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Summary:
Telomere shortening is associated with stem cell decline, fibrotic disorders and premature aging through mechanisms that are incompletely understood. Here, we show that telomere shortening in livers of telomerase knockout mice leads to a p53 - dependent repression of all seven sirtuins. P53 regulates non-mitochondrial sirtuins (Sirt1, 2, 6, & 7) post-transcriptionally through microRNAs (miR-34a, 26a & 145), while the mitochondrial sirtuins (Sirt3, 4 & 5) are regulated in a peroxisome proliferator-activated receptor gamma co-activator 1 alpha/beta - dependent manner at the transcriptional level. Administration of the NAD(+) precursor nicotinamide mononucleotide maintains telomere length, dampens the DNA damage response and p53, improves mitochondrial function and, functionally, rescues liver fibrosis in a partially Sirt1-dependent manner. These studies establish sirtuins as downstream targets of dysfunctional telomeres and suggest that increasing Sirt1 activity alone or in combination with other sirtuins stabilizes telomeres and mitigates telomere-dependent disorders.
eTOC
Telomere dysfunction is implicated in the promotion of tissue damage and fibrosis through mechanisms that are incompletely understood. Amano et al. show that telomere dysfunction in liver tissue downregulates sirtuins through p53 dependent mechanisms. Increasing NAD(+) stabilizes telomeres, dampens DNA damage response, and improves telomere-dependent fibrosis in a partially Sirt1-dependent manner.
Introduction
Telomeres, the repetitive ends of chromosomes, consist of double-stranded TTAGGG repeats that are coated with a specialized protein complex known as shelterin that plays a fundamental role in the regulation of telomere length and protection. Telomeres are maintained by a specialized reverse transcriptase, telomerase, which is repressed in the majority of human cells except in stem and progenitor cells and a small subset of other cells. Evidence for a role of telomerase and telomere length in human disease and aging comes from studies in patients with telomerase mutations and telomerase knock-out mice. Cumulatively, these studies have demonstrated that telomere shortening compromises the regenerative capacity of stem/ progenitor cells in highly proliferative tissues such as the hematopoietic system, intestine and skin. Another well-recognized pathological consequence of compromised telomeres is the increased risk of tissue fibrosis with liver and lung most commonly affected. In patients with the telomere-maintenance disorder Dyskeratosis congenita (DKC) approximately 20% and 7% develop lung and liver fibrosis respectively, although the factors that trigger and maintain fibrosis are not well understood(Armanios and Blackburn, 2012). However, following irradiation and cytotoxic treatment, DKC patients are at increased risk to develop secondary, treatment-associated complications including liver and lung fibrosis indicating that increased susceptibility to DNA damage is a risk factor for the development of fibrotic disorders. Clinical trials are underway to protect patients from long-term complications of treatment-associated DNA damage(Savage and Alter, 2009).
Liver disease is recognized to occur with higher frequency in patients with segmental telomere disorders such as aplastic anemia and bone marrow failure. Telomere shortening is also a hallmark of long standing liver disease due to acquired causes such as Hepatitis B/C viral infection or alcohol consumption, presumably as a consequence of insufficient activity of telomerase in proliferating hepatocytes. The accumulation of hepatocytes with critically short telomeres is associated with disease progression leading to liver cirrhosis, liver failure and elevated cancer risk(Hartmann et al., 2011). These human studies are experimentally supported by studies in telomerase knockout mice (TKO) with short telomeres, which are highly susceptible to liver cirrhosis after exposure to the DNA damage - inducing agent carbon-tetra chloride (CCL4)(Rudolph et al., 2000). TKO mice display increased susceptibility to DNA damage and cell death when challenged with CCL4 leading to increased tissue damage and, coupled with decreased proliferative capacity of surviving hepatocytes, to liver fibrosis(Rudolph et al., 2000). Currently, no therapies exist to prevent or treat telomere-associated liver fibrosis except for organ transplantation in patients with end-stage organ failure(Calado and Young, 2009). This lack of disease-modifying therapies is in part due to an incomplete understanding of the mechanisms that are operative downstream of dysfunctional telomeres and induce tissue compromise and, ultimately, liver failure. However, it has been demonstrated that the activation of DNA damage response pathway and its central transducer p53 promotes degenerative disorders by activating genes that induce growth arrest, apoptosis and senescence(Chin et al., 1999). This is indicated by studies in humans with liver cirrhosis in which decreased proliferation and accumulation of senescent hepatocytes is a common feature and tracks with degree of telomere dysfunction. This view is also supported by studies in mouse models where deletion of p53 curbs apoptosis/senescence and improves regenerative capacity across different tissues(Chin et al., 1999).
