Deficiency in the DNA repair protein ERCC1 triggers a link between senescence and apoptosis in human fibroblasts and mouse skin

ERCC1 (excision repair cross complementing‐group 1) is a mammalian endonuclease that incises the damaged strand of DNA during nucleotide excision repair and interstrand cross‐link repair. Ercc1^?/Δ mice, carrying one null and one hypomorphic Ercc1 allele, have been widely used to study aging due to accelerated aging phenotypes in numerous organs and their shortened lifespan. Ercc1^?/Δ mice display combined features of human progeroid and cancer‐prone syndromes. Although several studies report cellular senescence and apoptosis associated with the premature aging of Ercc1^?/Δ mice, the link between these two processes and their physiological relevance in the phenotypes of Ercc1^?/Δ mice are incompletely understood. Here, we show that ERCC1 depletion, both in cultured human fibroblasts and the skin of Ercc1^?/Δ mice, initially induces cellular senescence and, importantly, increased expression of several SASP (senescence‐associated secretory phenotype) factors. Cellular senescence induced by ERCC1 deficiency was dependent on activity of the p53 tumor‐suppressor protein. In turn, TNFα secreted by senescent cells induced apoptosis, not only in neighboring ERCC1‐deficient nonsenescent cells, but also cell autonomously in the senescent cells themselves. In addition, expression of the stem cell markers p63 and Lgr6 was significantly decreased in Ercc1^?/Δ mouse skin, where the apoptotic cells are localized, compared to age‐matched wild‐type skin, possibly due to the apoptosis of stem cells. These data suggest that ERCC1‐depleted cells become susceptible to apoptosis via TNFα secreted from neighboring senescent cells. We speculate that parts of the premature aging phenotypes and shortened health‐ or lifespan may be due to stem cell depletion through apoptosis promoted by senescent cells.     

Age‐related dystrophic changes in corneal endothelium from DNA repair–deficient mice

The corneal endothelium (CE) is a single layer of cells lining the posterior face of the cornea providing metabolic functions essential for maintenance of corneal transparency. Adult CE cells lack regenerative potential, and the number of CE cells decreases throughout life. To determine whether endogenous DNA damage contributes to the age‐related spontaneous loss of CE, we characterized CE in Ercc1^?/Δ mice, which have impaired capacity to repair DNA damage and age prematurely. Eyes from 4.5‐ to 6‐month‐old Ercc1^?/Δ mice, age‐matched wild‐type (WT) littermates, and old WT mice (24‐ to 34‐month‐old) were compared by spectral domain optical coherence tomography and corneal confocal microscopy. Histopathological changes in CE were further identified in paraffin tissue sections, whole‐mount immunostaining, and scanning electron and transmission electron microscopy. The CE of old WT mice displayed polymorphism and polymegathism, polyploidy, decreased cell density, increased cell size, increases in Descemet's thickness, and the presence of posterior projections originating from the CE toward the anterior chamber, similar to changes documented for aging human corneas. Similar changes were observed in young adult Ercc1^?/Δ mice CE, demonstrating spontaneous premature aging of the CE of these DNA repair–deficient mice. CD45⁺ immune cells were associated with the posterior surface of CE from Ercc1^?/Δ mice and the tissue expressed increased IL‐1α, Cxcl2, and TNFα, pro‐inflammatory proteins associated with senescence‐associated secretory phenotype. These data provide strong experimental evidence that DNA damage can promote aging of the CE and that Ercc1^?/Δ mice offer a rapid and accurate model to study CE pathogenesis and therapy.     

Soluble guanylate cyclase activator BAY 54–6544 improves vasomotor function and survival in an accelerated ageing mouse model

DNA damage is a causative factor in ageing of the vasculature and other organs. One of the most important vascular ageing features is reduced nitric oxide (NO)soluble guanylate cyclase (sGC)—cyclic guanosine monophosphate (cGMP) signaling. We hypothesized that the restoration of NO‐sGC‐cGMP signaling with an sGC activator (BAY 54–6544) may have beneficial effects on vascular ageing and premature death in DNA repair‐defective mice undergoing accelerated ageing. Eight weeks of treatment with a non‐pressor dosage of BAY 54–6544 restored the decreased in vivo microvascular cutaneous perfusion in progeroid Ercc1 ^∆/− mice to the level of wild‐type mice. In addition, BAY 54–6544 increased survival of Ercc1 ^∆/− mice. In isolated Ercc1 ^∆/− aorta, the decreased endothelium‐independent vasodilation was restored after chronic BAY 54–6544 treatment. Senescence markers p16 and p21, and markers of inflammation, including Ccl2, Il6 in aorta and liver, and circulating IL‐6 and TNF‐α were increased in Ercc1 ^∆/− , which was lowered by the treatment. Expression of antioxidant genes, including Cyb5r3 and Nqo1, was favorably changed by chronic BAY 54–6544 treatment. In summary, BAY 54–6544 treatment improved the vascular function and survival rates in mice with accelerated ageing, which may have implication in prolonging health span in progeria and normal ageing.     

Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice

Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (Vdr^ΔIEpC) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into Vdr^ΔIEpC mice to produce the Vdr^ΔIEpC/3A4 line. LCA was administered to control, transgenic-CYP3A4, Vdr^ΔIEpC, and Vdr^ΔIEpC/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the Vdr^ΔIEpC mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the Vdr^ΔIEpC/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity.     

Characterisation of Ercc1 deficiency in the liver and in conditional Ercc1-deficient primary hepatocytes in vitro

The ERCC1/XPF complex is responsible for incision at the 5' side of the lesion during nucleotide excision repair and is also involved in homologous recombination and interstrand cross-link repair. The aim of the current study was to set up a better model for examination of Ercc1 deficiency in the murine liver and to determine the DNA lesions responsible for the premature polyploidy observed. We used the Cre/lox system with an adenovirus carrying Cre recombinase to conditionally induce Ercc1 deficiency in murine hepatocytes in vitro. Increased levels of apoptosis were apparent in our Ercc1-deficient cultures, both spontaneously and after UV irradiation and oxidative DNA damage. Increased apoptosis was also observed in simple Ercc1-deficient livers and the time course of the development of polyploidy was characterised. Livers from simple Ercc1 knockout mice contained mitochondria with disrupted outer membranes. Lipid accumulation was observed in older Ercc1-deficient hepatocyte cultures and in young Ercc1-deficient and wild-type livers. Lipids disappeared from the wild-type livers with age, but persisted in Ercc1-deficient livers, suggesting that a reduced ability to repair oxidative DNA damage and a malfunction of oxidative pathways could be responsible for the Ercc1-deficient liver phenotype. Real-time RT-PCR was used to determine differences in expression of cell cycle regulation and survival genes between Ercc1-deficient and control livers. Higher mRNA levels of Igfbp2, a possible marker for polyploidy, and p21 were detected in Ercc1-deficient livers. The pro-apoptotic factor, Bax, showed increased levels of mRNA expression in young Ercc1-deficient livers. However, no elevation in the levels of reactive oxygen species, or of malondialdehyde DNA adducts, a product of oxidative DNA damage, were found in Ercc1-deficient liver and no elevated levels of genes involved in the oxidative damage response were seen.     

High levels of oxidatively generated DNA damage 8,5′-cyclo-2′-deoxyadenosine accumulate in the brain tissues of xeroderma pigmentosum group A gene-knockout mice

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, a pathway that eliminates a wide variety of helix-distorting DNA lesions, including ultraviolet-induced pyrimidine dimers. In addition to skin diseases in sun-exposed areas, approximately 25% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of an oxidatively generated type of DNA damage called purine 8,5′-cyclo-2′-deoxynucleoside (cyclopurine). However, that hypothesis has not been verified. In this study, we tested that hypothesis by using the XP group A gene-knockout (Xpa^−/−) mouse model. To quantify cyclopurine lesions in this model, we previously established an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (CdA-1) that specifically recognizes 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA). By optimizing conditions, we increased the ELISA sensitivity to a detection limit of ˜one cyclo-dA lesion/10⁶ nucleosides. The improved ELISA revealed that cyclo-dA lesions accumulate with age in the brain tissues of Xpa^−/− and of wild-type (wt) mice, but there were significantly more cyclo-dA lesions in Xpa^−/− mice than in wt mice at 6, 24 and 29 months of age. These findings are consistent with the long-standing hypothesis that the age-dependent accumulation of endogenous cyclopurine lesions in the brain may be critical for XP neurological abnormalities.     

Mitochondrial DNA Mutations in Mutator Mice Confer Respiration Defects and B-Cell Lymphoma Development

Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ⁰) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.     

