Limitations in stem cell number or function have been proposed to restrict longevity. However, we show here that in WT mice, stem cells decline in function, when measured per HSC, with a concomitant increase in their number, resulting in a minimal net change in overall HSC activity, strongly suggesting that stem cells are not likely to be a factor limiting hematopoietic regeneration with age. However, their functional deficits do show that HSCs are impacted by the forces of aging in a manner similar to that of differentiated cells [31
In our molecular analysis, we identified global age-related changes in gene expression in murine HSCs, with a view to identifying mechanisms that could be responsible for these age-associated declines in HSC function. Genes involved in inflammatory and stress responses dominated the group of up-regulated genes, whereas those participating in chromatin regulation and DNA repair were prominent among down-regulated genes.
Many of the observed expression changes are corroborated by a recent study of HSC aging [4
]. Although the number of genes that go up or down with age was similar between our study and the one by Rossi et al. [4
], the precise overlap between the genes is modest: only 343 genes were differentially expressed in both lists. We believe this is primarily due to a different purification strategy and array analysis that was used in our study. Remarkably, however, the top ten genes in both lists of genes that were Up-with-Age as well as those that were Down-with-Age were identical, and the magnitude of the changes was striking. For example, P-selectin was found highly up-regulated with age, for a mean fold-change of approximately 60 between the two studies. Likewise, both studies observed the up-regulation of specific protooncogenes, such as Runx1,
that we believe could contribute to the increased incidence of myeloid leukemia with age.
In both studies, the inflammation markers P-selectin and clusterin (both regulated by NF-κB) and protein-folding genes such as Hspa8 and Dnajc3 were found up-regulated. Additionally, we found genes involved in DNA repair and chromatin maintenance (Xab2, Rad52, Polb, and Lmna) to be down-regulated in both studies, supporting the notion that HSC become epigenetically dysregulated.
The marked stress response exhibited by the HSC suggests that a proinflammatory microenvironment exists within the aging marrow. Studies of aging arteries, brain, and kidney have also observed up-regulation of inflammatory markers [33
], but could not distinguish between immune cell infiltration and intrinsic inflammatory response of the tissue. The up-regulation of TLR4 (Toll-like receptor 4)
() is especially intriguing because one of its principal signaling targets is NF-κB, which regulates a variety of genes involved in the inflammatory response, including prostaglandin E2 synthase (Cox-2) and the adhesion molecules ICAM1 and P-selectin [36
] (). This molecular profile resembles the elevated inflammatory state previously described in older mice (“inflamm-aging” [37
]); however, our mice were raised in specific-pathogen–free conditions, and thus the inflamm-aging can be independent of antigenic load. Strikingly, Inflammatory Response was one of the few GO categories found “older” in the p53+/m
mice compared to the p53+/−
mice. This correlates with the early-aging phenotype observed at the organismal level in p53+/m
mice, and suggests that inflammation is an intrinsic effect of age.
The up-regulated inflammatory genes in aging HSCs may have mechanistic implications for the decline of HSC function. The ability of HSCs to engraft in bone marrow is influenced by their homing properties; since cell adhesion plays a critical role in the engraftment of HSCs, aberrant up-regulation of genes encoding P-selectin and ICAM1 might affect the ability of older HSCs to home to the bone marrow, disrupting repopulation. Likewise, down-regulation of genes in the TGFβ signaling pathway such as SMAD4, endoglin,
and spectrin b2
(), may explain the increased number of HSCs, because the TGFβ pathway influences HSC pool size [38
Equally striking was the increased expression of clusterin (clu) and amyloid beta precursor protein (App) in aged HSCs (), because these proteins aggregate within plaques in the brains of Alzheimer's patients [39
]. Cathepsin B, involved in APP [40
] processing, also increased in aged HSCs (). Clusterin, containing chaperone homology, along with several chaperonin subunits (beta, gamma, zeta, eta, and theta) increased with age, as did stress-response genes Hsc70/Hspa8, HSP90/Hspca, Bip/Hspa5,
and several Hsp40
homologs (Dnaja1, Dnaja2, Dnajb6, Dnajb10,
( and S1
). These changes, as well as the approximately 6-fold enrichment in NO-mediated signaling genes (), may be in response to oxidative stress, a common feature of aged cells [41
]. Some of these genes were previously noted to increase with age in murine hearts and skeletal muscle, and to decrease in animals on dietary restriction [31
Although inflammatory and stress-response genes increased with age, genes that ensure transcriptional fidelity declined with age. For instance, lamin A
declined with age and is linked to normal human aging [42
] and Hutchinson-Gilford progeria [43
] (). Likewise, Bloom syndrome homolog (Blm)
declined, mutations in which cause genomic instability, hypermutability, and cancer predisposition [44
]. Multiple DNA repair genes, including Rad52, Xrcc1,
were also down-regulated. Because DNA damage is thought to be a driving factor in aging [45
], a blunted DNA damage response could retard DNA repair, increasing the risk of retaining mutations, leading to malignant transformation, one of the hallmarks of old age.
