SIRT2 Plays Essential Role in Early Hematopoiesis through Deacetylation of LMO2 Protein

Nicotinamide phosphoribosyltransferase (NAMPT) is known as a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD⁺) and supplies NAD⁺ to sirtuins (SIRTs) that require NAD⁺ to activate their protein deacetylase functions. SIRTs are members of the NAD⁺-dependent class III histone deacetylase family. We already reported that NAMPT-NAD⁺-SIRT1 pathway is essential for granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis (Skokowa J, et al. Nat Med. 2009).

In the present study, we analyzed the mechanism of NAMPT-NAD⁺ -SIRTs-triggered hematopoiesis and myelopoiesis using human induced pluripotent stem (iPS) cells model. We applied a serum- and feeder-free monolayer hematopoietic differentiation system of iPS cells by sequential incubation of iPS cells with combinations of mesoderm- and hematopoiesis-inducing cytokines. We found that addition of NAMPT to granulopoiesis-inducing iPS cells culture medium resulted in a marked increase of CD15⁺ and CD16⁺ myeloid and granulocytic cell numbers. These results are compatible with our published data in healthy donor derived CD34⁺ cells. To evaluate at which stage of hematopoietic differentiation NAMPT plays a role, we cultured iPS cells in the presence or absence of the specific inhibitor of NAMPT, FK866. Interestingly, we found marked reduction of VEGFR2⁺ CD34⁺ early hematopoietic progenitor cells at day 6 of differentiation after inhibition of NAMPT. These data argue for the requirement of NAMPT and its downstream effectors at early stages of hematopoietic differentiation. To evaluate which of the SIRTs is essential in this process, we tested early hematopoietic differentiation of iPS cells in the presence of specific inhibitors of several SIRTs. We found that inhibition of SIRT2 using its specific inhibitor AC93253 significantly suppressed differentiation into VEGFR2⁺ CD34⁺ early hematopoietic progenitor fraction. Furthermore, the emergence of CD43⁺ hematopoietic progenitor cells at day 13 was completely suppressed after inhibition of SIRT2. These results indicate that NAMPT-NAD⁺-SIRT2 signaling is needed for early hematopoiesis. We further evaluated downstream targets of NAMPT-NAD⁺-SIRT2. qRT-PCR analysis of well-known early hematopoietic genes (GATA2, RUNX1, LMO2, TAL1) revealed that mRNA expression of these factors was not down-regulated after NAMPT or SIRT2 inhibition. We therefore tested whether hematopoietic functions of these proteins might be affected by post-transcriptional modifications via NAMPT-NAD⁺-SIRT2-triggered deacetylation. We tested protein-protein interaction between SIRT2 and these candidate proteins in differentiated iPS cells on day 6 of culture using Duolink technique, which allows identification of protein complexes in single cells. We found that RUNX1, LMO2 and TAL1 proteins interact with SIRT2 protein. To identify if these proteins could be deacetylated by SIRT2, we compared the signals from total- and de-/acetylated- candidate proteins in differentiated iPS cells on day6 treated or not with SIRT2 inhibitor using Duolink-FACS, Duolink technique in combination with flow cytometry, as a readout method. This analysis revealed that only LMO2 is deacetylated by SIRT2. LMO2 is known as an essential transcriptional regulator in early hematopoiesis. It is a component of TAL1 complex, which consist of LMO2, TAL1, LDB1, E47 and GATA1/2 proteins and has transcriptional activity as a complex on hematopoiesis-specific target genes as KIT, GATA1, GYPA, KLF1 and KDR. Interestingly, mRNA expression levels of all these genes were markedly downregulated after inhibition of NAMPT or SIRT2, which was in line with severely reduced numbers of early hematopoietic progenitors.

Overall, these results indicate that NAMPT, known to be essential for myeloid differentiation, induces early stages of hematopoietic differentiation by SIRT2-triggered deacetylation of LMO2.