Breaking the Cell Cycle of HSCs by p57 and Friends
2011; Elsevier BV; Volume: 9; Issue: 3 Linguagem: Inglês
10.1016/j.stem.2011.08.005
ISSN1934-5909
AutoresMelania Tesio, Andreas Trumpp,
Tópico(s)Acute Myeloid Leukemia Research
ResumoThe cell cycle regulators involved in maintaining the quiescence, and thereby the self-renewal capacity, of somatic stem cells have long been elusive. Two new Cell Stem Cell articles in this issue (Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Zou et al., 2011Zou P. Arai F. Yoshihara H. Tai I. Hosokawa K. Matsumoto Y. Shinmyozu K. Tsukahara F. Maru Y. Nakayama K. et al.p57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70.Cell Stem Cell. 2011; 9 (this issue): 247-261Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) now show that the CDK inhibitor p57 is a crucial brake for cycling HSCs, and links self-renewal activity to cell cycle quiescence. The cell cycle regulators involved in maintaining the quiescence, and thereby the self-renewal capacity, of somatic stem cells have long been elusive. Two new Cell Stem Cell articles in this issue (Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Zou et al., 2011Zou P. Arai F. Yoshihara H. Tai I. Hosokawa K. Matsumoto Y. Shinmyozu K. Tsukahara F. Maru Y. Nakayama K. et al.p57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70.Cell Stem Cell. 2011; 9 (this issue): 247-261Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) now show that the CDK inhibitor p57 is a crucial brake for cycling HSCs, and links self-renewal activity to cell cycle quiescence. The self-renewal activity of somatic stem cells often inversely correlates with cell cycle activity. This pattern has been shown for epidermal, muscle, and adult neuronal stem cells and has probably been the most extensively studied in bone marrow hematopoietic stem cells (HSCs) (Wilson et al., 2008Wilson A. Laurenti E. Oser G. van der Wath R.C. Blanco-Bose W. Jaworski M. Offner S. Dunant C.F. Eshkind L. Bockamp E. et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar). In a healthy adult mouse, the most potent HSCs are in a deep dormancy phase, a status in which the metabolism and S phase machinery are mostly downregulated. Mathematical modeling combined with functional assays suggests that dormant HSCs divide about five times per lifetime. While dormant HSCs form a silent reservoir of stem cells during homeostasis, they are efficiently activated to proliferate and self-renew in response to stress and injury signals such as lypopholisaccharides or alpha and gamma type interferons produced during bacterial or viral infections (Essers et al., 2009Essers M.A. Offner S. Blanco-Bose W.E. Waibler Z. Kalinke U. Duchosal M.A. Trumpp A. IFNalpha activates dormant haematopoietic stem cells in vivo.Nature. 2009; 458: 904-908Crossref PubMed Scopus (981) Google Scholar, King et al., 2011King K.Y. Baldridge M.T. Weksberg D.C. Chambers S.M. Lukov G.L. Wu S. Boles N.C. Jung S.Y. Qin J. Liu D. et al.Irgm1 protects hematopoietic stem cells by negative regulation of IFN signaling.Blood. 2011; 118: 1525-1533Crossref PubMed Scopus (62) Google Scholar, Takizawa et al., 2011Takizawa H. Regoes R.R. Boddupalli C.S. Bonhoeffer S. Manz M.G. Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.J. Exp. Med. 2011; 208: 273-284Crossref PubMed Scopus (219) Google Scholar, Wilson et al., 2008Wilson A. Laurenti E. Oser G. van der Wath R.C. Blanco-Bose W. Jaworski M. Offner S. Dunant C.F. Eshkind L. Bockamp E. et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar). Extrapolation of the cycling kinetics of dormant mouse HSCs to humans would indicate that some human HSCs would only divide about once every 18 years. However, in contrast to laboratory mice that are kept under semisterile, specific pathogen free (SPF) conditions, humans are constantly exposed to bacterial and viral infections; thus, at least part of the HSC pool is most likely persistently activated, making it unlikely that long-term dormant cells exist in humans. Nevertheless, in vivo clonal analysis of human HSCs revealed cell cycle dynamics similar to those observed in mice, as it was shown that HSCs can become quiescent for months followed by another productive cycling phase (McKenzie et al., 2006McKenzie J.L. Gan O.I. Doedens M. Wang J.C. Dick J.E. Individual stem cells with highly variable proliferation and self-renewal properties comprise the human hematopoietic stem cell compartment.Nat. Immunol. 