Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.
Highlights d RhoA/Cdc42 DKO mice display severe macrothrombocytopenia and defective hemostasis d DKO megakaryocytes show defective cytoplasmic maturation but normal endomitosis d Loss of RhoA/Cdc42 signaling abolishes proplatelet formation d RhoA/Cdc42 signaling regulates gene expression during megakaryocyte maturation
The release of neutrophils from the bone marrow into the blood circulation is essential for neutrophil homeostasis and the protection of the organism from invading microorganisms. Granulocyte colony-stimulating factor (G-CSF) plays a pivotal role in this process and guides granulopoiesis as well as the release of bone marrow neutrophils into the blood stream both during homeostasis and in case of infection through activation of the G-CSF receptor/signal transduction and activation of transcription 3 (STAT3) signaling pathway. Here, we investigated the role of the mammalian sterile 20-like kinase 1 (MST1) for neutrophil homeostasis and neutrophil mobilization. We found increased plasma levels of G-CSF in Mst1-/- mice compared to wild type mice both under homeostatic conditions as well as after stimulation with the proinflammatory cytokine TNF-α. In addition, G-CSF-induced mobilization of neutrophils from the bone marrow into the blood circulation in vivo was markedly reduced in the absence of MST1. Interestingly, this was not accompanied by differences in the number of blood neutrophils. Addressing the underlying molecular mechanism of MST1-regulated neutrophil mobilization, we found reduced STAT3 phosphorylation and impaired upregulation of CXCR2 in Mst1-/- bone marrow neutrophils compared to wild type cells, while JAK2 phosphorylation was not altered. Taken together, we identify MST1 as a critical modulator of neutrophil homeostasis and neutrophil mobilization from the bone marrow, which adds another important aspect to the complex role of MST1 in regulating innate immunity.
Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared to extrauterine life. By transcriptomic analysis of fetal and adult neutrophils we set out to shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the non-canonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNFRII in fetal neutrophils as well as elevated levels of LT-α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells under flow.Conversely, mice with a neutrophil specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the non-canonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life, but also restricts appropriate immune responses particularly in prematurely born infants. Rohwedder et al., Neutrophil function during fetal ontogeny 4
Megakaryocytes are large cells in the bone marrow, which give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics were not affected in the absence of RhoB. However, in vitro generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, non-redundant functions in the megakaryocyte lineage.
Megakaryocytes are large cells in the bone marrow, which give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of mature cells in close proximity to bone marrow sinusoids and the formation of protrusions, which are shed into the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics were not affected in the absence of RhoB. However, in vitro generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with pronouncedly reduced levels of acetylated α-tubulin. Our findings imply that absence of this tubulin posttranslational modification results in decreased microtubule stability leading to microthrombocytopenia in RhoB-deficient mice. Our data thus points to specifically impaired microtubule - but not actin - dynamics as a general mechanism underlying the manifestation of microthrombocytopenia in vivo. We furthermore demonstrate that RhoA and RhoB have specific, non-redundant functions in the megakaryocyte lineage.KEY POINTSRhoB-deficient mice display microthrombocytopeniaRhoB has different functions in the megakaryocyte lineage than RhoA and regulates microtubule dynamics
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