It was surprising that HSCs from 12-mo-old early-aging p53+/m
mice appeared molecularly younger than age-matched WT and p53+/−
HSC. This suggests that a genetically imposed lower rate of stem cell proliferation, as seen in the p53+/m
HSC, can reduce the apparent age of the HSC, despite their residence in an environment that exhibits other outward manifestations of aging [28
]. But this reduced proliferative capacity also results in poorer hematopoietic regeneration activity, when the stem cells are examined at the population level [28
]. In other words, lower HSC proliferation results in a more youthful stem cell, but poorer tissue regeneration, and consequently an aged phenotype; this indicates that stem cell proliferation and tissue regeneration are finely balanced to maximize longevity, so that cell cycle disruption results in an uncoupling of tissue and organismal aging from the aging of the resident stem cell.
Finally, three lines of evidence in our work indicate broad changes in epigenetic regulation with age. Several GO categories (A) and specific genes involved in transcriptional silencing via chromatin regulation are down-regulated with age, such as the SWI/SNF-related chromatin remodeling genes (Smarca4 and Smarcb1), as well as three histone deacetylases (Hdac1, -5, and -6) and a DNA methyltransferase (Dnmt3b). Because these changes largely occur mid-way through life (B), they could easily be envisioned to underlie inappropriate expression of additional genes. In addition, the CORE analysis revealed many chromosomal regions coordinately changing with age, and suggested an overall loss of transcriptional silencing. Finally, inappropriate transcription from the IgK locus, known to be driven by NF-κB activity following epigenetic modification enabling accessibility of the locus, was observed in old, but not young, HSCs ().
Together, these data suggest an epigenetic view of aging that readily explains how so many diverse effects of age are evident at molecular, cellular, and organismal levels, and contrasts with the assumption that accumulation of lesions in genomic DNA or mitochondria accounts for the major effects of aging. Chromatin dysregulation could be a primary force in aging; epigenetic changes in otherwise normal cells could drive the loss of overall cellular functionality, as well as lay a fertile ground for secondary genetic events that lead irreversibly to oncogenic transformation. In this model, inappropriate expression of protooncogenes, or down-regulation of tumor suppressors, could result in a pre-transformed state, similar to myelodysplastic syndrome, a notion corroborated by another study of aging in murine HSC [4
] in which Runx1, Pml,
and other protooncogenes were up-regulated with age. Likewise, the increased transcriptional accessibility of some loci may enable interactions between otherwise distant chromatin domains, enhancing the likelihood of chromosomal translocations. Of particular note, the IgK
locus that we show is transcriptionally active in aged HSC, is a well-established translocation partner with the Myc
protooncogene in the generation of hematopoietic malignancies [46
]. Moreover, IgK
alleles have been shown to be differentially located within the nucleus depending on their state of activation [47
], which could result in their juxtaposition to oncogenes, increasing the likelihood of translocation [48
]. The role of epigenetic changes in cancer formation is increasingly recognized [49
]; here, we suggest that chromatin dysregulation is a natural result of environmental insults with age, and a primary driver of secondary effects of age, including malignancy. A systems approach to the ways in which inflammation, stress response, and epigenetic regulation are linked may be essential to understanding aging and cancer.