2006; 7: 1225-1233Crossref PubMed Scopus (130) Google Scholar). Moreover, the biological state of dormancy is certainly critical for the maintenance of high self-renewal capacity, which simultaneously makes HSCs insensitive to antiproliferative chemotherapeutic drugs. Typically, proliferation-inducing cytokines or genetic mutations that decrease quiescence and induce HSC cycling almost always result in exhaustion of the HSC pool, and do so in transplantation settings in particular (Wilson et al., 2009Wilson A. Laurenti E. Trumpp A. Balancing dormant and self-renewing hematopoietic stem cells.Curr. Opin. Genet. Dev. 2009; 19: 461-468Crossref PubMed Scopus (160) Google Scholar). It is widely assumed that signals from the bone marrow stem cell niches control the balance between HSC dormancy and self-renewal (Ehninger and Trumpp, 2011Ehninger A. Trumpp A. The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in.J. Exp. Med. 2011; 208: 421-428Crossref PubMed Scopus (413) Google Scholar). One of the quiescence-inducing pathways initiated by niche cells is the THPO-cMPL pathway, which later on during ontogeny is also involved in differentiation of the megakaryocytic lineage. The link between this pathway and the cell cycle machinery of HSCs appeared to be at least in part mediated by regulating Cyclin-Dependent Kinase (CDK) inhibitors (CDKis) such as p57KIP2 (p57) (Yoshihara et al., 2007Yoshihara H. Arai F. Hosokawa K. Hagiwara T. Takubo K. Nakamura Y. Gomei Y. Iwasaki H. Matsuoka S. Miyamoto K. et al.Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche.Cell Stem Cell. 2007; 1: 685-697Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). Although p57 expression is known to correlate with quiescence and self-renewal status, functional proof that p57 actively participates in maintaining or inducing HSC quiescence has not been reported. In this issue of Cell Stem Cell, two studies genetically dissect the role of p57 in HSC function and address its impact on cell cycle control and self-renewal (Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Zou et al., 2011Zou P. Arai F. Yoshihara H. Tai I. Hosokawa K. Matsumoto Y. Shinmyozu K. Tsukahara F. Maru Y. Nakayama K. et al.p57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70.Cell Stem Cell. 2011; 9 (this issue): 247-261Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Within the four phases (G1/S/G2/M) of the cell division cycle, it is only in G1 phase where the decision to cycle is dependent on external stimuli. Progression to S phase and proliferation only occurs in the presence of growth factors, while in their absence, cells enter a quiescent G0 phase. The G1 phase is separated into an early and late G1 phase by the "restriction point" or "G1/S checkpoint." If cells pass this point, they are committed to continue through an entire cycle and no longer require growth factors to complete division (Figure 1A ). The early and late G1 phases are controlled by enzymatic complexes formed by CDKs and their associated cyclins. During the early G1 phase, mitogenic stimulation promotes expression of D-type cyclins that associate with CDK4 or CDK6. CyclinD-CDK4 and cyclinD-CDK6 complexes phosphorylate the transcriptional repressor Retinoblastoma (Rb), resulting in its inactivation (Figure 1A). Phosphorylated Rb no longer inhibits the E2F transcription factors that activate a complex program promoting S phase initiation and progression. The initial inactivating Rb phosphorylation corresponds to transition through the restriction point and progression to late G1 phase. In this phase, induction of cyclinE promotes activation of cyclinE-CDK2 complexes, which hyperphosphorylates Rb and leads to further activation of S phase genes (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5132) Google Scholar) (Figure 1A). An additional level of complexity is added by the presence of CDKis, which reversibly antagonize cell cycle progression. CDKis are classified into two families. The INK4 family comprising p16Ink4a, p15Ink4b, p18Ink4c, and p19Ink4 targets CDK4 or CDK6. The CDKis p21, p27, and p57 belong to the Cip/Kip family that exhibits a broader spectrum by inhibiting also CDK2 on top of CDK4 and CDK6 (Figure 1A). Interestingly, p21 and p27 can also facilitate the activation of cyclinD-CDKs (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5132) Google Scholar). Moreover, in contrast to INK4, the Cip/Kip family mediates cell cycle-independent functions by affecting cytoskeletal organization and cell migration via the regulation of Rho activity (Besson et al., 2004Besson A. Assoian R.K. Roberts J.M. Regulation of the cytoskeleton: an oncogenic function for CDK inhibitors?.Nat. Rev. Cancer. 