Regular ArticleComplementation of Ah Receptor Deficiency in Hepatoma Cells: Negative Feedback Regulation and Cell Cycle Control by the Ah Receptor
Abstract
The Ah receptor (AhR) is a ligand-dependent transcription factor subunit that heterodimerizes with the AhR nuclear translocator (Arnt) and mediates the predominant biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD activates target genes in xenobiotica metabolism in many cell lines and, more specifically, delays G1–S progression of 5L hepatoma cells. Here we describe transient and stable AhR-expression analysis in AhR-deficient subclones of the TCDD-sensitive 5L cells. We tested the integrity of the AhR-signaling system beyond the lack of the receptor in the variant subclone and analyzed the role of AhR in cell cycle regulation. Transiently expressed AhR has a high basal activity on promoters containing AhR-binding sites, so-called XREs, when transfected into receptor-deficient variant cells compared to wild-type cells. Single- and double-hybrid analysis dissociates AhR ligand responsiveness, transactivation, and heterodimerization with Arnt from receptor binding to an XRE. Hybrid receptors also show the high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that the endogenous AhR-activating signal acts directly on the receptor rather than XRE-dependent promoters or DNA binding of the receptor. Stable expression of AhR in variant cell clones by retroviral infection fully reconstitutes TCDD responsiveness, including target-gene induction and delay of cell cycle progression. These AhR-reconstituted cells, like AhR-containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid. Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity.In vitroligand-binding assays are compatible with the idea that the increased basal activity is due to the accumulation of an AhR-binding endogenous ligand. In conclusion, AhR is causally responsible for TCDD-dependent cell cycle regulation and feedback control of AhR activity.
References (0)
Cited by (152)
Loss of the aryl hydrocarbon receptor increases tumorigenesis in p53-deficient mice
2022, Toxicology and Applied PharmacologyThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cell fate via activation of a diverse set of genes. There are conflicting reports describing the role of AhR in cancer. AhR-knockout mice do not develop tumors spontaneously, yet the AhR can act as a tumor suppressor in certain contexts. Loss of tumor suppression by p53 is common in human cancer. To investigate AhR function in the absence of p53, we generated mice lacking both AhR and p53. Mice deficient for AhR and p53 had shortened lifespan, increased tumorigenesis, and an altered tumor spectrum relative to control mice lacking only p53. In addition, knockout of both AhR and p53 resulted in reduced embryonic survival and neonatal fitness. We also examined the consequences of loss of AhR in p53-heterozygous mice and observed a significantly reduced lifespan and enhanced tumor burden. These findings reveal an important role for the AhR as a tumor suppressor in the absence of p53 signaling and support the development of anti-cancer therapeutics that would promote the tumor suppressive actions of the AhR.
L-Tryptophan activates the aryl hydrocarbon receptor and induces cell cycle arrest in porcine trophectoderm cells
2021, TheriogenologyDuring implantation, the proliferation of trophectoderm cells (the outer epithelium of blastocysts) is related to conceptus elongation and placenta formation. Tryptophan (Trp) is a key regulator of embryogenesis and embryonic implantation during pregnancy. We sought to determine whether different concentrations of Trp alters porcine trophectoderm (pTr) cell proliferation. pTr cells were cultured in medium containing 40, 500, or 1000 μM Trp. The cell proliferation rate and the progression of the cells through the cell cycle were determined. To identify differentially expressed genes (DEGs) in the pTr cells, we compared mRNA transcriptomes by RNA-Seq after cell treatment with different concentrations of Trp. Some candidate DEGs were identified by quantitative reverse transcription PCR (qPCR). High L-Trp levels (500 and 1000 μM) inhibited cell proliferation and induced cell cycle arrest. We identified 19 DEGs between the 500 μM L-Trp and 40 μM L-Trp groups and 168 DEGs between the 1000 μM L-Trp and 40 μM L-Trp groups and subsequently used qPCR to validate some genes that were upregulated or downregulated. The functional gene networks in which the DEGs were most enriched included those associated with regulating DNA replication and the cell cycle, and the majority of the DEGs in both of these functional pathways was downregulated. The results showed that the addition of 500 and 1000 μM Trp significantly increased the abundance of proteins in the Aryl Hydrocarbon Receptor (AHR) signaling pathway. Collectively, these results indicate a novel and important role for Trp in mediating the proliferation of porcine placental cells largely via the AHR signaling pathway. Additionally, these findings help to explain the side effects of excessive Trp supplementation on placenta development and embryo growth in mammals.