Recent studies have suggested a novel mechanism through which telomere dysfunction and p53 drive degenerative disorders by impacting cellular metabolism via a p53-dependent repression of peroxisome proliferator-activated receptor gamma co-activator 1 alpha/beta (PGC-1α/β), co-activators that drive mitochondrial biogenesis and function. In line with the concomitant repression of PGC-1α and PGC-1β, mitochondrial number and oxidative phosphorylation capacity is impaired in tissues with short telomeres resulting in decreased ATP synthesis, elevated ROS levels and decreased metabolic capacity including impaired gluconeogenesis(Sahin et al., 2011). Mitochondrial biogenesis and function is more severely compromised when telomere attrition is accelerated by predisposing pathological conditions such as muscular dystrophy leading to severe cardiomyopathy(Chang et al., 2016). While these reports point to the importance of dysregulated metabolism as a functionally relevant mechanism downstream of telomeres, several open questions regarding this telomere-metabolism link remain to be addressed. Importantly, the molecular mechanisms by which short telomeres regulate metabolism are yet to be fully defined as restoration PGC-1α in telomerase knockout mice with short telomeres only partially rescues the metabolic defect(Sahin et al., 2011). This partial rescue indicates the existence of other metabolic pathways that are altered by dysfunctional telomeres.
Here we investigated the role of sirtuins in telomere-dependent liver disease. Sirtuins are a class of NAD(+)-dependent enzymes that impact different cellular processes including transcriptional silencing, DNA recombination and repair, apoptosis and cellular metabolism through deacetylation and other posttranslational modifications of multiple downstream targets. Sirtuins are highly implicated in metabolic, ageing and age-related disorders. In the liver, Sirt1 has been particularly well studied among the seven sirtuins and has been shown to play an important role in diverse metabolic processes as well as implicated in the development of liver disease(Houtkooper et al., 2012). Increasing the activity of Sirt1 through overexpression or use of small molecules protects against fatty liver disease and improves insulin resistance induced by a high fat diet, while lack of Sirt1 in the liver accelerates hepatic steatosis and insulin resistance and is associated with inflammation and oxidative stress. The protective effects of Sirt1 have been suggested to be in large part due to increased mitochondrial function and biogenesis and improved metabolic function(Houtkooper et al., 2012). Another strategy to activate sirtuins is rooted in the observation that increasing NAD(+) levels can increase the activity of several sirtuins. An increase of NAD(+) has been achieved through the inhibition of major NAD(+) consuming enzymes such as PARP-1, CD38 or direct dietary supplementation with NAD(+) precursors such as nicotinamide mononucleotide (NNM) or nicotinamide riboside (NR). Increasing NAD(+) levels has been shown to protect from high fat diet-induced liver disease, insulin resistance and improve liver regeneration. The amelioration of liver disease has been linked to the improvement of mitochondrial biogenesis and function as well as activation of the mitochondrial unfolded stress response(Lagouge et al., 2006, Mouchiroud et al., 2013).
