P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1.
The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34(+) cells, we observed that p19(INK4D) expression was increased both at the mRNA and protein levels during ploidization. p19(INK4D) knockdown led to a moderate increase (31.7% +/- 5%) in the mean ploidy of MKs suggesting a role of p19(INK4D) in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41(high)CD42(high)) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19(INK4D) overexpression in CD34(+) cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19(INK4D) KO mice exhibited an increase in mean ploidy level from 18.7N (+/- 0.58N) to 52.7N (+/- 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19(INK4D) promoter. Moreover, AML-1 inhibition led to the p19(INK4D) down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.
Gilles
Laure
L
Institut National de la Santé et de la Recherche Médicale U790, Villejuif, France.
Guièze
Romain
R
Bluteau
Dominique
D
Cordette-Lagarde
Véronique
V
Lacout
Catherine
C
Favier
Rémi
R
Larbret
Fréderic
F
Debili
Najet
N
Vainchenker
William
W
Raslova
Hana
H
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation; D050676:Core Binding Factor Alpha 2 Subunit / Q000378:metabolism*; D050764:Cyclin-Dependent Kinase Inhibitor p19 / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D008938:Mitosis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D011003:Ploidies; D011401:Promoter Regions, Genetic / Q000235:genetics
D050676:Core Binding Factor Alpha 2 Subunit; D050764:Cyclin-Dependent Kinase Inhibitor p19; D019038:Platelet Glycoprotein GPIb-IX Complex; D040201:Platelet Membrane Glycoprotein IIb