P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1

Abstract

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 Until now, the best-studied model of polyploidization concerns megakaryocytes (MKs) in which after several replicative rounds, the process of classic mitosis is replaced by an endomitotic process. MK endomitosis is very similar to mitosis, but MKs skip anaphase B, telophase, and cytokinesis giving rise to a polyploid cell with a single polylobulated nucleus. 1,2 Although polyploidization is a part of the MK differentiation program, terminal differentiation that is associated with an increased platelet protein synthesis and the development of specific organelles requires an arrest of the cell cycle. Both the mitotic and endomitotic processes require an amplification of the DNA content. However, while mitosis leads to an increase in cell number, polyploidization results in an increase in the cell size and protein mass. In the case of MK, the platelet precursor, polyploidization increases platelet production.DNA replication is a regulated mechanism that depends on a well-organized network of the cyclin-dependent kinases (CDKs) and cyclin-dependent inhibitors (CDKIs) of the cell cycle. Progression through G1 to S phase of cell cycle in mammalian cells requires the activity of G1-cyclins (D and E) associated with their catalytic subunits (CDK4 and CDK6). The mitogen-stimulated cyclin D and CDK4/6 complexes phosphorylate the retinoblastoma protein (Rb) allowing its dissociation from E2Fs transcription factors. Activated E2Fs then regulate the expression of genes necessary for DNA synthesis during the S phase of cell cycle. Cells exit cell cycle and accumulate in a quiescent G 0 /G 1 state either in the absence of mitogenic stimuli when the cyclin D-dependent activity is lost or after CDK4/6 inhibition. CDKs are regulated by inhibitory phosphorylations mediated by the WEE1 and MYT1 kinases 3 or by induction of specific inhibitors from the INK4 and CIP/KIP families. Till now, 4 members of the INK4 family (p19 INK4D , p18 INK4C , p16 INK4A , and p15 INK4B ) are described to specifically block the activity of CDK4/6 either by forming inactive ternary INK4-CDK4/6-cyclin D or binary INK4-CDK4/6 complexes. 4 The members of the CIP/KIP family (p21 CIP1/WAF1 , p27 KIP1 , and p57 KIP2 ) inhibit cyclin A-or cyclin E-associated CDK2 activity, but stabilize cyclin D-associated CDK4/6 activity. 5,6 In MKs, a high level of 2 CIP/KIP family members, p21 CIP1 and p27 KIP1 ,7,8 and 1 member of the INK4 family, p16 INK4A , 7 was detected...

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

“…7c). To better understand the regulatory role of RUNX1 in MK, we then transduced human CD34 + cells induced for MK differentiation at day 1 and day 2 of culture with a lentivirus encoding an shRNA-targeting RUNX1 (shRUNX1) 23 . As shown in Fig.…”

“…shRUNX1-containing lentivirus was previously described 23 . Two shMYH10, and control SCR containing lentiviruses were constructed as previously described 23 .…”

“…CD34 + cells were cultured with TPO and SCF. Lentiviral particles were added at a concentration of 10 7 infectious particles per 1×10 5 cells for 12 h followed by a second transduction, then cells were cultured in presence of TPO 23 .…”

“…The mRNA isolation, reverse-transcription, and RT-PCR analyses were performed as described 23 . The expression levels of all genes studied were expressed relatively to housekeeping genes, PPIA for human cells and HPRT for murine cells, where the PPIA level was not comparable to that of MYH10.…”

“…ChIP assays were performed with a ChIP assay kit (Millipore Upstate Biotechnology) using the anti-RUNX1 antibody (8 µg per sample, sc-65, Santa Cruz Biotechnology). Assays were performed using chromatin prepared from human MK as previously described 23 . Immunoprecipitated DNA was analysed on a PRISM ® 7700 sequence detection system using SYBR ® green (Applied biosystems) in duplicate.…”