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. 2022 May 26;14(10):4281-4304.
doi: 10.18632/aging.204103. Epub 2022 May 26.

Aryl hydrocarbon receptor blocks aging-induced senescence in the liver and fibroblast cells

Ana Nacarino-Palma  1   2 , Eva M Rico-Leo  1   2 , Judith Campisi  3   4 , Arvind Ramanathan  3 , Francisco J González-Rico  1   2 , Claudia M Rejano-Gordillo  1   2 , Ana Ordiales-Talavero  1   2 , Jaime M Merino  1   2 , Pedro M Fernández-Salguero  1   2
Affiliations

Aryl hydrocarbon receptor blocks aging-induced senescence in the liver and fibroblast cells

Ana Nacarino-Palma et al. Aging (Albany NY). .

Abstract

Aging impairs organismal homeostasis leading to multiple pathologies. Yet, the mechanisms and molecular intermediates involved are largely unknown. Here, we report that aged aryl hydrocarbon receptor-null mice (AhR-/-) had exacerbated cellular senescence and more liver progenitor cells. Senescence-associated markers β-galactosidase (SA-β-Gal), p16Ink4a and p21Cip1 and genes encoding senescence-associated secretory phenotype (SASP) factors TNF and IL1 were overexpressed in aged AhR-/- livers. Chromatin immunoprecipitation showed that AhR binding to those gene promoters repressed their expression, thus adjusting physiological levels in AhR+/+ livers. MCP-2, MMP12 and FGF secreted by senescent cells were overproduced in aged AhR-null livers. Supporting the relationship between senescence and stemness, liver progenitor cells were overrepresented in AhR-/- mice, probably contributing to increased hepatocarcinoma burden. These AhR roles are not liver-specific since adult and embryonic AhR-null fibroblasts underwent senescence in culture, overexpressing SA-β-Gal, p16Ink4a and p21Cip1. Notably, depletion of senescent cells with the senolytic agent navitoclax restored expression of senescent markers in AhR-/- fibroblasts, whereas senescence induction by palbociclib induced an AhR-null-like phenotype in AhR+/+ fibroblasts. AhR levels were downregulated by senescence in mouse lungs but restored upon depletion of p16Ink4a-expressing senescent cells. Thus, AhR restricts age-induced senescence associated to a differentiated phenotype eventually inducing resistance to liver tumorigenesis.

Keywords: aryl hydrocarbon receptor; hepatocarcinogenesis; metabolism; senescence.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest related to this study.

