduction. Whereas the early peak, occurring approximately after 15 min after treatment, this seems to be due to a direct c-Src-mediated phosphorylation of EGFR at residue Y845 ( Fig. 1 ), the second temporally delayed peak of EGFR/ERK activation (approximately 2 h after treatment) involves extracellular events, i.e. the release of growth factors. Specifically, this process is driven by the c-Src-dependent sequential activation of PKC and metalloproteinases resulting in the ectodomain shedding of cell surface-bound EGFR ligands, in particular of amphiregulin (AREG) and transforming growth factor (TGF)-α ( Fig. 1 ). These polypeptide growth factors then bind to the ECD of EGFR and initiate dimerization and autophosphorylation of the RTK at various tyrosine residues, including Y1068 and Y1137 [ 48 ]. Importantly, bulk RNAseq analyses indicated that this second wave of EGFR-dependent signal transduction seems to be responsible for the major differences in the gene expression profile in BaP- versus polychlorinated biphenyl (PCB) 126-exposed keratinocytes [ 48 ]. In fact, we were able to show that binding of dioxin-like compounds, including PCB126 and TCDD, induces similar AHR-dependent and c-Src-driven signaling events culminating in the shedding of EGFR ligands from the plasma membrane. However, dioxin-like compounds stimulate EGFR signal transduction only early (15 min) but not late (2 h) after treatment. In silico as well as further experimental work revealed that dioxin-like compounds bind to the ECD of EGFR directly and thereby inhibit an activation of the RTK by growth factors [ 48 , 49 ]. Apart from non-canonical signaling events, the ligand-activated AHR has been found to induce the expression of several EGFR ligands ( Fig. 1 ), including epiregulin and AREG, in an XRE-dependent manner [ 50 , 51 ]. Other investigators reported that tobacco smoke induces the expression of AREG in oral epithelial cells via a non-canonical AHR pathway involving the cAMP – protein kinase A signaling axis [ 52 ]. However, Lemjabbar et al . identified tobacco smoke to stimulate the proliferation of lung epithelial cells by promoting the metalloprotease-mediated ectodomain shedding of AREG [ 53 ]. Hence, depending on ligand, cell type and context, AHR may affect EGFR signal transduction via canonical and non-canonical signaling events or a combination of both. The laboratory of Thomas Sutter reported an inhibition of the TCDD-induced expression of CYP1A1 and CYP1B1 upon co-treatment of epidermal keratinocytes with EGF. Results from further mechanistic experiments indicated that both pathways compete for the common transcriptional co-activator CBP/p300. Accordingly, the AHR-mediated keratinocyte differentiation was also inhibited by EGF while co

The NF-κB family of transcription factors regulates the expression of numerous cytokines and immune response genes [ 110 ]. NF-κB is critically involved in biological processes, including apoptosis, cell differentiation, immunity and diseases such as autoimmunity or cancer [ 111 ]. Considering the physiological and pathological roles of AHR, a significant overlap with the NF-κB signaling pathway becomes evident [ 112 ]. When considering AHR as a transcription factor interacting with NF-κB signaling and modulating immune responses, it is crucial to understand its role in diseases based on immune modulatory effects. The activity of NF-κB is induced by many cytokines and inflammatory signals in a time- and concentration-dependent manner [ 113 ]. Early studies have shown that cytokines also affect the activity of AHR-regulated drug-metabolizing enzymes. For instance, treatment with TNF-α clearly suppressed the activity of hepatic CYP-dependent drug metabolism in mice [ 114 ]. Subsequent studies noticed suppression of TCDD-induced Cyp1a1 and Cyp1a2 mRNA expression after treatment of isolated rat hepatocytes with IL-1β [ 115 ]. In line with this, the acute phase protein IL-6 suppressed the gene expression of CYP1A1 , CYP1A2 and CYP3A3 in human hepatoma cells [ 116 ]. These early observations strongly indicate that NF-κB activated by inflammatory cytokines interferes with AHR signaling and thereby shapes target gene expression and the associated biological outcome. The activation of NF-κB changes the expression and activity of AHR and vice versa . One of the first studies reporting a crosstalk between AHR and proteins of the NF-κB family was published