The Role of Microtubule Regulatory Protein Stathmin (STMN1) in Megakaryopoiesis.

Abstract 4591

Megakaryopoiesis is a complex process in which hematopoietic progenitor cells proliferate and acquire megakaryocyte (MK)-specific markers then undergo polyploidization (i.e. acquisition of DNA content >2n) and cytoplasmic maturation, and start producing platelets. Polyploidization and platelet formation are highly dependent on microtubule (MT) function. To become polyploid, MK undergo abortive mitosis that is mediated by a mitotic spindle that consists of MT. Mature polyploid MK extend cytoplasmic extensions (i.e. proplatelets) into the vascular space and release platelets into the circulation. MT provide the structural scaffold for the proplatelets and mediate the transport of organelles and specific granules into nascent platelets. Despite the critical role of MT in MK biology, the regulation of MT in MK is poorly defined. Stathmin (STMN1) is a cytosolic phosphoprotein whose major function is to regulate MT function by promoting their depolymerization. We had previously shown that STMN1 is expressed at high levels early during megakaryopoiesis and is downregulated later during MK maturation. We also showed that inhibition of STMN1 expression increased ploidy while its overexpression decreased ploidy of MK-like cell lines. Thus, we hypothesized that the dynamic regulation of STMN1 expression may be necessary for megakaryopoiesis and that perturbing its expression may impair MK polyploidization and platelet production. To test this hypothesis, we developed feline immunodeficiency virus (FIV)-based lentiviruses that express STMN1 to investigate the effects of overexpression in primary MK. Since the depolymerizing activity of STMN1 can be inactivated by a variety of cellular kinases, we generated a STMN1 vectors that expresses wild-type (WT) and another that expresses a contitutively active phosphorylation-deficient mutant of STMN1 (MT). We also developed a vector that expresses GFP as a negative control. Human MK generated ex vivo in liquid culture from CD34+ cells were infected with these different lentiviruses. After ectopic STMN1 expression by RT-PCR and flow cytometry was confirmed, MK differentiation was assessed in the presence or absence of STMN1 overexpression. Uninfected MK and MK infected with GFP lentiviruses differentiated and matured into large, easily recognizable cells with typical nuclear morphology and expressed similar levels of CD41 and CD42b by flow cytometry. The numbers of MK generated in the presence of WT-STMN1 expressing lentiviruses was similar to that generated in the cultures infected with control lentiviruses, while the number of MK generated in the presence of phosphorylation-deficient MT-STMN1 was drasticaly reduced. Similarly, the numbers of CD41⁺ and CD42b⁺ MK generated in the presence of MT-STMN1 was reduced two and three times, respectively, suggesting that overexpression of a contitutively active form of STMN1 prevents MK differentiation and maturation. We then evaluated the effects of STMN1 overexpression on MK polyploidization by determining the number X and Y chromosomes by FISH analysis. While a normal diploid cell has one copy of each chromosome, cells with ploidy levels of 4N, 8N and 16N will have 2, 4 and 8 copies, respectively. There was no significant difference between the fraction of polyploid MK infected with control-GFP and those infected with WT-STMN1 lentiviruses. In contrast, the fraction of polyploid MK infected with MT-STMN1 lentiviruses was reduced by approximately 50%, suggesting that STMN1 overexpression impairs the ability of MK to become polyploid. In conclusion, we demonstrated that perturbing the normal downregulation of STMN1 in primary human MK impairs differentiation and polyploidization. Since STMN1 is expressesd at extremely high levels in a variety of human leukemias, we have started assessing STMN1 expression expression in patients with hematological malignancies characterized by striking abnormalities in their MK lineage. Such studies might validate the role of MT regulation in MK biology in vivo and support the development of potential therapeutic strategies to target MT and/or STMN1 function in MK and platelet disorders.