While both telomeres and sirtuins are independently implicated in aging and disease, how they are interconnected in driving disorders is not well understood, although previous studies in different model systems have demonstrated that both are tightly linked. In yeast, Sir2, the homolog of mammalian Sirt1, binds to telomeres and is required for the establishment and maintenance of silent chromatin formation at telomeres(Gottschling et al., 1990). This Sir2 – telomere interaction is dynamic as Sir2 and other Sir proteins redistribute from telomeres to other loci during yeast aging and in response to DNA damage(Guarente, 2000). The translocation of Sir2 from telomeres to DNA break sites is dependent on the DNA damage-checkpoint response(Martin et al., 1999, Mills et al., 1999). This re-localization is thought to promote DNA repair by different mechanisms including de-repression of genes involved in DNA repair and direct chromatin modifications at DNA breaks through the recruitment of repair factors(Guarente, 2000). In mice, a similar Sirt1 redistribution in response to DNA damage has been reported and thought to be important in the aging process(Oberdoerffer et al., 2008). Besides Sirt1, Sirt6 has also been shown to bind to telomeres and contribute to telomere integrity as evidenced by the accumulation of DNA damage foci at telomeres and rampant genomic instability with emergence of chromosomal end-to-end fusions after loss of Sirt6(Tennen and Chua, 2011). While these studies point to an important interplay between telomeres and sirtuins, how telomere dysfunction, in turn, impacts sirtuins, the molecular pathways that link telomeres to sirtuins and the relevance of sirtuins for telomere-dependent disease remain to be defined. Here, we find that telomere dysfunction induces the repression of all seven sirtuins in liver tissue of TKO mice, which is accompanied by hyperacetylation of many sirtuin targets including transcription factors, histones, mitochondrial proteins and metabolic enzymes. We demonstrate that the observed downregulation of sirtuins in the context of dysfunctional telomeres is p53-dependent as genetic ablation of p53 in TKO mice normalizes sirtuin expression and acetylation levels of their targets. We further demonstrate that telomere-induced sirtuin repression is functionally relevant as increasing sirtuin activity with the NAD(+) precursor NMN ameliorates telomere-dependent liver cirrhosis in a partially Sirt1- dependent manner.
Results
To assess the impact of telomere dysfunction on sirtuins we made use of mice lacking the reverse transcriptase component of telomerase, TERT. TERT knockout mice display no overt phenotype but develop multi-system premature aging when telomeres become progressively shorter in successive generations (G1 - G4). Unless otherwise stated, male G4 mice (“G4”) between 8-16 weeks of age were used in these studies. G4 mice display hallmarks of telomere dysfunction including stem cell compromise, regenerative defects in high turnover tissues, tissue atrophy, cardiomyopathy and shortened lifespan as reported previously(Sahin et al., 2011). To probe the relationship between telomere dysfunction and sirtuin expression, we determined sirtuin protein levels in liver tissue where both telomere dysfunction and sirtuin repression are implicated in organ pathology. Compared to age- and sex-matched wild type (WT) mice, all seven sirtuin members are down-regulated in G4 liver tissue (Fig. 1a; 9 mice per group analyzed; shown are 3 representatives per group; p <0.05). This sirtuin repression is dependent on the degree of telomere dysfunction as G1 and G2 mice with intermediate telomere length display attenuated Sirtuin repression compared to G4 mice (data not shown). We assessed the acetylation of Sirt1 (p53, PGC-1α, FOXO1), Sirt2 (H3K56, H4K16), Sirt3 (SOD2, majority of mitochondrial proteins), Sirt5 (succinylation of mitochondrial proteins), Sirt6 (H3K9 and H3K56) and Sirt7 (H3K18) targets. Combined immunoprecipitation-western blotting demonstrates pronounced hyperacetylation and succinylation of these sirtuin targets in G4 whole tissue lysates or lysates from isolated mitochondria (Fig. 1b and Supp. Fig. 1a and data not shown; 9 mice per group; p <0.05). Sirtuin expression in G4 mouse embryonic fibroblasts (MEFs) is also significantly reduced and several sirtuin targets (p53, FOXO1, SOD2 and CPS1) are hyperacetylated indicating that the observed repression of sirtuins upon telomere dysfunction is cell-autonomous (Supp. Fig. 1b, c; 3 different MEF cell lines were analyzed per group; p <0.05). Telomerase reintroduction in G4 mice (Fig. 1c and and1d;1d; n= 9) and G4 MEFs (Supp. Fig. 1d, e; two independent cell lines; p <0.05) normalizes sirtuin expression and acetylation levels of sirtuin targets in liver tissue and MEFs while telomerase overexpression in WT mice and WT MEFs has no effect on sirtuin expression and acetylation levels of their targets (Supp. Fig. 1d and data not shown).