Figures

Figure 1
Figure 1
AhR depletion increases liver tumorigenesis with aging. (A) Representative tumors developed by AhR+/+ and AhR−/− at the indicated ages. (B) Haematoxylin and Eosin staining of liver tumor sections from AhR+/+ and AhR−/− mice at 22 months of age. Note the abundance of pycnotic nuclei in AhR−/− tumors. (C) Quantification of the number of liver tumors in mice of both genotypes at two age intervals. (D) AhR mRNA levels in AhR+/+ livers at the indicated ages using RT-qPCR and the oligonucleotides indicated in Supplementary Table 1. (E) AhR protein levels were analyzed in liver extracts at the indicated ages by immunoblotting. β-Actin was used to normalize protein levels. (F) AhR+/+ mice were injected i.p. with 4 mg/kg FICZ and mRNA levels of the AhR canonical target gene Cyp1a1 were determined by RT-qPCR using the oligonucleotides indicated in Supplementary Table 1. (G) Liver progenitor cells were analyzed by FACS using antibodies against CD133-PE and EPCAM-APC. Distribution of cell subpopulations and gating from representative experiments are shown. (H) Bone marrow progenitor cells were analyzed by FACS using the markers CD44-FITC and Sca1-APC. Distribution of cell subpopulations and gating from representative experiments are shown. Gapdh was used to normalize target gene expression (△Ct) and 2−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (*P < 0.05; **P < 0.01).
Figure 2
Figure 2
Cell senescence increases with age in AhR-deficient liver. (A) SA-β-Gal activity was analyzed in AhR+/+ and AhR−/− liver sections by staining with the chromogenic substrate X-Gal. (B) SA-β-Gal activity was analyzed by FACS in isolated liver cells (gentleMACS) using the fluorescent substrate C12FDG. (C–F) mRNA levels of senescence driver genes p16Ink4a (C) and p21Cip1 (D) and SASP-related genes IL1 (E) and TNFα (F) were analyzed by RT-qPCR in AhR+/+ and AhR−/− livers at the indicated ages. Oligonucleotides used are indicated in Supplementary Table 1. Gapdh was used to normalize target gene expression (△Ct) and 2−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 3
Figure 3
AhR−/− livers overexpress senescence and undifferentiation/stemness markers. Protein levels of senescence markers p16Ink4 and p21Cip1 and pluripotency/stemness inducers NANOG and OCT4 were analyzed by immunofluorescence in liver sections of aged AhR+/+ and AhR−/− mice and in hepatocarcinomas from AhR−/− mice. Expression of α-SMA was also analyzed as indicator of vasculogenesis. Conjugated secondary antibodies labelled with Alexa 488, Alexa 550 and Alexa 633 were used for detection. DAPI staining was used to label cell nuclei. An Olympus FV1000 confocal microscope and the FV10 software (Olympus) were used for the analysis. Scale bar corresponds to 50 μm.
Figure 4
Figure 4
AhR modulates the senescence-associated secretory phenotype with aging. (A). Chromatin immunoprecipitation (ChIP) for AhR binding to XRE binding sites located in the promoters of p16Ink4a (A), p21Cip1 (B) and TNFα (C). qPCR was used to quantify changes in DNA binding and the results were normalized to the corresponding inputs. Amounts of MCP-2 (D), MMP12 (E), FGF (F) and HGF (G) were analyzed in liver homogenates from AhR+/+ and AhR−/− mice at the indicated ages. Levels of HGF (H) and VEGF (I) VEGF were also determined in sera from mice of the same genotypes and ages. Bio-Plex Multiplex immunoassays kits were used. Oligonucleotides for qPCR are indicated in Supplementary Table 1. Gapdh was used to normalize target gene expression (△Ct) and 2−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 5
Figure 5
Senescence increases with aging in adult AhR-null fibroblasts. (A) AhR+/+ and AhR−/− fibroblasts were stained with the SA-β-Gal fluorescent substrate C12FDG to determine senescence levels by confocal microscopy. (B) SA-β-Gal activity was also measured by flow cytometry analyzing the percentage of C12FDG positive cells. (C–E) p16Ink4a (C), p21Cip1 (D) and Cyclin E (E) were analyzed by florescence confocal microscopy using specific antibodies in young and aged fibroblast cells. Conjugated secondary antibodies used were Alexa 488 and Alexa 633. DAPI staining was used to label cell nuclei. An Olympus FV1000 confocal microscope with FV10 software (Olympus) were used. (F) Percentage in each cell cycle phase. Cell cycle analysis was performed by FACS using propidium iodide staining. (G) Mitochondrial membrane potential (MMP) was quantified by the percentage of TMRM positive cells analyzed by Cytoflex S cytometer (Beckman Coulter). (H) MMP was also calculated using the JC10 kit for mitochondrial membrane polarization (Sigma-Aldrich). The red/green fluorescence intensity ratio was used to determine MMP activity. Data are shown as mean + SD (*P < 0.05; **P < 0.01).
