The yin and yang of the Cdkn2a locus in senescence and aging

Abstract

Senescence of cultured cells involves activation of the p19(Arf)-p53 and the p16(Ink4a)-Rb tumor suppressor pathways. This, together with the observation that p19(Arf) and p16(Ink4a) expression increases with age in many tissues of humans and rodents, led to the speculation that these pathways drive in vivo senescence and natural aging. However, it has been difficult to test this hypothesis using a mammalian model system because inactivation of either of these pathways results in early death from tumors. One approach to bypass this problem would be to inactivate these pathways in a murine segmental progeria model such as mice that express low amounts of the mitotic checkpoint protein BubR1 (BubR1 hypomorphic mice). These mice have a five-fold reduced lifespan and develop a variety of early-aging associated phenotypes including cachetic dwarfism, skeletal muscle degeneration, cataracts, arterial stiffening, (subcutaneous) fat loss, reduced stress tolerance and impaired wound healing. Importantly, BubR1 hypomorphism elevates both p16(Ink4a) and p19(Arf) expression in skeletal muscle and fat. Inactivation of p16(Ink4a) in BubR1 mutant mice delays both cellular senescence and aging specifically in these tissues. Surprisingly, however, inactivation of p19(Arf) has the opposite effect; it exacerbates in vivo senescence and aging in skeletal muscle and fat. These mouse studies suggest that p16(Ink4a) is indeed an effector of aging and in vivo senescence, but p19(Arf) an attenuator. Thus, the role of the p19(Arf)-p53 pathway in aging and in vivo senescence seems far more complex than previously anticipated.

Figure 1

Senescence controlled by the p16^Ink4a-Rb and p19^Arf-p53 pathways. Various intrinsic and extrinsic stress-inducing signals are able to engage the tumor suppressor pathways containing p53, p16^Ink4a-Rb or both. Activation of p53 triggers cell death by inducing expression of a number of downstream target genes implicated in apoptosis (e.g., Bim, Puma, Noxa, Bid, Bax and Apaf1) or a transient or permanent (though reversible) cell cycle arrest via induction of p21. On the other hand, Rb is capable of inducing an irreversible state of cellular senescence via repression of E2F target genes and alterations in chromatin structure.

Figure 2

Summary of tissues affected with age-related pathologies in BubR1 hypomorphic mice. Shown is a 5-month-old BubR1 insufficient animal. Highlighted are several of the progeroid features commonly observed in this model.

Figure 3

The Cdkn2a locus and aging. (A) In young mice, the amount of p16^Ink4a and p19^Arf is quite low and in balance. (B) As animals age, p16^Ink4a and p19^Arf accumulate in the same tissue, with p19^Arf attempting to counter-balance the pro-aging effects of p16^Ink4a. With time, the balance shifts towards a state of aging. (C) In scenarios where p19^Arf is not present to counteract p16^Ink4a, the absolute levels of p16^Ink4a increase dramatically, further promoting a pro-aging microenvironment. (D) When p16^Ink4a is absent, p19^Arf does not accumulate. Aging is delayed and the induction of p19^Arf is not required.

Figure 4

Working model for regulation of aging by the p19^Arf-p53 pathway. Various stresses engage and stabilize p53. Acute situations mobilize an intense p53 response, leading to apoptosis or cellular senescence and the establishment of early aging. Chronic/low amounts of stress on the other hand, might activate p53 to provide a transient cell cycle arrest that provides an opportunity to repair damage or relieve cellular stress, thereby improving cell function in the presence of stress. Stress encountered with natural aging might engage a response similar to that of chronic stress.

Figure 5

BubR1 deficient cells have normal DNA damage responses. (A) Passage 5 mouse embryonic fibroblasts (MEFs) of the indicated genotypes were treated with doxorubicin (Dox) for the indicated times as described. Cell lysates were generated and western blotting was performed as described. Blots were probed for BubR1, Parp-1 and γH2AX. Position of p89, the major degradation product of Parp-1 is indicated. Antibodies for BubR1, Parp-1, and γH2AX were as described. Actin was used as a loading control. These results suggest an intact response to doxorubicin-induced damage in MEFs expressing low amounts of BubR1. (B) Additional DNA damage-induced cell death analysis in BubR1 hypomorphic cells. Passage 5 MEFs were treated with actinomycin D (Act D), or a combination of Act D and anti-Fas or TNFα and compared to non-treated MEFs as described. These blots suggest that signals that activate apoptosis are interpreted similarly in wildtype and BubR1 hypomorphic MEFs. (C) Comet assay results from 5-month-old tissues of wildtype and BubR1 hypomorphic mice. Comet assays were performed according to manufacturer’s protocol (Trevigen). In this assay, both the percentage of nuclei demonstrating DNA damage as well as the amount of damage reflected by tail length, are equal irrespective of genotype, suggesting that BubR1 hypomorphic mice exhibit a normal DNA damage response to in vivo stresses. γ-irradiation was used as a positive control in the Comet assays.