Rho kinase inhibition drives megakaryocyte polyploidization and proplatelet formation through MYC and NFE2 downregulation

Abstract

SummaryThe processes of megakaryocyte polyploidization and demarcation membrane system (DMS) formation are crucial for platelet production, but the mechanisms controlling these processes are not fully determined. Inhibition of Rho kinase (ROCK) signalling leads to increased polyploidization in umbilical cord blood-derived megakaryocytes. To extend these findings we determined the effect of ROCK inhibition on development of the DMS and on proplatelet formation. The underlying mechanisms were explored by analysing the effect of ROCK inhibition on the expression of MYC and NFE2, which encode two transcription factors critical for megakaryocyte development. ROCK inhibition promoted DMS formation, and increased proplatelet formation and platelet release. Rho kinase inhibition also downregulated MYC and NFE2 expression in mature megakaryocytes, and this down-regulation correlated with increased proplatelet formation. Our findings suggest a model whereby ROCK inhibition drives polyploidization, DMS growth and proplatelet formation late in megakaryocyte maturation through downregulation of MYC and NFE2 expression.

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B

“…Given that all markers of MK maturity (such as CD41, CD42b, and the TPO receptor) were present, this underdeveloped DMS seems to be the consequence of a reduced capacity to generate these membranes. Consecutive stages of DMS organization do rely on F-actin dynamics to polarize membranes (24) and are also controlled by ROCK (34). It seems thus conceivable that the insufficiently developed membrane reservoir is in part responsible for the reduced platelet count and/or the increased platelet size in these 2B mice through dysregulation of F-actin dynamics and RhoA activity, both found to be involved in proplatelet formation defects in 2B MKs.…”

Sex-specific differences in genetic and nongenetic determinants of mean platelet volume: results from the Gutenberg Health Study

“…rs10506328 (located on chromosome 12q13.13) has been associated with the NFE2 gene which is critical for megakaryocyte development and proplatelet formation, whereas the COPZ1 gene, which encodes a subunit of coatomer protein complex 1 (COPI), is involved in intracellular traffic and autophagy. 45,46 The DOCK8 gene on chromosome 9p24.3 (rs2296828) regulates interstitial dendritic cell migration and is critical for the survival and function of natural killer T cells. 47 Family history of MI and/or stroke is known to be a major cardiovascular risk factor.…”

Kinase signaling and targeted therapy for primary myelofibrosis

“…Megakaryocyte endomitosis is precisely regulated by several signaling pathways including Aurora kinase, and the RhoA/ROCK/myosin light chain [13,14]. RhoA/ROCK signaling contributes to demarcation system (DMS) formation by regulating the remodeling and assembly of cell skeleton structure that is also necessary for megakaryocyte functions [15]. …”

“…Megakaryocyte maturation requires both polyploidization and formation of the demarcation membrane system, which facilitates platelet production. [14, 15, 46] The Rho GTPase/ROCK/Myosin pathway plays an important role in these two processes.…”

“…In MKs, the down-regulated guanine exchange factors (GEFs), including ECT2 and GEF-H1, causes the deactivation of RhoA and leads to contractile ring disassembly and cleavage furrow regression, resulting polyploidization [51]. During proplatelet formation, RhoAdriven cytoskeleton plays important roles during both early and late stages of platelet biogenesis by control the actin cytoskeleton [14, 15]. …”