2004; 4: 948-955Crossref PubMed Scopus (169) Google Scholar). The apparently inverse relationship between cell cycle activity and self-renewal capacity in HSCs puts CDKis at the heart of the pool of candidates that induce quiescence and possibly preserve self-renewal. Indeed, in various previous studies, high expression of p57 was observed in normal mouse HSCs. In more general terms, expression of p57 is highly restricted in adult tissues, but is expressed during fetal organogenesis, particularly in differentiating cells. Mice lacking p57 show perinatal lethality and display a large number of developmental defects. Interestingly, these defects are strikingly similar to those observed in Beckwith-Wiedemann syndrome patients, and various studies demonstrate direct involvement of the p57 locus in the pathogenesis of this syndrome (Pateras et al., 2009Pateras I.S. Apostolopoulou K. Niforou K. Kotsinas A. Gorgoulis V.G. p57KIP2: "Kip"ing the cell under control.Mol. Cancer Res. 2009; 7: 1902-1919Crossref PubMed Scopus (122) Google Scholar, Zhang et al., 1997Zhang P. Liégeois N.J. Wong C. Finegold M. Hou H. Thompson J.C. Silverman A. Harper J.W. DePinho R.A. Elledge S.J. Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome.Nature. 1997; 387: 151-158Crossref PubMed Scopus (651) Google Scholar) Moreover, transcriptional and translational downregulation of p57 is frequent in many human cancers, including lymphoid malignancies, strongly suggesting that this protein has tumor-suppressing-like effects in various cancers (Pateras et al., 2009Pateras I.S. Apostolopoulou K. Niforou K. Kotsinas A. Gorgoulis V.G. p57KIP2: "Kip"ing the cell under control.Mol. Cancer Res. 2009; 7: 1902-1919Crossref PubMed Scopus (122) Google Scholar). p57 transcripts and protein are highly expressed in primitive, quiescent HSCs, defined phenotypically by the expression of Sca1 and cKit, and by the absence of CD34 and Lineage markers (CD34-LSK). This expression progressively declines during differentiation toward less self-renewing multipotent progenitors (Figure 1B) (Passegué et al., 2005Passegué E. Wagers A.J. Giuriato S. Anderson W.C. Weissman I.L. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates.J. Exp. Med. 2005; 202: 1599-1611Crossref PubMed Scopus (485) Google Scholar, Yamazaki et al., 2006Yamazaki S. Iwama A. Takayanagi S. Morita Y. Eto K. Ema H. Nakauchi H. Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells.EMBO J. 2006; 25: 3515-3523Crossref PubMed Scopus (215) Google Scholar, Yamazaki et al., 2009Yamazaki S. Iwama A. Takayanagi S. Eto K. Ema H. Nakauchi H. TGF-beta as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation.Blood. 2009; 113: 1250-1256Crossref PubMed Scopus (233) Google Scholar). It is thought that dormant/quiescent HSCs are maintained in specialized niches that provide signals that maintain their stem cell fate (Ehninger and Trumpp, 2011Ehninger A. Trumpp A. The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in.J. Exp. Med. 2011; 208: 421-428Crossref PubMed Scopus (413) Google Scholar). These signals include TPO and TGF-beta, both of which increase p57 expression, thus linking p57 function to niche-mediated maintenance of functional HSCs (Yamazaki et al., 2006Yamazaki S. Iwama A. Takayanagi S. Morita Y. Eto K. Ema H. Nakauchi H. Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells.EMBO J. 2006; 25: 3515-3523Crossref PubMed Scopus (215) Google Scholar, Yoshihara et al., 2007Yoshihara H. Arai F. Hosokawa K. Hagiwara T. Takubo K. Nakamura Y. Gomei Y. Iwasaki H. Matsuoka S. Miyamoto K. et al.Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche.Cell Stem Cell. 2007; 1: 685-697Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). Although these previous studies hypothesized a critical role for p57 in regulating HSC quiescence and self-renewal, genetic data on the function of this CDKi in HSCs were still missing. In this issue of Cell Stem Cell, the groups of Toshio Suda and Keiichi Nakayama provide new insights into this topic by analyzing HSCs lacking p57 (Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Zou et al., 2011Zou P. Arai F. Yoshihara H. Tai I. Hosokawa K. Matsumoto Y. Shinmyozu K. Tsukahara F. Maru Y. Nakayama K. et al.p57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70.Cell Stem Cell. 2011; 9 (this issue): 247-261Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). In the first manuscript, Matsumoto and colleagues circumvented the perinatal lethality of p57−/− mice by generating a conditional p57flox allele, which in combination with the MxCre transgene can be inducibly deleted in the entire hematopoietic system by treatment with poly(I)-poly(C) (pIC). Four weeks after deletion of p57, mutant mice showed a 2-fold reduction in hematopoietic progenitors (CD34+LSK, CD150+CD48-LSK) and in primitive CD34-LSK HSCs, highly enriched in functional HSCs. This phenotype was associated with an equivalent decrease in quiescent cells. In addition, p57-deficient stem/progenitors showed a 2-fold increase in p53-mediated apoptosis. At a functional level, mutant HSCs showed a 5-fold reduction in their capacity to reconstitute lethally irradiated recipients, which further decreased in secondary transplants. Interestingly, HSC repopulating capacity was intrinsically affected by p57 deficiency, since pIC-mediated deletion of p57 in mice previously reconstituted with a 1:1 mixture of undeleted MxCre;p57flox/flox and control BM cells resulted in the robust decrease of mutant HSCs over time (Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The dependency of HSCs on p57 was specific to this CDKi because mutants for the other two CIP/KIP members, p21 and p27, did not show defective repopulating potential, confirming earlier reports (Cheng et al., 2000Cheng T. Rodrigues N. Dombkowski D. Stier S. Scadden D.T. Stem cell repopulation efficiency but not pool size is governed by p27(kip1).Nat. Med. 2000; 6: 1235-1240Crossref PubMed Scopus (294) Google Scholar, van Os et al., 2007van Os R. Kamminga L.M. Ausema A. Bystrykh L.V. Draijer D.P. van Pelt K. Dontje B. de Haan G. A Limited role for p21Cip1/Waf1 in maintaining normal hematopoietic stem cell functioning.Stem Cells. 2007; 25: 836-843Crossref PubMed Scopus (91) Google Scholar). The authors also addressed the question of redundancy and analyzed HSCs lacking both p57 and p21. While the double mutants showed virtually identical phenotypes to p57 singles in vivo, the colony-forming capacity was strongly reduced in double mutant cells, suggesting that p21 is an important regulator in culture stress-induced cell activity, but is likely not involved in normal HSC function. Finally, the authors wondered whether the specific role of p57 is indeed due to its specific biological activity compared with the other CDKIs. To address this point, they analyzed HSCs from a knockin (KI) mouse line in which the p57 coding region was replaced by that for p27. Strikingly, expression of four alleles of p27 (two endogenous and two from the p57 locus) generated normal HSCs even in the absence of any p57 protein (Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). This suggests that the two proteins are highly similar with respect to their biological activity, and that the importance of p57 in HSCs is rather due to its specific expression pattern, i.e., p57 being highly expressed in quiescent/dormant HSCs but downregulated in progenitors. In the second report, Zou et al. circumvent the newborn lethality of p57−/− mice by transplanting mutant fetal liver (FL) HSCs into lethally irradiated adult recipients and compared their repopulation activity to that of wild-type FL cells. While p57−/− FL HSCs could normally reconstitute adult hematopoiesis in primary and secondary transplants, they showed a 2-fold impaired repopulating capacity in tertiary recipients. This relatively subtle self-renewal deficiency was not associated with alteration of HSC quiescence. As it seemed likely that other CDKi factors compensate for p57 deficiency in the mutant FL-donor-derived HSCs, the authors determined the expression of p18, p21, and p27 in bone marrow LSK stem/progenitor cells after each round of transplantation. This revealed a significant compensatory upregulation of p18 and p27 transcripts and protein, but not of p21. To study whether this compensatory upregulation is functionally significant, Zou and colleagues performed shRNA-mediated knockdown of p57 on a p27 mutant background, and also analyzed mice transplanted with FL HSCs derived from p57/p27 double mutant mice. In both cases, the reconstitution capacity was significantly reduced after serial transplantation. In this setting the phenotype was associated with a decrease in HSC quiescence while progenitor proliferation was unaffected. These data strongly suggest that the compensatory upregulation of p27 in p57-deficient FL HSCs preserves their quiescence and most of their functional capacity. Although both studies demonstrate the relevance of p57 in HSC maintenance, the phenotypes are not absolute, as self-renewal and reconstitution activity are still present (albeit drastically reduced) in cells lacking both p57 and p27. Do the genetic data really address whether p57 is necessary for the maintenance of dormant/quiescent HSCs, or do the experiments show that p57 upregulation is a critical mechanism to establish quiescence of initially cycling HSCs? Evidence for the latter comes from the fact that Zou and colleagues study FL HSCs that are inherently cycling, and only achieve quiescence later on in the adult in specialized niches of the bone marrow. Similarly, the conditional knockout (KO) approach used by Matsumoto and colleagues induces Cre-mediated deletion of the p57 gene by pIC, which is known to rapidly activate all dormant HSCs (Essers et al., 2009Essers M.A. Offner S. Blanco-Bose W.E. Waibler Z. Kalinke U. Duchosal M.A. Trumpp A. IFNalpha activates dormant haematopoietic stem cells in vivo.Nature. 