NAD-Dependent Histone Deacetylase, SIRT1, Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells.

Sir2 (silent information regulator 2) initially isolated as a transcriptional silencer was shown to be essential for the longevity caused by the calorie restriction in yeast and C. elegans. Also, SIRT1, a murine homologue of Sir2, was reported to play pivotal roles in cellular senescence as a calorie sensor. SIRT1 deficiency in mice was reported to result in early postnatal lethality because of the incomplete development of embryos and/or heart malformation. However, in these studies, the abnormality in hematopoiesis was not precisely analyzed. So, in the present study, we investigated the role of SIRT1 in hematopoietic stem cells.

At first, we examined the expression profile of SIRT1 in various hematopoietic cells by RT-PCR analysis, and found that it was ubiquitously expressed in all of the hematopoietic lineages from immature to differentiated cells. Next, we examined the effects of SIRT1 inhibitor, nicotinamide(NA), and its activator, resveratol, on murine hematopoietic stem/progenitor cells. We isolated Lineage(−) Sca-1(+)(LS) cells from murine bone marrow and cultured with the cytokine cocktail containing SCF, IL-6, Flt3L, and TPO, which is utilized for the expansion of stem cells, together with NA or resveratol. As a result, NA significantly reduced LS cell population from 21.5% to 5.7%, while resveratol increased this fraction up to 37.4%. Also, we performed colony assays using LS cells cultured with or without NA for two days. The numbers of CFU-mix, BFU-E, CFU-E, CFU-G, CFU-M, and CFU-Meg yielded from NA-treated cells were all reduced about 50–80% as compared with those from untreated cells. We also examined the effects of NA on terminal differentiations of LS cells. For this purpose, we cultured LS cells using the following cytokine combinations: SCF and G-CSF for inducing granulocytic differentiation; SCF and EPO for erythroid differentiation, or with SCF and TPO for megakaryocytic differentiation. NA accelerated differentiation toward all lineages. To inhibit SIRT1 activity more specifically, we introduced SiRNA for SIRT1 into murine LS cells using the retrovirus system. In consistent with the results obtained from the experiments using NA, the proportion of immature LS cells was reduced from 8.0% to 1.8%, and terminal differentiation was promoted in SIRT1 SiRNA-infected cells in comparison with MOCK-infected cells.

We further examined the mechanisms through which SIRT1 keeps hematopoietic cells undifferentiated. Since p38MAPK cascade is also reported to promote differentiation of hematopoietic stem cells, we analyzed whether p38MAPK is a downstream effector of SIRT1 using its inhibitor SB202190. Even if SB202190 was added to the culture medium of LS cells, it did not affect NA-induced acceleration of differentiation, suggesting that p38MAPK does not act as a downstream effector of SIRT1. In a recent paper, SIRT1 was reported to regulate cellular resistance against stress and apoptosis by suppressing p53 activity through its deacetylation. So, we examined the effects of NA using LS cells isolated from p53 knockout mice. However, NA accelerated differentiation of p53−/− LS cells as was the case with those from normal mice, indicating that SIRT1 suppresses the differentiation of hematopoietic stem/progenitor cells independently of p53. Thus, SIRT1 was supposed to regulate the fate and differentiation of hematopoietic cells through a novel system as a calorie sensor.