by Tian et al . describing the physical interaction of AHR with NF-κB RelA causing a mutual repression of both signaling pathways [ 117 ]. In their study, the authors treated mouse hepatoma cells with TNF-α to activate NF-κB and focused on the AHR-mediated induction of CYP1A1 which was significantly repressed after TNF-α treatment. Further, they found that TNF-α-induced NF-κB binding activity was suppressed when Hepa1c1c7 cells were treated with TCDD. Mechanistically, the ligand-dependent activation of AHR induces histone H4 acetylation at the TATA box of the Cyp1a1 promoter region, which was inhibited by TNF-α-induced NF-κB activity [ 118 ]. Further studies have shown that AHR interacts with the NF-κB component RelA which supported the TCDD-mediated induction of IL-6, plasminogen activator inhibitor-2, FAS ligand and c-myc [ 119 – 122 ]. Dinatale et al . found that the AHR-mediated amplifying effects on NF-κB signaling requires the AHR/ARNT-heterodimer for the synergistic activation of IL-6 following IL-1β and TCDD treatment. This may involve the dismissal of co-repressors by DNA-bound AHR [ 119 ]. The AHR-dependent expression of FasL in thymic stromal cells was found to be regulated through TCDD-mediated activation of the NF-κB subunits p50 and p65 on the Fasl promoter [ 123 ]. Another mechanism was described for the expression of C-MYC in breast cancer cells, where RelA and AHR together bind to NF-κB elements and induce the transcription of C-MYC [ 122 ]. Conversely, AHR was also identified to suppress NF-κB-mediated gene expression. For instance, the expression of the Ig heavy chain was repressed through the interactions of NF-κB and AHR at a DNA replication-related element and an overlapping κB element [ 124 ]. The acute phase protein serum amyloid A was found to be suppressed by the interaction of the activated AHR with RelA, but DNA binding was not involved [ 125 ]. Alvaro Puga’s group concluded that TCDD-mediated activation of AHR and induction of CYP1A1 leads to the generation of an oxidative stress signal which enhances NF-κB and AP-1 DNA-binding activity. [ 126 ]. Interestingly, the enhanced binding activity of the NF-κB complex was found to be formed by p50/p50 complexes which could be responsible for the inhibitory effect of AHR on NF-κB activity. Another study reported an inhibition of IgM expression by TCDD in LPS-activated B cells, which was associated with an AHR-dependent decreased DNA-binding activity of AP-1 but not of NF-κB [ 127 ]. Besides RelA, several studies reported an interaction of AHR with the NF-κB member RelB [ 128 – 131 ]. We identified RelB to physically interact with AHR and bind on a previously unrecognized RelB/AHR-responsive element in the promoter of the IL-8 gene to induce its expression [ 132 ]. Further, a ligand-independent activation of AHR via protein kinase A triggers its nuclear translocation and the induction of IL-8. Therefore, this mechanism was described as the non-canonical AHR pathway which involves ligand-independent activation and interaction with proteins other than ARNT, such as RelB. Similarly, the expression of other chemokines including the B-cell activating factor of the TNF family, B-lymphocyte chemoattractant and chemokine (C–C–motif) ligand (CCL) 1 was also found to involve the binding of RelB/AHR-complexe

The Nobel Prize in Physiology or Medicine 2019 was awarded to Gregg Semenza, Sir Peter Ratcliffe and William Kaelin for their joint discovery of hypoxia-inducible factor (HIF)-1α and its functionality under cellular oxygen shortage [ 174 – 176 ]. Of note, the identified co-factor HIF-1β turned out identical to the already known AHR co-factor ARNT. AHR, ARNT, and HIF-1α all belong to the group of basic helix-loop-helix/Per-Arnt-Sim proteins which act as environment-sensing heterodimeric transcription factors [ 177 ] governing adaptation to changing external conditions. HIF-1α function is crucial under physiologic conditions including embryogenesis [ 178 – 180 ] or during anaerobic activity [ 181 , 182 ], but also under pathologic conditions like infection and cancer [ 180 , 183 , 184 ]. Under normoxic conditions (~21 % O 2 ), the HIF-1α protein is constantly degraded by the proteasome: Oxygen-dependent HIF-prolyl hydroxylases permit binding of the von Hippel–Lindau protein to hydroxylated HIF-1α, leading to ubiquitination and subsequent degradation by the proteasome [ 174 , 176 , 185 ]. Under