2009; 458: 904-908Crossref PubMed Scopus (981) Google Scholar). Because activation of HSCs is likely faster than deletion of p57, the protein is lost in already-cycling HSCs. Thus, in both cases, the data show that p57 is important for the establishment of quiescence starting from cycling HSCs. Future experiments using other inducible Cre systems that do not activate HSCs, such as the SCL-CreERT2 allele, will be necessary to address whether p57 is critical to maintain quiescence of HSCs in vivo. Nevertheless, the data show that failure of HSCs to enter quiescence is associated with decreased self-renewal (Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Zou et al., 2011Zou P. Arai F. Yoshihara H. Tai I. Hosokawa K. Matsumoto Y. Shinmyozu K. Tsukahara F. Maru Y. Nakayama K. et al.p57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70.Cell Stem Cell. 2011; 9 (this issue): 247-261Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Moreover, it is interesting to observe that conditional ablation of p57 produced a more profound phenotype than that observed in straight KO mice. Whereas this pattern might be explained by compensatory mechanisms that become active in germline KO mice, it also raises the question of whether conditional approaches targeting the other CDKi family members will reveal more severe phenotypes as compared with the reported germline KO models. In both studies, p57-deficient HSCs compensatorily upregulated p18 and p27 (Figure 1B). Although p27 protein is expressed in normal HSCs, it is not differentially regulated within the various HSC and progenitor populations (Figure 1B) (Yamazaki et al., 2006Yamazaki S. Iwama A. Takayanagi S. Morita Y. Eto K. Ema H. Nakauchi H. Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells.EMBO J. 2006; 25: 3515-3523Crossref PubMed Scopus (215) Google Scholar, Matsumoto et al., 2011Matsumoto A. Takeishi S. Kanie T. Susaki E. Onoyama I. Tateishi Y. Nakayama K. Nakayama K.I. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem Cells.Cell Stem Cell. 2011; 9 (this issue): 262-271Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Zou et al., 2011Zou P. Arai F. Yoshihara H. Tai I. Hosokawa K. Matsumoto Y. Shinmyozu K. Tsukahara F. Maru Y. Nakayama K. et al.p57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70.Cell Stem Cell. 2011; 9 (this issue): 247-261Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Genetic deficiency of p27 affects mostly progenitor cells, resulting in their increased number and cycling. Moreover, progenitor cells lacking p27 outcompete their wild-type counterparts in serial and competitive transplantation, and they enhance survival of mice in short-term radioprotection assays. However, lack of p27 does not perturb HSC number, cycling, or repopulation potential (Cheng et al., 2000Cheng T. Rodrigues N. Dombkowski D. Stier S. Scadden D.T. Stem cell repopulation efficiency but not pool size is governed by p27(kip1).Nat. Med. 2000; 6: 1235-1240Crossref PubMed Scopus (294) Google Scholar). Thus, p27 negatively regulates the hematopoietic progenitor pool without playing a specific major role in HSC function. However, it may be important for fine-tuning the cell cycle of HSCs, because p27 interacts not only with p57, but also with the Myc-Max-Mad transcription factors, and p27/Mad1 double mutants display increased HSC number (Walkley et al., 2005Walkley C.R. Fero M.L. Chien W.M. Purton L.E. McArthur G.A. Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells.Nat. Cell Biol. 2005; 7: 172-178Crossref PubMed Scopus (94) Google Scholar). The second CDKi upregulated in p57 mutant HSCs was p18, which has previously been shown to regulate HSC function. Mice deficient for p18 displayed an expansion of stem and progenitor cells, which showed a competitive advantage in serial bone marrow transplantation assays compared with that of normal controls (Yu et al., 2006Yu H. Yuan Y. Shen H. Cheng T. Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners.Blood. 2006; 107: 1200-1206Crossref PubMed Scopus (108) Google Scholar, Yuan et al., 2004Yuan Y. Shen H. Franklin D.S. Scadden D.T. Cheng T. