Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD+ levels and SIRT inhibition

Abstract

Objective-Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood (mPB) CD34 + cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy.Methods-We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mPB CD34 + cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD + precursors. We tested additional inhibitors of the SIRT1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets.Results-NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure. 6.25 mM NIC increased NAD + levels 5-fold. Quinolinic acid increased NAD + similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest DisclosureNo financial interest/relationships with financial interest relating to the topic of this article have been declared. Nicotinamide has diverse functions in cells. NIC inhibits the activity of the silent information regulator 2 (Sir2) family of histone/protein deacetylases (sirtuins or SIRTs) [9][10][11][12]. Sirtuins catalyze a unique NAD + -dependent deacetylation reaction and are important for a wide variety of biological processes including transcriptional silencing, lifespan regulation, and the regulation of apoptosis [13][14][15]. NIC is also a precursor of NAD + via the salvage pathway [16,17]. NAD + regulates a variety of intracellular activities such as the DNA-binding specificity of p53 [18,19]. We have previously established that the p53-mediated Mk apoptotic program is intimately linked with terminal maturation and polyploidization, possibly through the downstream effects of p53 on MDM2 and BCL2 expression [20,21]. NIH Public AccessThe aim of this study was to examine the generality of the NIC effects, NIC's ...

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

“…In one-phase mPB CD34 + cell cultures carried out at 20% O 2 and pH 7.4 with Tpo as the only cytokine, Mks mature quickly such that NIC addition at day 5 is optimal. 18,40 However, expression of CD41 and CD42b on Mks was delayed in the multi-phase culture system, so that NIC addition was optimal between days 7 and 8. Delaying NIC addition until day 9 did not allow enough time for extensive Mk polyploidization before apoptosis.…”

“…NIC greatly increases Mk polyploidization, but also inhibits cell expansion. 18,29,40 Thus, one must optimize the timing of NIC addition to produce the greatest number of high-ploidy Mks. In one-phase mPB CD34 + cell cultures carried out at 20% O 2 and pH 7.4 with Tpo as the only cytokine, Mks mature quickly such that NIC addition at day 5 is optimal.…”

“…38,39 We also investigated shifting to supervascular pH 7.6 during terminal Mk differentiation, as well as the timing for adding nicotinamide (NIC), which greatly increases Mk ploidy. 18,29,40 …”

Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation

“…Finally, we investigated the dynamic TF activity associated with NIC promotion of MK maturation using the CHRF-288-11 (CHRF) cell line, which resembles MK progenitors (Fuhrken et al, 2007). The previously established NIC-mediated inhibition of SIRTs, as well as changes in metabolism due to increased NAD + concentration (Giammona et al, 2009), were expected to influence TF activities. TF activity arrays can provide unique perspectives on cell differentiation, which may ultimately be translated into strategies to more effectively promote production of cells in specific lineages.…”

“…MK cells are unique in that they dramatically increase their DNA content and volume by undergoing multiple rounds of endomitosis (DNA replication without cell division) to become polyploid (>4N) cells. Treatment of MK progenitor cells with nicotinamide (NIC) dramatically increases MK polyploidization in ex vivo cultures (Giammona et al, 2006, 2009; Leysi-Derilou et al, 2012), which is due, in part, to inhibition of sirtuin deacetylases (SIRTs). Nevertheless, the mechanism of NIC action remains unclear (Giammona et al, 2009).…”