hypoxia (~0,1–1 % O 2 /0 % O 2 is termed anoxia), these enzymes are inactive, allowing accumulation of HIF-1α and activation via dimerization with ARNT to induce transcription by binding to hypoxia-responsive elements (HRE) on nuclear DNA ( Fig. 4 ) [ 175 , 186 ]. There is clear long-standing evidence from independent studies that HIF-1α and AHR activities are interdependent [ 187 – 190 ] in different tissues, indicating a functional crosstalk between their pathways in conditions as diverse as obesity, glioblastoma, prostate cancer, nephrocalcinosis, autoimmune hepatitis and affecting processes like T cell and macrophage differentiation as well as blood–brain barrier function [ 87 , 191 – 199 ]. However, this issue has rarely been addressed systematically [ 200 ]: While a majority of studies suggests downregulation of AHR function through HIF-1α activation and vice versa [ 201 – 203 ], they almost exclusively investigated the function of TCDD, and more physiologic means of AHR activation have been even more scarcely addressed. The modes of interaction can be competitive or counteracting, but could possibly also be additive or compensatory under certain circumstances ( Fig. 4 ). The specific effect may depend on contextual factors ranging from the specific tissue or cell type or pathway trigger to biochemical properties like ARNT availability, binding preference and other parameters. Importantly, it has been reported that ARNT is influenced by both hypoxia and hypoxia mimetics [ 204 , 205 ]. The skin has been a subject to hypoxia research, since especially the avascular epidermis is mildly hypoxic with constant low HIF-1α activity [ 206 – 208 ]. Of note, loss of ARNT in epidermal keratinocytes causes skin barrier failure in neonatal mice [ 209 – 211 ] associated with dysregulation of processes like synthesis of ceramides [ 212 ], a group of lipids implicated in oxidative stress balance [ 213 ] which in turn is also mediated by ARNT [ 214 ]. Since both keratinocyte-specific HIF-1α −/− and AHR −/− mice are viable without an overt phenotype [ 215 – 217 ], these findings underline (I) the importance of cooperative function of HIF-1α and AHR in epidermal keratinocytes and (II) may hint toward the involvement of other ARNT-dependent factors and/or (III) possible compensatory or aberrant pathway activation. Though a majority of studies demonstrates interaction of HIF-1α and AHR via ARNT ( Fig. 4 ), others have argued that cells harbor a relative excess of ARNT not limiting its availability to dimerization partners [ 218 ], which also include HIF-2α, AHRR and others [ 177 ]. Other molecules proposed in HIF-1α-AHR crosstalk are microRNAs [ 197 ] and the proteins NCoA-2 [ 219 ] and p23 [ 203 ]. Among prototypical targets induced by HIF-1α activity are the vascular endothelial growth factor (VEGF), erythropoietin (EPO) and Glut-1 [ 220 , 221 ] which contribute to metabolic adaptation to hypoxia and improvement of oxygen availability: VEGF induces endothelial vessel growth, EPO is a growth factor for erythrocytes and Glut-1, encoded by the SLC2A1 gene, is an important membrane transport protein enabling the cellular import of glucose and other monosaccharides as well as ascorbic acid. Further, numerous other genes are modulated which can be grouped into inflammation/immune defense, proliferation, tumor promotion/cancer therapy resistance [ 222 ]. Taken together, HIF-1α-dependent processes promote cellular survival under stress through oxygen shortage. The normal oxygen level is tissue-specific and low tissue oxygen availability is not necessarily pathologic: Intermediate O 2 levels are termed physoxia (range ~ 1–13 % O 2 , medium values ~ 5 % O 2 ) [ 223 ]. HIF-1α is typically stabilized under hypoxic conditions (below 1 % O 2 ) which arise during high cell proliferation or metabolic activity including embryogenic development, infla