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C.Nat. Cell Biol. 2004; 6: 436-442Crossref PubMed Scopus (221) Google Scholar). This was associated with an unexpected increase in HSC cycling, because typically, increased HSC cycling is almost always associated with stem cell exhaustion in serial transplant settings. As a mechanism for this novel phenotype, it has been proposed that p18 mutant HSCs have a shorter G1 phase due to a lower cyclinD/CDK4 threshold activity. According to this model, mutant cells transit more rapidly through late G1, and thus would be less exposed to differentiation-inducing cytokine stimuli, resulting in increased self-renewal capacity (Orford and Scadden, 2008Orford K.W. Scadden D.T. Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation.Nat. Rev. Genet. 2008; 9: 115-128Crossref PubMed Scopus (662) Google Scholar). Hence, p18 appears to be a CDKi that balances HSC self-renewal and differentiation by regulating the kinetics of early G1 transit. Because p57-controlled exit from G0 and p18-controlled G1 length are consecutive biological processes, it is likely that p18 and p57 function are highly coordinated or possibly even dependent on each other. Future genetic studies analyzing the phenotype of p57/p18 double mutant HSCs will likely provide further insights into the relationship between quiescence/dormancy and self-renewing divisions in functional HSCs. Although p21CIP1 (p21) was initially indicated to be an important regulator of normal HSC function, more recent data suggest that the role of p21 is mainly restricted to genotoxically stressed HSCs (Choudhury et al., 2007Choudhury A.R. Ju Z. Djojosubroto M.W. Schienke A. Lechel A. Schaetzlein S. Jiang H. Stepczynska A. Wang C. Buer J. et al.Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation.Nat. Genet. 2007; 39: 99-105Crossref PubMed Scopus (345) Google Scholar, van Os et al., 2007van Os R. Kamminga L.M. Ausema A. Bystrykh L.V. Draijer D.P. van Pelt K. Dontje B. de Haan G. A Limited role for p21Cip1/Waf1 in maintaining normal hematopoietic stem cell functioning.Stem Cells. 2007; 25: 836-843Crossref PubMed Scopus (91) Google Scholar, Viale et al., 2009Viale A. De Franco F. Orleth A. Cambiaghi V. Giuliani V. Bossi D. Ronchini C. Ronzoni S. Muradore I. Monestiroli S. et al.Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells.Nature. 2009; 457: 51-56Crossref PubMed Scopus (255) Google Scholar). Unlike other members of the CDKi families, p21 is a p53 target gene and is a key mediator of p53-mediated G1 arrest and apoptosis (el-Deiry et al., 1994el-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. Pietenpol J.A. Burrell M. Hill D.E. Wang Y. et al.WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis.Cancer Res. 1994; 54: 1169-1174PubMed Google Scholar). In response to DNA damage provoked by telomerase dysfunction, CD34-KLS HSCs were shown to upregulate p21, detrimentally affecting their function. In this setting, HSC self-renewal was rescued by p21 deletion (Choudhury et al., 2007Choudhury A.R. Ju Z. Djojosubroto M.W. Schienke A. Lechel A. Schaetzlein S. Jiang H. Stepczynska A. Wang C. Buer J. et al.Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation.Nat. Genet. 2007; 39: 99-105Crossref PubMed Scopus (345) Google Scholar). Intriguingly, while p21 impairs normal HSC self-renewal during the DNA damage response induced by telomerase dysfunction, it maintains leukemic stem cell self-renewal during oncogene-induced DNA damage. In a PML-RAR leukemia model, it was shown that p21 prevents leukemic stem cell exhaustion by limiting cell proliferation and accumulation of DNA damage (Viale et al., 2009Viale A. De Franco F. Orleth A. Cambiaghi V. Giuliani V. Bossi D. Ronchini C. Ronzoni S. Muradore I. Monestiroli S. et al.Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells.Nature. 2009; 457: 51-56Crossref PubMed Scopus (255) Google Scholar). p16INK4a mRNA levels are high in primitive CD34-LSK HSCs and progressively decrease during progenitor commitment (Figure 1B) (Passegué et al., 2005Passegué E. Wagers A.J. Giuriato S. Anderson W.C. Weissman I.L. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates.J. Exp. Med. 2005; 202: 1599-1611Crossref PubMed Scopus (485) Google Scholar). Interestingly, increased p16 expression has been observed in aged HSCs or in response to stress such as reactive oxygen production (Ito et al., 2004Ito K. Hirao A. Arai F. Matsuoka S. Takubo K. Hamaguchi I. Nomiyama K. Hosokawa K. Sakurada K. Nakagata N. et al.Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells.Nature. 