Actin reorganization and proplatelet formation in murine megakaryocytes: the role of protein kinase Cα

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 154-161 ◽  
Author(s):  
Ponlapat Rojnuckarin ◽  
Kenneth Kaushansky
Keyword(s):  
Protein Kinase ◽  
Actin Polymerization ◽  
Map Kinases ◽  
Molecular Mechanisms ◽  
Marrow Cell ◽  
Actin Dynamics ◽  
Actin Reorganization ◽  
Murine Bone Marrow ◽  
Proplatelet Formation

Abstract With the recent cloning and characterization of thrombopoietin, appreciation of the molecular events surrounding megakaryocyte (MK) development is growing. However, the final stages of platelet formation are less well understood. Platelet production occurs after the formation of MK proplatelet processes. In a study to explore the molecular mechanisms underlying this process, mature MKs isolated from suspension murine bone marrow cell cultures were induced to form proplatelets by exposure to plasma, and the role of various cell-signaling pathways was assessed. The results showed that (1) bis-indolylmaleimide I, which blocks protein kinase C (PKC) activation; (2) down-modulation of conventional or novel classes of PKC by phorbol myristate acetate; and (3) ribozymes specific for PKCα each inhibited proplatelet formation. Inhibition of several MAP kinases, PI3 kinase, or protein kinase A failed to affect MK proplatelet formation. To gain further insights into the function of PKCα in proplatelet formation, its subcellular localization was investigated. In cultures containing active proplatelet formation, cytoplasmic polymerized actin was highly aggregated, its subcellular distribution was reorganized, and PKCα colocalized with the cellular actin aggregates. A number of MK manipulations, including blockade of integrin signaling with a disintegrin or inhibition of actin polymerization with cytochalasin D, interrupted actin reorganization, PKC relocalization, and proplatelet formation. These findings suggest an important role for PKCα in proplatelet development and suggest that it acts by altering actin dynamics in proplatelet-forming MKs. Identification of the upstream and downstream pathways involved in proplatelet formation should provide greater insights into thrombopoiesis, potentially allowing pharmacologic manipulation of the process.

The Molecular Mechanisms of Serotonin in the Regulation of Megakaryocytopoiesis and TPO Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3347-3347
Author(s):  
Mo Yang ◽  
Fanyi Meng ◽  
Jie Yu Ye ◽  
Yue Xu ◽  
Bin Xiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Promoting Effect ◽  
Actin Reorganization ◽  
Cord Blood Cd34 ◽  
5Ht2 Receptors ◽  
Proplatelet Formation ◽  
Subsequent Activation

Abstract Abstract 3347 We have reported that serotonin (5-HT) show a promoting effect on cord blood CD34+ stem/progenitor cells (Yang et al, Stem Cells 2007). We also demonstrated that serotonin enhances murine megakaryopoiesis via 5-HT2 receptors (Yang et al. Blood Coagul Fibrinol 1996). In this present study, we explored how serotonin regulated human megakaryocytopoiesis, proplatelet formation, and thrombopoietin (TPO) production. Our results indicated that serotonin significantly promoted human CFU-MK formation and reduced apoptosis in megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression. Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryocytopoiesis through stimulating release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO by q-PCR, ELISA and cytokine-array assays. Our findings demonstrated an important role of serotonin played on megakaryocytopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs, which indirectly promoted megakaryopoiesis. In present studies, we have demonstrated a positive “feed-back” control loop between MK-derived granule- serotonin and megakaryocytopoiesis. These findings improved our knowledge on megakaryocytopoiesis regulation and provided new clues on identifying novel thrombopoietic agents. It also deepened our understandings on how TPO production is regulated. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of Long Non-Coding RNAs and the Molecular Mechanisms Involved in Insulin Resistance

2021 ◽  
Vol 22 (14) ◽  
pp. 7256
Author(s):  
Vianet Argelia Tello-Flores ◽  
Fredy Omar Beltrán-Anaya ◽  
Marco Antonio Ramírez-Vargas ◽  
Brenda Ely Esteban-Casales ◽  
Napoleón Navarro-Tito ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Biological Species ◽  
Necrosis Factor Alpha ◽  
Polypyrimidine Tract Binding Protein ◽  
Non Coding Rnas

Long non-coding RNAs (lncRNAs) are single-stranded RNA biomolecules with a length of >200 nt, and they are currently considered to be master regulators of many pathological processes. Recent publications have shown that lncRNAs play important roles in the pathogenesis and progression of insulin resistance (IR) and glucose homeostasis by regulating inflammatory and lipogenic processes. lncRNAs regulate gene expression by binding to other non-coding RNAs, mRNAs, proteins, and DNA. In recent years, several mechanisms have been reported to explain the key roles of lncRNAs in the development of IR, including metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), imprinted maternal-ly expressed transcript (H19), maternally expressed gene 3 (MEG3), myocardial infarction-associated transcript (MIAT), and steroid receptor RNA activator (SRA), HOX transcript antisense RNA (HOTAIR), and downregulated Expression-Related Hexose/Glucose Transport Enhancer (DREH). LncRNAs participate in the regulation of lipid and carbohydrate metabolism, the inflammatory process, and oxidative stress through different pathways, such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), polypyrimidine tract-binding protein 1/element-binding transcription factor 1c (PTBP1/SREBP-1c), AKT/nitric oxide synthase (eNOS), AKT/forkhead box O1 (FoxO1), and tumor necrosis factor-alpha (TNF-α)/c-Jun-N-terminal kinases (JNK). On the other hand, the mechanisms linked to the molecular, cellular, and biochemical actions of lncRNAs vary according to the tissue, biological species, and the severity of IR. Therefore, it is essential to elucidate the role of lncRNAs in the insulin signaling pathway and glucose and lipid metabolism. This review analyzes the function and molecular mechanisms of lncRNAs involved in the development of IR.

