Proplatelet formation is regulated by the Rho/ROCK pathway

Abstract

Platelets are released by megakaryocytes (MKs) via cytoplasmic extensions called proplatelets, which require profound changes in the microtubule and actin organization. Here, we provide evidence that the Rho/ROCK pathway, a well-known regulator of actin cytoskeleton, acts as a negative regulator of proplatelet formation (PPF). Rho is expressed at a high level during the entire MK differentiation including human CD34 ؉ cells. Thrombopoietin stimulates its activity but at a higher extent in immature than in mature MKs. Overexpression of a dominantnegative or a spontaneously active RhoA leads to an increase or a decrease in PPF indicating that Rho activation inhibits PPF. This inhibitory effect is mediated through the main Rho effector, Rho kinase (ROCK), the inhibition of which also increases PPF. Furthermore, inhibition of Rho or ROCK in MKs leads to a decrease in myosin light chain 2 (MLC2) phosphorylation, which is required for myosin contractility. Interestingly, inhibition of the MLC kinase also decreases MLC2 phosphorylation while increasing PPF. Taken together, our results suggest that MLC2 phosphorylation is regulated by both ROCK and MLC kinase and plays an important role in platelet biogenesis by controlling PPF and fragmentation. IntroductionMegakaryocytes (MKs) are the highly specialized precursor cells that lead to platelet production. MK differentiation is a continuous process characterized by sequential steps. 1 First, MKs increase their ploidy via endomitosis and begin to increase their size. 2 Then, the synthesis of storage organelles is enhanced, as well as the synthesis of plasma membrane to form the demarcation membranes. This cytoplasmic maturation is associated with a marked increase in the MK size. Finally, mature MKs release platelets probably through cytoplasmic fragmentation at the tips of long and thin extensions called proplatelets (PPTs) that contain all the platelet organelles. 3,4 The mechanisms controlling proplatelet formation (PPF) are still incompletely understood. However, PPF is associated with remarkable morphologic changes that require a profound reorganization of the cytoskeleton. 5 Increasing evidence indicates that PPTs arise from the unfolding of demarcation membranes. The microtubule cytoskeleton provides the sliding power to unfold demarcation membranes and thus to induce the pseudopodial elongations corresponding to PPTs. 6 In addition, microtubules permit the organelle transport in the PPTs and maintain the platelet discoid shape. [7][8][9][10] Although not studied in detail, the actin cytoskeleton may also participate in PPF because cytoplasmic polymerized actin is associated with demarcation membranes and actin is highly aggregated in cultured MKs when PPF occurs. 11 In addition, a crucial role of the actin cytoskeleton has been reported in platelet functions since it regulates platelet shape in unstimulated and activated platelets. 12 Evidence suggests that actin cytoskeleton may play important roles during PPT formation at 2 different stages: (1) at early stages,...

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

“…Depending on the experiments, MKs were sorted according to CD41, or CD41 and CD42 expression using Influx flow cytometer (BD, Mountain View, CA) 42 .…”

“…Immunofluorescence was performed on CD41 + MK sorted at day 6 of human culture 42 or day 2 of murine culture derived from Lin − liver fetal cells. Cells were examined under the Zeiss LSM.…”

Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

“…31,32 Recent findings have shown that NMMHC-IIA attenuates proplatelet formation, 33,34 which occurs in terminally mature megakaryocytes where most of the cytoplasm is converted into lengthy beaded extensions. 35 Thus, NMMHC-IIA might restrain thrombopoiesis until megakaryocytes accumulate sufficient quantities of the materials required for optimal platelet assembly; loss of myosin-IIA function could trigger precocious proplatelet formation, whereas gain-of-function mutations might limit platelet production.…”

Caspase-activated ROCK-1 allows erythroblast terminal maturation independently of cytokine-induced Rho signaling

“…In the erythroblast, myosin II could function in an actomyosin contractile apparatus responsible for maintaining a normal cell shape during cell condensation. Rho/ROCK-1 pathway has been shown to be involved in other differentiation systems, including proplatelet formation through MLC2 phosphorylation, 30 and keratinocyte differentiation through stratification and enlargement of cell size. 31 Recent findings also define a cytoskeleton-independent function for ROCK in regulating morphogenesis and cell size Figure 7 ROCK-1 is the main MLC2-regulating kinase.…”

“…Retrovirus MIGR-GFP and the retrovirus expressing the dominant-negative form of Rho MIGR RhoA-N19, were produced as described. 30 Erythroid cell infection. CD34 þ cells isolated from cord blood were cultured for 5 days as described then infected by shRNA GIPZ lentiviruses or MIGR-GFP retroviruses.…”