2004; 431: 997-1002Crossref PubMed Scopus (963) Google Scholar, Janzen et al., 2006Janzen V. Forkert R. Fleming H.E. Saito Y. Waring M.T. Dombkowski D.M. Cheng T. DePinho R.A. Sharpless N.E. Scadden D.T. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.Nature. 2006; 443: 421-426Crossref PubMed Scopus (911) Google Scholar). Consistently, p16 plays a major role in regulating HSC function during aging. In young mice p16 deficiency is dispensable for HSC self-renewal (Stepanova and Sorrentino, 2005Stepanova L. Sorrentino B.P. A limited role for p16Ink4a and p19Arf in the loss of hematopoietic stem cells during proliferative stress.Blood. 2005; 106: 827-832Crossref PubMed Scopus (67) Google Scholar). However, in old mice, lack of p16 increased the number of long-term HSCs. Moreover, in serial transplantation assays, p16-deficient old HSCs showed increased cell cycle entry and outcompeted p16-deficient young HSCs (Janzen et al., 2006Janzen V. Forkert R. Fleming H.E. Saito Y. Waring M.T. Dombkowski D.M. Cheng T. DePinho R.A. Sharpless N.E. Scadden D.T. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.Nature. 2006; 443: 421-426Crossref PubMed Scopus (911) Google Scholar). Furthermore, activation of the p16-CDK4/CDK6-Rb signaling pathway has been shown to be involved in defective stem cell maintenance in response to intracellular reactive oxygen species (ROS) elevation, a situation associated with aging (Ito et al., 2004Ito K. Hirao A. Arai F. Matsuoka S. Takubo K. Hamaguchi I. Nomiyama K. Hosokawa K. Sakurada K. Nakagata N. et al.Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells.Nature. 2004; 431: 997-1002Crossref PubMed Scopus (963) Google Scholar). In summary, p16 plays a major role in the age-related decline of HSC function. At the molecular level, Zou et al. showed that HSCs lacking p57 and p27 had increased Rb phosphorylation. To address the mechanism behind this phenomenon, the authors analyzed the expression of the various cell cycle components by immunofluorescence of FACS-sorted HSCs. This revealed a surprising finding: p57 and cyclinD1 are localized to the cytoplasm of unstimulated HSCs, while the CDKs are all localized to the nucleus. In response to stimulation by stem cell factor (SCF), p57 is degraded, expression of CDKs increased, cyclinD1 moves to the nucleus, and Rb becomes phosphorylated (Zou et al., 2011Zou P. Arai F. Yoshihara H. Tai I. Hosokawa K. Matsumoto Y. Shinmyozu K. Tsukahara F. Maru Y. Nakayama K. et al.p57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70.Cell Stem Cell. 2011; 9 (this issue): 247-261Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). These data suggest that in HSCs, p57 might not inhibit nuclear cyclinD/CDK complexes directly, but rather, indirectly, by retaining cyclinD1 in the cytoplasm. To shed light on this observation, Zou et al. used a coimmunoprecipitation approach to identify proteins that were bound to Myc-tagged p57 in the hematopoietic progenitor cell line EML. Unexpectedly, Hsc70, a member of the heat shock protein 70 (HSP70) family, immunoprecipitated with p57. This molecular chaperone is known as a factor that shuttles proteins from the cytoplasm to the nucleus, and is also known to bind cyclinD1. Subsequent binding studies revealed that p57 and p27 formed complexes with Hsc70 in the cytoplasm, possibly retaining a cyclinD1-Hsc70 complex in this compartment, thus preventing G1 cell cycle progression via Rb phosphorylation. To show that Hsc70-mediated retention of cyclinD1 is a key mechanism in preserving cell quiescence, Hsc70 or an Hsc70 mutant that lacked the nuclear localization-related signal (NLRS) was transduced into LSK stem/progenitors and transplanted into lethally irradiated mice. HSCs overexpressing Hsc70 showed an increase in proliferation and a decrease in quiescence and were less able to form colonies in vitro. These effects were blunted in HSCs overexpressing the mutant form of Hsc70 that is unable to shuttle cyclinD1 to the nucleus. The ability of both p57 and p27 to interact with Hsc70 might be the molecular basis for the functional overlap of these CDKis that includes the p27 compensatory role in the absence of p57. However, the exact biochemical composition of the complexes that bring Hsc70-cyclinD1 together with p27 and p57 remains unclear. Several scenarios are plausible. First, a quaternary complex where both p27 and p57 are bound to Hsc70-cyclinD1 might exist. Alternatively, Hsc70-cyclinD1 may only bind either p27 or p57, resulting in two distinct complexes (p57-Hsc70-cyclinD1 and p27-Hsc70-cyclinD1). In a third possibility, the p57-Hsc70-cyclinD1 complex would be the predominant complex in quiescent HSCs, while finally p27 is only part