While the AHR is infamous for its function as a ligand-activated transcription factor, some studies regarding the toxicity of dioxins and related compounds have shown that the AHR also mediates estrogenic [ 303 , 304 ], anti-estrogenic [ 305 , 306 ] and androgenic effects [ 307 ]. The underlying mechanisms were unclear and could not be explained exclusively by CYP1-mediated degradation of steroid hormones or the CYP19A (aromatase)-catalyzed synthesis of the estrogen receptor (ER)-ligand 17β-estradiol. In 2003, Ohtake and co-workers showed that dioxins exert estrogenic effects through the association of ligand-activated AHR/ARNT to unliganded ER [ 308 ]. Additionally, Wormke et al . revealed that TCDD induces the proteasome-dependent degradation of endogenous ER-α [ 309 ]. Based on this observation, a novel ubiquitin ligase complex was identified. This complex is initiated by ligand activation of the AHR and targets sex steroid receptors [ 310 ]. Nevertheless, mechanistic insights into the regulation of both AHR functions remained unclear until 2017, when Luecke-Johansson and co-workers described how the AHR switches between its functions as a transcription factor and an E3 ubiquitin ligase [ 311 ]. One of the most important regulators of cellular homeostasis is the ubiquitin proteasome system which specifically degrades targeted proteins [ 312 , 313 ]. In a first step, an activating enzyme (E1) transfers a ubiquitin molecule to a ubiquitin-conjugating enzyme (E2). Ubiquitin protein ligases (E3) recognize specific degradation signals and are therefore responsible for substrate specificity. With the help of an E3 ligase, the E2 enzyme conjugates one or more ubiquitin molecules to the target protein, followed by proteasomal degradation [ 312 , 313 ]. The AHR-associated E3 ligase complex, denoted CUL4B AHR , is composed of the scaffold protein Cullin 4B (CUL4B), damaged-DNA binding protein 1, RING-box protein 1, transducin-β-like protein 3 and the proteasomal 19S particle [ 310 ]. Upon ligand-activation of the AHR, it initiates assembly of the CUL4B AHR complex and serves as a substrate-specific adapter protein within the complex ( Fig. 6 ). Then, the availability of ARNT determines the functionality of AHR: when ARNT is available, the AHR functions as a ligand-activated transcription factor and activates the canonical AHR signaling pathway. However, when ARNT is occupied by other proteins, such as the AHRR, the AHR functions as an E3 ligase and induces assembly of the CUL4B AHR complex [ 311 ]. Subsequently, substrate proteins are targeted for proteasomal degradation ( Fig. 6 ). Target proteins of the CUL4B AHR complex include ER-α, ER-β and androgen receptor (AR) [ 310 , 314 ]. It was also shown that PPARγ, a transcription factor and important regulator of adipogenesis, is targeted by the CUL4B AHR complex for proteasomal degradation [ 315 ]. Moreover, β-catenin, which is a transcription factor downstream from the WNT signaling pathway, has been reported as target protein [ 316 ]. Canonical WNT/β-catenin signaling pathway is a key regulator of embryonic development and adult tissue homeostasis [ 317 ]. While β-catenin degradation by the CUL4B AHR complex was shown in mouse intestine, other working groups could not find evidence for β-catenin degradation in mouse hepatoma cells despite evidence of physical interaction of AHR and β-catenin. This suggests that AHR/β-catenin interaction, or rather the AHR function as an E3 ligase, may not only be ligand-dependent but also tissue-specific. Interestingly, the AHR was found to induce the ubiquitin-proteasomal and lysosomal degradation of RelA/p65 in mouse peritoneal macrophages [ 140 ]. However, whether this process involves AHR’s function as a E3 ubiquitin ligase requires further investigation. Importantly, upon its ligand-induced nuclear translocation and the subsequent transactivation of target genes, AHR itself becomes degraded [ 318 , 319 ]. Even though this has been known for many years, details about the degradation process only became evident in 2021. Rijo and co-workers demonstrated that the CUL4B AHR complex and TCDD-inducible poly(ADP-ribose) polymerase collaborate in ligand-induced AHR degradation. Knockdown of CUL4B in mouse embryonic fibroblasts partially prevented AHR degradation by TCDD, while additional knockdown of TCDD-inducible poly(ADP-ribose) polymerase completely abolished ligand-induced AHR degradation [ 320 ]. Due to the fact that the canonical transcriptional activity and E3-ligase activity of the AHR are in a close relation with each other, it should be considered that molecular targeting of the AHR by classical chemical ligands does also affect other signaling pathways, such as ER, AR, PPARγ and WNT/β-catenin signaling. A dysregulation of these major signaling pathways may contribute to the development of diseases, such as cancer [ 321 – 324 ]. However, the bifunctionality of the AHR gives rise to the opportunity of utilizing the E3 ligas