Fig. 1
Telomere dysfunction leads to sirtuin repression and hyperacetylation of sirtuin targets
(a) Western blot demonstrates that Sirt1-7 are significantly down-regulated in G4 liver tissue (9 mice per group analyzed; shown are 3 representative mice per group); (b) IP-western blot analysis shows acetylation of targets of Sirt1 (p53, Foxo1), Sirt2 (H3K56, H4K16) Sirt3 (mitochondrial protein acetylation), Sirt5 (mitochondrial protein succinylation), Sirt6 (H3K9 and H3K56) and Sirt7 (H3K18) are increased in G4 liver tissue (shown are 3 representative results per group; a total of 9 mice per group were analyzed); © Western blot analysis of liver tissue from G4 mice infected with adenovirus expressing either telomerase (“Tert”) or GFP control shows that reactivation of telomerase increases sirtuin protein levels in G4 liver tissues (shown are 3 representatives per group; a total of 9 mice per group were analyzed); (d) telomerase reactivation decreases acetylation levels of Sirt targets compared to GFP-Adenovirus control group (9 mice per group were analyzed); Results are quantified by densitometry and expressed as mean ± s.e.m.; t-test was used to determine statistical significance with p <0.05 considered as significant, as indicated by (*).
To gain mechanistic insight into how telomere dysfunction leads to the down-regulation of all sirtuin members, we focused on the DNA damage response pathway and p53, which plays a pivotal role in inducing transcriptional changes, metabolic alterations and cellular regeneration upon telomere dysfunction(Sahin et al., 2011, Chin et al., 1999). We analyzed WT, p53 deficient (p53 −/−) and G4 mice either proficient or deficient for p53 (G4/p53 +/+ and G4/p53 −/−). RT-qPCR analysis of liver tissue demonstrates that telomere dysfunction leads to decreased mRNA abundance for mitochondrial sirtuins in G4/p53 +/+ mice, which is reversed when p53 is deleted in G4 mice (compare G4/p53 +/+ with G4/p53 −/− mice, Fig. 2a; n = 8 mice per group; p <0.05). In contrast to the mitochondrial sirtuins, the mRNA abundance of non-mitochondrial sirtuins is not impacted by either telomere dysfunction or p53 status in G4/p53 +/+ mice (Fig. 2a, n = 8 mice per group). At the protein level, the expression of all sirtuins is significantly increased in G4/p53 −/− compared to G4/p53 +/+ mice (Fig. 2b, 8 mice per group; p <0.05). The increase in sirtuin expression in G4/p53 −/− mice is accompanied by a decreased acetylation of sirtuin targets, including FOXO1, PGC-1α, SOD2, CPS1 and liver mitochondrial proteins (Fig. 2c, ,d;d; 6 mice per group analyzed; p <0.05).
Figure 2p53 Regulates Sirtuins in Telomere Dysfunctional Mice
(A) qRT-PCR analysis of sirtuin transcripts in WT, p53−/−, G4/p53+/+, and G4/p53−/− liver tissue demonstrates that mitochondrial sirtuins (Sirt3, 4, and 5) are repressed in G4/p53+/+ mice and p53 deficiency in G4 mice rescues their expression (9 mice per group analyzed).
(B) Western blot analysis using total liver tissue or isolated liver mitochondria shows elevated sirtuin protein expression in G4/p53−/− mice compared to G4/p53+/+ mice (9 mice per group were analyzed).
© Combined IP-western blot analysis of liver tissues derived from WT, p53−/−, G4/p53+/+, and G4/p53−/− mice demonstrates decreased acetylation levels of PGC-1α, FOXO1, SOD2, and CPS1 in G4/p53−/− compared to G4/p53+/+ mice (6 mice per group were analyzed).
(D) Analysis of acetylation levels of mitochondrial proteins from WT, p53−/−, G4/p53+/+, and G4/p53−/− mice shows that G4/p53−/− have decreased acetylation compared to G4/p53+/+ mice (6 mice per group were analyzed). Results are quantified by densitometry and expressed as mean ± SEM; t test was used to determine statistical significance, with ∗p < 0.05 considered as significant.
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Edited by Engadin, 09 July 2020 - 07:52 PM.
