Atg5-mediated autophagy controls apoptosis/anoikis via p53/Rb pathway in naked mole-rat fibroblasts
Introduction
The naked mole-rat (NMR, Heterocephalus glaber) is a eusocial subterranean rodent, native to Africa [1]. NMRs are the longest-living rodent species, with a maximum lifespan of over 30 years [2]. While the body size of NMRs is similar to that of the house mouse (Mus musculus), NMRs live 10 times longer than the house mouse [3]. Furthermore, NMRs generally experience a greatly extended healthy lifespan within their total lifespan of 30 years [4]. These extraordinary animals also exhibit profound resistance to both spontaneous and experimentally induced cancer [[4], [5], [6]]. A previous study identified that NMR fibroblasts exhibit hypersensitive contact inhibition, termed early contact inhibition, which is regulated by the p16INK4a, p53, and Rb pathways [6]. Moreover, NMRs have increased levels of basal macroautophagy compared with mouse [7].
Macroautophagy (hereafter, autophagy) is the evolutionarily conserved pathway that degrades intracellular components, including aggregated protein, organelles, macromolecules, and invading pathogens via lysosomal degradation. Autophagy contributes to the maintenance of cellular homeostasis and fitness in both basal state and stressed state. Studies in C. elegans have suggested that autophagy is deeply implicated in animal aging [8] and lifespan extension [9]. In addition, brain-specific overexpression of Atg8a [10] and neuron-specific upregulation of Atg1 [11] activate autophagy and extend the lifespan of Drosophila. Atg5 overexpression in mice contributes to activation of autophagy and extension of the lifespan [12]. However, the molecular mechanisms underlying the high basal autophagy activity of NMRs and the physiological significance of this phenomenon remain to be elucidated.
In the present study, we identified that NMR skin fibroblasts (NSFs) expressed higher levels of the Atg12-Atg5 conjugate, a critical component for autophagosome formation, than did mouse skin fibroblasts (MSFs). Phenotypic analyses of Atg5 knockdown NSFs revealed that increased levels of the Atg12-Atg5 conjugate contributed to high levels of basal autophagy in NSFs. Furthermore, Atg5 knockdown in NSFs induced the accumulation of dysfunctional mitochondria, and suppressed cell proliferation and adhesion to substrates, enhancing induction of apoptosis/anoikis. However, inhibition of the pro-apoptotic p53/Rb pathway with SV40 large T antigen abolished the apoptotic phenotypes induced by Atg5 knockdown. These results suggest that high basal autophagy levels in NMR cells, mediated in part by Atg5, contribute to suppression of apoptosis by inhibiting activation of the pro-apoptotic p53/Rb pathway.
Section snippets
Reagents and antibodies
Anti-LC3b (D11) and anti-Atg5 (D5F5U) were purchased from Cell Signaling Technology (MA). anti-β-tubulin (H-235), anti-Actin (C-11), and anti-Atg7 (B-9) were from Santa Cruz Biotechnology (TX). Anti-Pcna (Ab-1) was purchased from Oncogene science (NY). Chloroquine and Hoechst33342 were purchased from Nacalai Tesque (Kyoto, Japan). Puromycin and Blasticidin S were purchased from InvivoGen (CA). Mitotracker Green FM and Mitotracker Orange CMTMRos were purchased from Invitrogen/Thermo Fisher
High basal autophagy in NSFs was associated with abundant Atg12-Atg5 conjugate
To compare basal autophagic activity among mouse NIH3T3 cells, MEFs, MSFs, and NSFs, we measured LC3b-II levels by immunoblotting [13]. LC3b-II accumulation is indicative of autophagic activity, when lysosomal degradation of autophagosomes is blocked by lysosomal inhibitors, such as CQ [14]. NSFs had substantially higher LC3b-II levels than mouse fibroblasts, particularly when treated with CQ (Fig. 1A). Flow cytometric analysis using DAPGreen [15] also revealed higher autophagic activity in
Discussion
In the present study, we found that the high basal autophagy activity in NMR fibroblasts was attributable to increased levels of the Atg12-Atg5 conjugate. To address the molecular mechanisms and physiological roles of basal Atg5-meidated autophagy in NMR cells, we examined the impact of Atg5 knockdown on the cellular phenotypes of NSFs. Atg5 knockdown induced various phenotypic changes, including robust suppression of autophagosome formation, accumulation of dysfunctional mitochondria, growth
Declaration of competing interest
The authors have no conflicts of interest to declare.
Acknowledgments
We thank Dr. Kyoko Miura (Kumamoto University) and Prof. Hideyuki Okano (Keio University School of Medicine) for kindly providing NSFs, and Prof. Noboru Mizushima (The Tokyo University) for kindly providing Atg5 KO MEFs. This work was supported by JPSP KAKENHI (Grant Numbers: 19H03504 and 19H04962).
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