Figure 6
Figure 6
Lack of AhR enhances in vitro cellular senescence in mouse embryonic fibroblasts. (A) Senescence profiles of AhR+/+ and AhR−/− MEFs at the indicated passages as determined by the level of SA-β-gal activity analyzed by FACS using the β-galactosidase fluorescent substrate C12FDG. Results are normalized to wildtype MEFs at passage 2. (B) Representative flow cytometric profiles of senescent cells stained with C12FDG in AhR+/+ and AhR−/− MEFs at passage 5. (C) SA-β-Gal activity in AhR+/+ and AhR−/− MEFs at passage 5 as determined by staining using X-Gal as substrate. (D) AhR mRNA expression was determined by RT-qPCR at the indicated cell culture passages. (E–G) mRNA expression of senescence driver genes p16Ink4a (E), p21Cip1 (F) and Trp53 (G) were determined in AhR+/+ and AhR−/− MEFs by RT-qPCR using the oligonucleotides indicated in Supplementary Table 1. Gapdh was used to normalize target gene expression (△Ct) and 2−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (**P < 0.01; ***P < 0.001).
Figure 7
Figure 7
Senolytic agent Navitoclax restores wild-type mRNA levels of senescence driver genes in AhR−/− MEFs. Embryonic fibroblasts at P4 or P5 were treated with vehicle or 10 μM Navitoclax for 48 h. (A) Bright-field microscopy of AhR+/+ and AhR−/− MEFs untreated or treated with Navitoclax. (B) Cell senescence measured as percentage of SA-β-Gal activity in MEF cells of both genotypes. SA-β-Gal activity was analyzed by FACS using the β-galactosidase fluorescent substrate C12FDG. Results are normalized to vehicle-treated wild-type MEFs. (C) X-Gal staining in untreated and Navitoclax-treated AhR+/+ and AhR−/− MEFs. (D) AhR mRNA expression was determined by RT-qPCR in both experimental conditions using the oligonucleotides indicated in Supplementary Table 1. (E–G) mRNA expression of senescence driver genes p16Ink4a (E), p21Cip1 (F) and Trp53 (G) was determined in AhR+/+ and AhR−/− MEFs by RT-qPCR using oligonucleotides indicated in Supplementary Table 1. Gapdh was used to normalize target gene expression (△Ct) and 2−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 8
Figure 8
CDK4/6 inhibitor Palbociclib induces senescence in MEF cells and mimicks AhR deficiency. Embryonic fibroblasts were cultured for 48 h, then plated at 4 x 105 cells per 10-cm plate. Two days later, cells were treated with 4 μM CDK4/6 inhibitor Palbociclib/PD-0332991 for 8 days. (A) Bright-field microscopy of AhR+/+ and AhR−/− MEFs untreated or treated with Palbociclib. (B) Cell senescence measured as SA-β-Gal activity in AhR+/+ and AhR−/− MEFs. SA-β-Gal activity was analyzed by FACS using the β-galactosidase fluorescent substrate C12FDG. Results are normalized to vehicle-treated wild-type MEFs. (C) X-gal staining in untreated and Palbociclib treated AhR+/+ and AhR−/− MEFs. (D) AhR mRNA level was determined by RT-qPCR in AhR+/+ MEFs under both experimental conditions using oligonucleotides indicated in Supplementary Table 1. Determinations were done after 8 days of treatment with Palbociclib or vehicle (control). (E–G) mRNA levels of senescence driver genes p16Ink4a (E), p21Cip1 (F) and Trp53 (G) was determined in AhR+/+ and AhR−/− MEFs by RT-qPCR using oligonucleotides indicated in Supplementary Table 1. Gapdh was used to normalize target gene expression (△Ct) and 2−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 9
Figure 9
AhR expression varies with the eradication of senescence in vivo in p16Ink4a-3MR transgenic mice. (A) Schematic representation of the construct used to generate the p16-3MR transgenic mice to deplete senescent cells in vivo by ganciclovir treatment. p16-3MR mice at 4–7 months of age were treated with doxorubicin (DOXO), doxorubicin + ganciclovir (DOXO+GCV) or vehicle PBS (vehicle). mRNA expression of AhR (B), p16Ink4a (C), p21Cip1 (D), p53 (E) and Mmp3 (F) was determined by RT-qPCR in lung tissue using the oligonucleotides indicated in Supplementary Table 1. Gapdh was used to normalize target gene expression (△Ct) and 2−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (*P < 0.05; **P < 0.01).
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