Cip Kip proteins are expressed at higher levels in HSCs

Cip/Kip proteins are expressed at higher levels in HSCs than in progenitor cells (Passegué et al., 2005). The role of p21Cip1 in HSCs is restricted to cell cycle regulation under stress conditions (van Os et al., 2007). p27Kip1 deficiency does not affect HSC numbers or HSC self-renewal, but alters the proliferation of progenitor cells (Cheng et al., 2000a). p57Kip2 is an important regulator of hematopoiesis in the zolmitriptan gonads mesonephros region, where HSCs emerge (Mascarenhas et al., 2009). Inducible loss of p57 in hematopoietic cells has demonstrated the critical role of this CDKI in the maintenance of HSC quiescence (Matsumoto et al., 2011). More recent studies have implicated INK4 members in the control of HSC functions. p16INK4a expression is repressed by EZH1 in young animals (Hidalgo et al., 2012). Its expression increases with age, contributing to the decreased self-renewal, homing, and repopulating activities of HSCs in response to stress (Janzen et al., 2006). However, the role of p16INK4a in regulating steady-state HSC aging in vivo appears to be less important (Attema et al., 2009). p18INK4c is also involved in the senescence of HSCs. In its absence, the number of cycling HSCs increases, although the overall self-renewal capacity of the HSC compartment remains unchanged (Yuan et al., 2006). In a sense, p18 deletion mimics HSC aging, and it may, paradoxically, have an opposite role to p16INK4a and p21Cip1. Prior evidence for the importance of p19INK4d in HSC cell cycle regulation was reported using the Thpo mouse model. These mice exhibit a significant decrease in HSC numbers that correlates with decreased expression of p19INK4d and p57Kip2 (Qian et al., 2007; Yoshihara et al., 2007). p19INK4d plays a role in the development of the cerebral cortex (Zindy et al., 1999), controls mouse spermatogenesis (Zindy et al., 2001), and is involved in macrophage differentiation (Adachi et al., 1997). We previously demonstrated that by linking endomitotic arrest and terminal maturation p19INK4d is implicated in megakaryopoiesis (Gilles et al., 2008). In addition to its role in cell cycle and differentiation, in neuroblastoma cells, p19INK4d is also important for DNA repair and resistance to apoptosis in response to diverse forms of genotoxic stress (Ceruti et al., 2009). Interestingly, sensory hair cells lacking p19INK4d aberrantly re-enter the cell cycle and subsequently undergo apoptosis. This supports the notion that p19INK4d is essential for maintenance of their postmitotic state (Chen et al., 2003) and that p19INK4d therefore acts as an antiapoptotic regulator. Although a number of studies suggest that p19INK4d is implicated in HSC biology, its precise role remains unclear. Using a p19 mouse model, we demonstrate that p19INK4d is involved in the regulation of HSC quiescence. Under conditions of genotoxic stress, the absence of p19INK4d leads to HSC apoptosis during the S/G2-M phases of the cell cycle. We also report that p19INK4d participates in control of the HSC microenvironment during aging, as its absence results in BM and spleen fibrosis, and loss of HSCs.
Results
Discussion We report here that the p19INK4d CDKI plays at least two important roles in regulation of the HSC pool. One of these is cell autonomous and manifests itself under conditions of stress. The second role relates to the microenvironment and underscores the role of p19INK4d in mediating age-related effects on the HSC pool.
Experimental Procedures
Acknowledgments
Introduction Embryonic stem (ES) cells derived from the inner cell mass of the blastocyst have been used to understand early embryonic development (Keller, 2005). The notable characteristic of ESCs is self-renewal that is critically involved in the stimulation of rapid proliferation. In fact, rapid proliferation might protect ESCs from external signals inducing differentiation (Ruiz et al., 2011). However, rapid proliferation would be harmful because it causes successive mitotic division with a long S phase in which DNA is replicating most of the time (Fluckiger et al., 2006; Savatier et al., 2002), and the successive rounds of DNA replication causes many replication errors that may lead to DNA damage (Strumberg et al., 2000; Tichy and Stambrook, 2008). In addition, there is no G1 checkpoint in ESCs (White and Dalton, 2005), which might exacerbate DNA damage during rounds of replication without enough time for repair (Hong and Stambrook, 2004). Consistent with these characteristics, there is a high level of double-strand break (DSB) damage, which is the most toxic type of DNA damage (Valerie and Povirk, 2003), and DSB damage is indicated by the γ-H2AX marker (H2AX becomes phosphorylated on serine 139) in both human ES (hES) cells and mouse ES (mES) cells (Banáth et al., 2009; Chuykin et al., 2008; Momcilovic et al., 2010). Similar to irradiated fibroblast cells, normal mESCs also contain a high frequency of single-strand break (SSB) (Chuykin et al., 2008). However, ESCs still have an integrated genome and stable pluripotency during rapid proliferation (Tichy and Stambrook, 2008; Wang et al., 2008). Additionally, the mutation frequency and mitotic recombination frequency are lower in mESCs than in adult somatic or isogenic mouse embryonic fibroblasts cells (Tichy and Stambrook, 2008). Thus, ESCs must have unique regulatory mechanisms that counteract DNA damage both quickly and efficiently.