SIRT1 Deficiency Suppresses the Maintenance of Hematopoietic Stem Cell Pool.

Abstract

Sir2 (silent information regulator 2) is a member of a gene family (sirtuins) encoding NAD(+)-dependent histone deacetylases, which leads to increased DNA stability and prolonged lifespan in Saccharomyces cerevisiae and Caenorhabditis elegans. In mammalians, SIRT1 has also been found to function as a deacetylase for numerous protein targets involved in various cellular pathways, including stress responses, apoptosis, and neural axonal degeneration. However, the effects of SIRT1 on hematopoiesis remains unknown. We previously reported that the SIRT1 inhibitor, nicotinamide(NA), promoted the differentiation of murine hematopoietic stem/progenitor cells, and its activator, Resveratrol, suppressed the differntiation. In this report, we analysed the differentiation of stem/progenitor cells derived from SIRT1 KO mice. Because SIRT1 KO mice can survive less than a week after birth, we performed experiments using c-Kit(+)Lineage(−)Sca-1(+) cells (KSLs) derived from KO mice fetal liver. At first, we cultured KSLs with the cytokine cocktail containing SCF, IL-6, Flt3L, and TPO, which is utilized for the expansion of stem cells. After five day culture, we estimated the population which remains in KSL fraction. As a result, percentage of KSLs from KO fetal liver was less than 5%, while that from WT was about 15%. We also examined the colony formation of KO and WT fetal liver KSL cells using replating assays. At the first plating, total number of colonies developed from KO fetal liver KSLs was smaller than that from WT by 30–40%, and at the third plating, there could be detected no colonies from KO, while 20–30 colonies were observed from WT. Furthermore, we performed serial transplantation assays using WT and KO fetal liver KSLs. Although after primary transplant, we detected no significant difference in repopulation from KO KSLs compared to WT controls, three weeks after secondary transplant, % chimerism from KO KSLs was reduced to 1/2 compared with that from WT KSLs. These results suggested that Sirt1 suppresses the differentiation and promotes self-renew of hematopoietic stem/progenitor cells. To dissect the roles of target molecules of Sirt1 in suppression of differentiation, we first examined the mRNA expressions of some cell cycle-relating molecules in KO and WT fetal liver KSLs. As consequence, p16Ink4A and p19Arf were detected only in KO KSLs. Then we analyzed the roles of molecules which may effect those expressions. First, we examined the effects of MAPkinases inhibitors on the differentiation of KO and WT fetal liver KSLs. During the culture with SCF, IL-6, Flt3L, and TPO, the addition of p38 inhibitor(SB202190), or MEK1 inhibitor(PD98059), or JNK inhibitor did not change the effects of the SIRT1 targeting. Then it was suggested that MAPkinase pathways have little relation with the SIRT1-induced suppression of differentiation. Next we examined the role of p53, which was reported to combine with SIRT1 and to be deacetylated and repressed by SIRT1. KO and WT fetal liver KSLs were cultured with p53 inhibitor (pifithrin?), which partially cancelled the promotion of differntiation in SIRT1 KO KSLs. This result suggested that SIRT1 might inhibit differentiation of KSLs partially by antagonizing p53 activity. Next we examined the role of Foxo3a, a downstream molecule of SIRT1. Enforced expression of constitutive active form of Foxo3a(FKHRL1TM) also cancelled the promotion of differentiation in SIRT1 KO KSLs. As conclusion, we demonstrate that SIRT1 suppresses the differentiation of hematopoietic stem/progenitor cells by antagonizing p53 and enhancing Foxo3a activities, and contributes to maintenance of stem cell properties and stem cell pool.