Depletion of SIRT1, but Not SIRT2, Inhibits PMA-Stimulated Megakaryocytic Differentiation of the K562 Cell Line

Depletion of SIRT1, but not SIRT2, inhibits PMA-stimulated megakaryocytic differentiation of the K562 cell line

Mark T. Duncan*, Zachary Mays, Nitin Kini, William M. Miller

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, United States of America

*email: mtduncan@northwestern.edu

Sirtuins (SIRT1-7 in humans) are class III NAD-dependent histone/protein deacetylases that have been implicated in modulating the differentiation of many cell types including adipocytes and endothelial, neural, and skeletal muscle cells.  Previously, we have demonstrated that treatment with the pan-SIRT inhibitor Nicotinamide (NIC) greatly enhances the polyploidization of human megakaryocytes (MKs) derived from CD34+ cells in culture.  This effect was found to be, at least in part, due to inhibition of SIRT1/2 deacetylases, as similar effects were recapitulated by the SIRT1/2 specific inhibitor cambinol.  We are currently using the cell line K562 to further investigate the roles of SIRT1/2 in MK differentiation. Addition of phorbol 12-myristate 13-acetate (PMA) induces K562 cells to express the MK antigen CD41 and exhibit morphological characteristics of MK maturation, including enlargement of cell size, polyploidization, and the appearance of cytoplasmic vacuoles.  Similar to primary cells, NIC treatment of differentiating K562 cells led to an increased fraction of polyploid cells.  In addition, NIC treatment reduced cell expansion and appeared to increase/accelerate apoptosis.  Surprisingly, silencing of SIRT1 with lentiviral shRNAs strongly inhibited PMA-mediated differentiation.  We consistently observed a moderate reduction in CD41 expression, and a >50% reduction in polyploidization in SIRT1-silenced cells. SIRT2 silencing did not appear to affect differentiation.  Thus, we hypothesized that SIRT1 may have an important role in PMA-induced cell signaling, and confirmed its high expression in K562 cells via immunostaining.  Unexpectedly, SIRT1 appeared to be predominantly localized to the cytosol (both pre- and post-PMA stimulation). We are currently investigating a possible role for cytosolic SIRT1 in PMA-induced MAP kinase signaling, as a previous report suggests SIRT1 can enhance ERK1/2 activation.¹  In addition, we have begun to examine the effects of SIRT1 silencing on the differentiation of human MKs derived from CD34+ cells in culture.  In an initial experiment, SIRT1 silencing reduced MK expansion, again suggesting SIRT1's importance in regulating the cell cycle (though whether this is through a similar or distinct mechanism from the K562 model cell line remains to be determined).  We are also currently exploring knockdown of SIRT7, which is upregulated during primary Mk differentiation. 

References

1          Huang, J. et al. SIRT1 Overexpression Antagonizes Cellular Senescence with Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts. PLoS ONE 3, e1710, doi:10.1371/journal.pone.0001710 (2008).