Ink4a/Arf expression is a biomarker of aging

Abstract

The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16INK4a and Arf, which are principal mediators of cellular senescence. To study the links between senescence and aging in vivo, we examined Ink4a/Arf expression in rodent models of aging. We show that expression of p16INK4a and Arf markedly increases in almost all rodent tissues with advancing age, while there is little or no change in the expression of other related cell cycle inhibitors. The increase in expression is restricted to well-defined compartments within each organ studied and occurs in both epithelial and stromal cells of diverse lineages. The age-associated increase in expression of p16INK4a and Arf is attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlates with diminished expression of an in vivo marker of senescence, as well as decreased pathology of those organs. Last, the age-related increase in Ink4a/Arf expression can be independently attributed to the expression of Ets-1, a known p16INK4a transcriptional activator, as well as unknown Ink4a/Arf coregulatory molecules. These data suggest that expression of the Ink4a/Arf tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.

Figure 1

Expression of the Ink4a/Arf locus increases with aging. (A) Relative expression. The ratios (log₂ scale) of the expression of cell cycle inhibitors – old (26 months)/young (2.5 months) – from 15 tissues is graphed ± SEM. Each estimate represents the mean of 8–32 quantitative RT-PCR reactions on independent RNA samples derived from 4–6 mice. *Minimum estimate of old/young ratio. (B) Absolute expression. The absolute copy number of p16^INK4a and Arf mRNA molecules (log₁₀ scale) per 90 ng total RNA RT-PCR from 15 tissues of young (2.5 months) and old (26 months) mice is graphed ± SEM. Murine embryo fibroblasts (MEFs) at early (P4) and late (P7) passage are shown for comparison. ^#Maximum estimated expression is indicated, as expression was below the level of detection.

Figure 2

p16^INK4a expression in specific compartments by immunohistochemistry and cell purification. (A) Immunoperoxidase staining performed on paraffin-embedded sections of germ-line p16^INK4a-deficient (KO), WT young (3.5 months), and WT old (25 months) murine tissues using an anti-p16^INK4a antibody. Positively staining cells demonstrate both nuclear and cytoplasmic expression. GC, germinal center. (B) Relative expression ratios (old/young, log₂ scale) of p16^INK4a in specific compartments (average purity >94% for all fractions) of bone marrow (lin^–, 2%; lin⁺, 97%), spleen (B220⁺, 48%; Mac1⁺, 9%; B220^–Mac1^–, 22%), and lymph node. Asterisks indicate that p16^INK4a expression was undetectable in these cell populations from young mice, and therefore a minimum estimate of the fold increase is shown.

Figure 3

Effects of caloric restriction and GHR deficiency on gene expression and aging. (A) Relative expression ratios (old/young, log₂ scale) of cell cycle inhibitors in 7 tissues derived from old (28 months) and young (3 months) AL or CR F344 rats. The relative ratios are graphed ± SEM. Each estimate represents the mean of 8–16 quantitative RT-PCR reactions on independent RNA samples derived from 4 rats. (B) Immunoperoxidase staining on paraffin-embedded kidney sections from young, old AL, and old CR F344 rats using an anti-p16^INK4a antibody. G, glomeruli seen in cortical sections. (C) SA-β-gal staining in AL and CR mouse and rat kidney. C, renal cortex; M, renal medulla. Thin tissue slices were stained for mice, as opposed to small tissue wedges for rats. SA-β-gal activity is predominantly restricted to the renal cortex. (D) Relative expression ratios (old/young, log₂ scale) of Ets-1 in kidneys derived from AL and CR rats and mice. Results from the kidneys from AL and CR mice with and without GHR deficiency are also shown. Each estimate represents the mean of 8–32 quantitative RT-PCR reactions on independent RNA samples derived from 8 mice or 4 rats.

Figure 4

p16^INK4a expression with aging strongly correlates with Arf and Ets-1 expression. (A) A scatter plot (log₂ scale, both axes) of the ratios (old/young) of p16^INK4a expression versus the expression ratios (old/young) of Arf, Ets-1, and Id1 seen in the corresponding tissue (n = 22–70 data pairs per gene from up to 15 tissues in both mouse and rat). Each ratio represents a mean value of multiple measurements per tissue as described in Methods. A best-fit line determined by linear regression is shown for each data series, with Pearson correlation coefficient and 2-tailed P value. No significant correlation was seen between Arf and Ets-1, or between Arf or p16^INK4a and Bmi-1 (not shown). (B) Arrows show known or inferred transcriptional relationships, and numbers indicate the covariances (r²) for the linked elements as determined in A. As p16^INK4a and Arf do not regulate one another, it seems reasonable to assume that an unknown coregulator (X) modulates the expression of both transcripts with aging, explaining their strong correlation (r² = 48%). Furthermore, as Arf and Ets-1 do not covary, X and Ets-1 must be independent. X need not represent a single transcription factor: it may represent the combined activity of several genes (e.g., the PcG family members) or genes that affect other transcript properties, such as message stability. This model suggests that the majority (87%) of the variance in p16^INK4a expression with aging in the analyzed tissues can be attributed to the activity of X and Ets-1.