Aryl hydrocarbon receptor (AhR) a possible target for the treatment of skin disease
2018, Medical HypothesesAryl hydrocarbon receptor (AhR) is a transcription factor expressed in all skin cells type. It responds to exogenous and endogenous chemicals by inducing/repressing the expression of several genes with toxic or protective effects in a wide range of species and tissues. In healthy skin, AhR signalling contributes to keratinocytes differentiation, skin barrier function, skin pigmentation, and mediates oxidative stress. In the last years, some studies have shown that AhR seems to be involved in the pathogenesis of some skin diseases, even if the currently available data are contradictory. Indeed, while the blocking the AhR signalling activity could prevent or treat skin cancer, the AhR activation seems to be advantageous for the treatment of inflammatory skin diseases. Therefore, for its multifaceted role in skin diseases, AhR seems to be an attractive therapeutic target. Indeed, recently some molecules have been identified for the prevention of skin cancer and the treatment of inflammatory skin diseases
The role of aryl hydrocarbon receptor (AhR) in the pathology of pleomorphic adenoma in parotid gland
2016, Archives of Oral BiologyPleomorphic adenoma (benign mixed tumor) is one of the most common salivary gland tumors. However, molecular mechanisms implicated in its development are not entirely defined. Therefore, the study aimed at definition of aryl hydrocarbon receptor (AhR) involvement in pleomorphic adenoma pathology, as the AhR controlled gene system was documented to play a role in development of various human tumors.
The study was carried out in pleomorphic adenoma and control parotid gland tissues where gene expression of AHR, AhR nuclear translocator (ARNT), AhR repressor (AHRR), as well as AhR controlled genes: CYP1A1 and CYP1B1, at mRNA and protein (immunohistochemistry) levels were studied. Functional evaluation of AhR system was evaluated in HSY cells (human parotid gland adenocarcinoma cells) using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as AhR specific inducer.
Pleomorphic adenoma specimens showed cytoplasmic and nuclear AhR expression in epithelial cells as well as in mesenchymal cells. In parotid gland AhR was expressed in cytoplasm of duct cells. Quantitative expression at mRNA level showed significantly higher expression of AHR, ARNT and CYP1B1, and comparable levels of CYP1A1 in pleomorphic adenoma tissue in comparison to healthy parotid gland. The HSY cell study revealed significantly higher expression level of AHRR in HSY as compared with MCF-7 cells (human breast adenocarcinoma cell line used as reference). Upon TCDD stimulation a drop in AHRR level in HSY cells and an increase in MCF-7 cells were observed. The HSY and MCF-7 cell proliferation rate (measured by WST-1 test) was not affected by TCDD.
Summarizing both in vitro and in vivo observations it can be stated that AhR system may play a role in the pathology of pleomorphic adenoma.
Aryl hydrocarbon receptor-dependent cell cycle arrest in isolated mouse oval cells
2013, Toxicology LettersThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which mediates toxic responses to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Besides its well known role in induction of xenobiotic metabolizing enzymes, for instance CYP1A1, the AhR is also involved in tumor promotion in rodents although the underlying mechanisms are still poorly understood. Additionally, the AhR is known to regulate cellular proliferation, which might result in either inhibition or stimulation of proliferation depending on the cell-type studied. Potential targets in hepatocarcinogenesis are liver oval (stem/progenitor) cells. In the present work we analyzed the effect of TCDD on proliferation in oval cells derived from mouse liver. We show that TCDD inhibits proliferation in these cells. In line, the amount of G0/G1 cells increases in response to TCDD. We further show that the expression of cyclin D1 and cyclin A is decreased, while p27 is increased. As a result, the retinoblastoma protein is not phosphorylated thereby inducing G0/G1 arrest. Pharmacological inhibition of the AhR and knock-down of AhR expression by RNA interference decreased the inhibitory effect on cell cycle and protein expression, indicating that the AhR at least partially mediates cell cycle arrest.
Previous quantitative proteomic studies on the actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells, a cell model frequently used for investigating the mechanisms of TCDD toxicity, had indicated that dioxin exposure reduced the abundance of numerous proteins which are regulated at the level of protein synthesis initiation. In the present study, we have analysed the mechanism mediating this inhibition. TCDD treatment of the cells largely prevented the activation of eukaryotic translation initiation factor 4E-binding protein 1, a regulator of translation initiation and substrate of the mammalian target of rapamycin (mTOR). By “working upwards” from mTOR, we observed that TCDD inhibited endogenous and IGF-I-induced AKT and ERK activation by interfering with tyrosine phosphorylation of insulin receptor substrate 1. This inhibition was mediated by a TCDD-induced secreted factor which was identified as insulin-like growth factor binding protein 4 (IGFBP-4). The induction of IGFBP-4 protein was dependent on a functional aryl hydrocarbon receptor and was preceded by a rapid increase in the level of IGFBP-4 mRNA indicating that IGFBP-4 is a previously unknown transcriptional target of TCDD in 5L cells. IGFBP-4 was not induced by TCDD in the parental cell line of 5L cells, Fao, and in various closely related rat hepatoma cell lines as well as in other unrelated cell types. Analysis of 5L cell chromosomes by multicolour spectral karyotyping (SKY) revealed that the cells carry several hitherto uncharacterised chromosomal translocations. The observations suggest that in 5L cells the Igfbp-4 gene may have got under the control of a promoter containing dioxin responsive element(s) leading to the induction of IGFBP-4 by TCDD. These findings emphasise a particular caution when interpreting and extrapolating results on the action mechanisms of TCDD obtained in studies using 5L cells as a model system.
- 1
To whom correspondence and reprint requests should be addressed. Fax: -49-7247-82-3354.