Exploring the Role of Sirtuin Deacetylases in Regulating Megakaryocyte and Erythrocyte Differentiation

Exploring the role of sirtuin deacetylases in regulating megakaryocyte and erythrocyte differentiation

Mark T. Duncan, Minchang Yi, William M. Miller

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

Previously, our group has reported that nicotinamide potently enhances the polyploidization of megakaryocytes (MK) in ex vivo culture, and that this phenomenon is at least in part a consequence of its inhibition of sirtuin deacetylase activity.^1,2  We further investigated the functional role of three of the sirtuins that are found in the nucleus (SIRT1, SIRT2, and SIRT7) by silencing them in two leukemic cell lines, K562 and CHRF-288-11 (CHRF), that undergo MK differentiation in response to PMA treatment.  Despite their similar response to PMA, CHRF cells are already committed to the MK lineage, while K562 cells are bipotent for the erythroid (E) and MK lineages.  In K562 cells, only SIRT1 silencing significantly repressed MK differentiation, as indicated by moderately reduced CD41 expression and greatly reduced polyploidization.  However, since this effect was not recapitulated in CHRF cells, we considered three possible explanations for the differential dependence on SIRT1:  1) that the effect was dependent on the absence of p53 (K562 cells are p53 null, while CHRF cells express functional p53), 2) that SIRT1 is important for commitment of uncommitted progenitor cells, or 3) that K562 cells' dependence on SIRT1 was a consequence of its bcr-abl+ status.  To test the importance of the presence of p53, we performed SIRT1 silencing experiments in K562 cells with a stably integrated p53 expression vector.  Silencing of SIRT1 in these cells had a similar effect as in wildtype cells, suggesting the observed effects were not related to p53-status.  To test the possibility that SIRT1 was involved in the commitment of immature progenitor cells, we transduced CD34+ cells from mobilized peripheral blood with a SIRT1 silencing vector.  We did not observe any significant difference in the commitment or rate of differentiation of the transduced cells, as indicated by acquisition of the identifying MK antigen CD41 or CD42, which is expressed by mature MKs.  However, we did find a slight (typically ~25%) but consistent enhancement in the polyploidization of SIRT1-silenced MKs, suggesting a fundamental difference between the endomitotic programs of primary MKs and K562 cells.  Thus, our current results suggest the SIRT1-dependence of K562 cell differentiation by PMA is related to its expression of bcr-abl.

Since megakaryocytes and erythrocytes are closely related lineages, derived from a common progenitor cell, we are further investigating whether SIRT1 has a role in regulating aspects of erythropoiesis.  Preliminary experiments performed in K562 cells demonstrated that SIRT1 overexpression strongly suppresses cell proliferation, while stimulating spontaneous erythroid differentiation (indicated by increased glycophorin A expression and hemoglobinization).  This observation could have potential clinical implications for diseases including chronic myeloid leukemia, and hereditary hemoglobin disorders such as sickle cell anemia and beta-thalassemia.  We are currently investigating the mechanism of this effect, and also exploring whether it is recapitulated in primary erythrocyte differentiation.

References

1          Giammona, L. M., Fuhrken, P. G., Papoutsakis, E. T. & Miller, W. M. Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes. British Journal of Haematology 135, 554-566, doi:10.1111/j.1365-2141.2006.06341.x (2006).

2          Giammona, L. M. et al. Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD+ levels and SIRT inhibition. Experimental Hematology 37, 1340-1352.e1343, doi:10.1016/j.exphem.2009.08.004 (2009).