of the complex in the absence of p57 (Figure 2). Future biochemical studies will need to determine the stochiometric relationships between the various complexes and their roles in cell cycle activation. In summary, the data predict a model in which niche-secreted quiescence-inducing growth factors such as TPO and TGF-beta induce and maintain the expression of cytoplasmic p57. This factor forms a complex with Hsc70-cyclinD1 and prevents cyclinD1 from entering the nucleus and activating Rb-mediated G1 progression (Figure 2). Activating cytokines such as SCF or IFNa (interferon-alpha) are likely associated with an exit of HSCs from the niche environment that maintains HSC quiescence. This move leads to the transcriptional downregulation and degradation of p57. CyclinD1 enters the nucleus via Hsc70 and activates CDK4/6 complexes, which in turn phosphorylate Rb. Subsequently, hyperphosphorylation of this transcription factor by cyclinE/CDK2 promotes E2F activation and initiation of the S phase program. The identification of Hsc70-cyclinD1-mediated regulation of cell cycle entry of quiescent HSCs is novel and fascinating. It will be interesting to see whether this is a mechanism also used in other types of dormant stem cells such as those present in the brain or bulge region of the skin epidermis, and whether this is a general mechanism to exit from a deeply quiescent G0 stage. The roles played by the classic nuclear inhibitory mechanisms of CDKIs in HSCs still remain to be explored. While p57 seems to function predominantly in the cytoplasm by preventing cyclinD1 shuttling in the nucleus, other CDKis such as p18 are strictly nuclear and thus potentially inhibit CDKs in that compartment via mechanisms independent of Hsc70. Thus HSC quiescence is likely regulated in a concerted manner by nuclear and cytoplasmic CDKis acting via distinct cellular and molecular pathways. This new data on p57 and HSC quiescence are of major significance, because cell cycle quiescence and dormancy is one of the mechanisms that allow cancer stem cells to escape antiproliferative chemotherapy and to possibly mediate resistance to targeted therapies such as imatinib in BCR-Abl driven CML. Along these lines, the identification of Hsc70 may allow the design of novel therapeutic strategies to drive cells out of quiescence into a more drug-sensitive stage. Indeed, Hsc70 is upregulated in imatinib-resistant CML and pharmaceutical inhibition of this protein has already been shown to decrease the survival of CML cells in combination with imatinib (José-Enériz et al., 2008José-Enériz E.S. Román-Gómez J. Cordeu L. Ballestar E. Gárate L. Andreu E.J. Isidro I. Guruceaga E. Jiménez-Velasco A. Heiniger A. et al.BCR-ABL1-induced expression of HSPA8 promotes cell survival in chronic myeloid leukaemia.Br. J. Haematol. 2008; 142: 571-582Crossref PubMed Scopus (30) Google Scholar). Future approaches will need to uncover further players in the machinery that links quiescence and self-renewal activity in normal and leukemic stem cells to allow the development of more efficient strategies to target the entire pool of leukemic stem cells within a patient. The authors would like to thank Drs. Irène Baccelli, Nina Cabezas, and Anne Wilson for comments on the manuscript. This work was supported by the Haematosys Program funded by the German Bundesministerium für Bildung und Forschung (BMBF), the EU-FP7 Program "EuroSyStem," the SFB 873 funded by the Deutsche Forschungsgemeinschaft (DFG), and the Dietmar Hopp Foundation. p57 Is Required for Quiescence and Maintenance of Adult Hematopoietic Stem CellsMatsumoto et al.Cell Stem CellSeptember 02, 2011In BriefQuiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained uncharacterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G0 phase. Full-Text PDF Open Archivep57Kip2 and p27Kip1 Cooperate to Maintain Hematopoietic Stem Cell Quiescence through Interactions with Hsc70Zou et al.Cell Stem CellSeptember 02, 2011In BriefCell cycle regulators play critical roles in the balance between hematopoietic stem cell (HSC) dormancy and proliferation. In this study, we report that cell cycle entry proceeded normally in HSCs null for cyclin-dependent kinase (CDK) inhibitor p57 due to compensatory upregulation of p27. HSCs null for both p57 and p27, however, were more proliferative and had reduced capacity to engraft in transplantation. We found that heat shock cognate protein 70 (Hsc70) interacts with both p57 and p27 and that the subcellular localization of Hsc70 was critical to maintain HSC cell cycle kinetics. Full-Text PDF Open Archive
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