Klotho lacks a vitamin D independent physiological role in glucose homeostasis, bone turnover, and steady-state PTH secretion in vivo

Apart from its function as co-receptor for fibroblast growth factor-23 (FGF23), Klotho is thought to regulate insulin signaling, intracellular oxidative stress, and parathyroid hormone (PTH) secretion in an FGF23 independent fashion. Here, we crossed Klotho deficient (Kl^−/−) mice with vitamin D receptor (VDR) mutant mice to examine further vitamin D independent functions of Klotho. All mice were fed a rescue diet enriched with calcium, phosphorus, and lactose to prevent hyperparathyroidism in VDR mutants, and were killed at 4 weeks of age after double fluorochrome labeling. Kl^−/− mice displayed hypercalcemia, hyperphosphatemia, dwarfism, organ atrophy, azotemia, pulmonary emphysema, and osteomalacia. In addition, glucose and insulin tolerance tests revealed hypoglycemia and profoundly increased peripheral insulin sensitivity in Kl^−/− mice. Compound mutants were normocalcemic and normophosphatemic, did not show premature aging or organ atrophy, and were phenocopies of VDR mutant mice in terms of body weight, bone mineral density, bone metabolism, serum calcium, serum phosphate, serum PTH, gene expression in parathyroid glands, as well as urinary calcium and phosphate excretion. Furthermore, ablation of vitamin D signaling in double mutants completely normalized glucose and insulin tolerance, indicating that Klotho has no vitamin D independent effects on insulin signaling. Histomorphometry of pancreas islets showed similar beta cell volume per body weight in all groups of animals. In conclusion, our findings cast doubt on a physiologically relevant vitamin D and Fgf23 independent function of Klotho in the regulation of glucose metabolism, bone turnover, and steady-state PTH secretion in vivo.     

Premature aging is associated with higher levels of 8‐oxoguanine and increased DNA damage in the Polg mutator mouse

Mitochondrial dysfunction plays an important role in the aging process. However, the mechanism by which this dysfunction causes aging is not fully understood. The accumulation of mutations in the mitochondrial genome (or “mtDNA”) has been proposed as a contributor. One compelling piece of evidence in support of this hypothesis comes from the Polg ^D257A/D257A mutator mouse (Polg ^mut/mut ). These mice express an error‐prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations, accelerated aging, and premature death. In this paper, we have used the Polg ^mut/mut model to investigate whether the age‐related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome. Our results show that mutator mouse has significantly higher levels of 8‐oxoguanine (8‐oxoGua) that are correlated with increased nuclear DNA (nDNA) strand breakage and oxidative nDNA damage, shorter average telomere length, and reduced mtDNA integrity. Based on these results, we propose a model whereby the increased level of reactive oxygen species (ROS) associated with the accumulation of mtDNA mutations in Polg ^mut/mut mice results in higher levels of 8‐oxoGua, which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening. These results suggest that mitochondrial play a central role in aging and may guide future research to develop potential therapeutics for mitigating aging process.     

Thyroid Hormone Signaling In Vivo Requires a Balance between Coactivators and Corepressors

Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1^−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1^−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1^−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR^ΔID/ΔID Src-1^−/− mice have normal TH and TSH levels and are triiodothryonine (T₃) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T₃ activation of key hepatic gene targets, NCoR^ΔID/ΔID Src-1^−/− mice reacquired hepatic T₃ sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR^ΔID/ΔID Src-1^−/− mice, suggesting that SRC-2 is responsible for T₃ sensitivity in the absence of NCoR1 and SRC-1. Thus, T₃ targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1^−/− mice through increased SRC-2 recruitment to T₃ target genes.     

Role of nucleotide excision repair deficiency in intestinal tumorigenesis in multiple intestinal neoplasia (Min) mice

Mice deficient in the Xeroderma pigmentosum group A (Xpa) gene are defective in nucleotide excision repair (NER) and highly susceptible to skin carcinogenesis after dermal exposure to UV light or chemicals. Min (multiple intestinal neoplasia) mice, heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli (Apc), develop intestinal tumors spontaneously and show additional intestinal tumors after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In this study, we investigated the impact of loss of XPA function on PhIP-induced intestinal tumorigenesis in F1 offspring of Min/+ (Apc^+/−) mice crossed with Xpa gene-deficient mice. Apc^+/− mice lacking both alleles of Xpa had higher susceptibility towards toxicity of PhIP, higher levels of PhIP–DNA adducts in the middle and distal small intestines, as well as in liver, and a higher number of small intestinal tumors at 11 weeks, compared with Apc^+/− mice with one or two intact Xpa alleles. Localization of tumors was not affected, being highest in middle and distal small intestines in all genotypes. At 11 weeks of age, the number of spontaneous intestinal tumors was not significantly increased by homozygous loss of Xpa, but untreated Apc^+/−/Xpa^−/− mice had significantly shorter life-spans than their XPA-proficient littermates. Heterozygous loss of Xpa did not affect any of the measured end points. In conclusion, the Xpa gene and the NER pathway are involved in repair of bulky PhIP–DNA adducts in the intestines and the liver, and most probably of DNA lesions leading to spontaneous intestinal tumors. These results confirm a role of the NER pathway also in protection against cancer in internal organs, additional to its well-known importance in protection against skin cancer. An effect of Apc^+/− on adduct levels, additional to that of Xpa^−/−, indicates that the truncated APC protein may affect a repair pathway other than NER.     

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1-/- mice

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.     

Different responses to DNA damage determine ageing differences between organs

Organs age differently, causing wide heterogeneity in multimorbidity, but underlying mechanisms are largely elusive. To investigate the basis of organ‐specific ageing, we utilized progeroid repair‐deficient Ercc1^{Δ /−} mouse mutants and systematically compared at the tissue, stem cell and organoid level two organs representing ageing extremes. Ercc1^{Δ /−} intestine shows hardly any accelerated ageing. Nevertheless, we found apoptosis and reduced numbers of intestinal stem cells (ISCs), but cell loss appears compensated by over‐proliferation. ISCs retain their organoid‐forming capacity, but organoids perform poorly in culture, compared with WT. Conversely, liver ages dramatically, even causing early death in Ercc1‐KO mice. Apoptosis, p21, polyploidization and proliferation of various (stem) cells were prominently elevated in Ercc1^{Δ /−} liver and stem cell populations were either largely unaffected (Sox9+), or expanding (Lgr5+), but were functionally exhausted in organoid formation and development in vitro. Paradoxically, while intestine displays less ageing, repair in WT ISCs appears inferior to liver as shown by enhanced sensitivity to various DNA‐damaging agents, and lower lesion removal. Our findings reveal organ‐specific anti‐ageing strategies. Intestine, with short lifespan limiting time for damage accumulation and repair, favours apoptosis of damaged cells relying on ISC plasticity. Liver with low renewal rates depends more on repair pathways specifically protecting the transcribed compartment of the genome to promote sustained functionality and cell preservation. As shown before, the hematopoietic system with intermediate self‐renewal mainly invokes replication‐linked mechanisms, apoptosis and senescence. Hence, organs employ different genome maintenance strategies, explaining heterogeneity in organ ageing and the segmental nature of DNA‐repair‐deficient progerias.     

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16^Ink4a and p21^Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1^?/? and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16^Ink4a and p21^Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1^?/? mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16^Ink4a and p21^Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21^Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1^?/Δ mice with a p16^Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1^?/? and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.     

Aryl Hydrocarbon Receptors in Osteoclast Lineage Cells Are a Negative Regulator of Bone Mass

Aryl hydrocarbon receptors (AhRs) play a critical role in various pathological and physiological processes. Although recent research has identified AhRs as a key contributor to bone metabolism following studies in systemic AhR knockout (KO) or transgenic mice, the cellular and molecular mechanism(s) in this process remain unclear. In this study, we explored the function of AhR in bone metabolism using AhR^{RANKΔOc/ΔOc} (RANK^Cre/+;AhR^flox/flox) mice. We observed enhanced bone mass together with decreased resorption in both male and female 12 and 24-week-old AhR^{RANKΔOc/ΔOc} mice. Control mice treated with 3-methylcholanthrene (3MC), an AhR agonist, exhibited decreased bone mass and increased bone resorption, whereas AhR^{CtskΔOc/ΔOc} (Ctsk^Cre/+;AhR^flox/flox) mice injected with 3MC appeared to have a normal bone phenotype. In vitro, bone marrow-derived macrophages (BMDMs) from AhR^{RANKΔOc/ΔOc} mice exhibited impaired osteoclastogenesis and repressed differentiation with downregulated expression of B lymphocyte-induced maturation protein 1 (Blimp1), and cytochrome P450 genes Cyp1b1 and Cyp1a2. Collectively, our results not only demonstrated that AhR in osteoclast lineage cells is a physiologically relevant regulator of bone resorption, but also highlighted the need for further studies on the skeletal actions of AhR inhibitors in osteoclast lineage cells commonly associated with bone diseases, especially diseases linked to environmental pollutants known to induce bone loss.