scholarly journals Role of Calcium and EPAC in Norepinephrine-Induced Ghrelin Secretion

Endocrinology ◽  
2014 ◽  
Vol 155 (1) ◽  
pp. 98-107 ◽  
Author(s):  
Bharath K. Mani ◽  
Jen-Chieh Chuang ◽  
Lilja Kjalarsdottir ◽  
Ichiro Sakata ◽  
Angela K. Walker ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Intracellular Signaling ◽  
Endocrine Cells ◽  
Molecular Mechanisms ◽  
Phosphodiesterase Inhibitor ◽  
Intracellular Signaling Pathways ◽  
Ghrelin Secretion ◽  
Kinase A

Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to β1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca2+ and cAMP. Several voltage-gated Ca2+ channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca2+ levels both in the presence and absence of extracellular Ca2+. Ca2+-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca2+ influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca2+. Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.

Withaferin A binds covalently to the N-terminal domain of annexin A2

2012 ◽  
Vol 393 (10) ◽  
pp. 1151-1163 ◽  
Author(s):  
Gabriel Ozorowski ◽  
Christopher M. Ryan ◽  
Julian P. Whitelegge ◽  
Hartmut Luecke
Keyword(s):  
Actin Polymerization ◽  
Inhibitory Effect ◽  
Annexin A2 ◽  
Actin Dynamics ◽  
Withaferin A ◽  
Core Domain ◽  
Cancer Pathways ◽  
Terminal Domain ◽  
C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

scholarly journals Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

2010 ◽  
Vol 21 (20) ◽  
pp. 3529-3539 ◽  
Author(s):  
Tim Ting Chiu ◽  
Nish Patel ◽  
Alisa E. Shaw ◽  
James R. Bamburg ◽  
Amira Klip
Keyword(s):  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Muscle Cells ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Glut4 Translocation ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Rac Gtpase ◽  
Skeletal Myoblasts

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

scholarly journals The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Céline Michard ◽  
Daniel Sperandio ◽  
Nathalie Baïlo ◽  
Javier Pizarro-Cerdá ◽  
Lawrence LeClaire ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Actin Polymerization ◽  
Secretion System ◽  
Endocytic Pathway ◽  
Specific Chemical ◽  
Actin Remodeling ◽  
Content Type

ABSTRACTLegionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role inLegionellainfection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation byListeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to theLegionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses.IMPORTANCEDeciphering the individual contribution of each Dot/Icm type 4 secretion system substrate to the intracellular life-style ofL. pneumophilaremains the principal challenge in understanding the molecular basis ofLegionellavirulence. Our finding that LegK2 is a Dot/Icm effector that inhibits actin polymerization on theLegionella-containing vacuole importantly contributes to the deciphering of the molecular mechanisms evolved byLegionellato counteract the endocytic pathway. Indeed, our results highlight the essential role of LegK2 in preventing late endosomes from fusing with the phagosome. More generally, this work is the first demonstration of local actin remodeling as a mechanism used by bacteria to control organelle trafficking. Further, by characterizing the role of the bacterial protein kinase LegK2, we reinforce the concept that posttranslational modifications are key strategies used by pathogens to evade host cell defenses.

ADF/n-cofilin–dependent actin turnover determines platelet formation and sizing

Blood ◽  
2010 ◽  
Vol 116 (10) ◽  
pp. 1767-1775 ◽  
Author(s):  
Markus Bender ◽  
Anita Eckly ◽  
John H. Hartwig ◽  
Margitta Elvers ◽  
Irina Pleines ◽  
...  
Keyword(s):  
Actin Filament ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Actin Dynamics ◽  
Complex Process ◽  
First Time ◽  
Late Stages ◽  
Platelet Formation

Abstract The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin–mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.

scholarly journals Mechanisms for actin reorganization in chemotactic factor-activated polymorphonuclear leukocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2750-2757
Author(s):  
RG Watts ◽  
TH Howard
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Actin Polymerization ◽  
Insoluble Fraction ◽  
Chemotactic Factor ◽  
Actin Dynamics ◽  
Cross Linking ◽  
Actin Reorganization ◽  
Cytoskeletal Structure ◽  
Cross Links ◽  
Human Pmns

Cytoskeletal structure in polymorphonuclear leukocytes (PMNs) is thought to reflect a simple equilibrium between two actin pools (globular [G]- and filamentous [F] actin). Recent description of two distinct F-actin pools in PMNs (Triton-insoluble [stable] and Triton- soluble [labile] F-actin pools) (Watts and Howard, Cell Motil Cytoskeleton, 21:25, 1992) suggest a tripartite equilibrium between these F-actin pools and G-actin and multiple possible mechanisms for polymerization. To study the contribution of each actin pool to actin dynamics in PMNs, changes in actin content of the Triton-soluble and - insoluble F-actin pools and G-actin in chemotactic factor (CTF)- activated PMNs were measured by NBDphallacidin binding and by gel scans of Triton-lysed PMNs. From 0 to 30 seconds after CTF activation, PMNs rapidly increase total (Triton-soluble + Triton-insoluble) F-actin content (maximum = 1.7- +/- 0.10-fold basal at 30 seconds). Concurrent measures of the actin content of individual actin pools (Triton-soluble and -insoluble F-actin and G-actin) show that at all times (0 to 30 seconds) only the Triton-insoluble F-actin pool grows (maximum = 2.81- +/- 0.73-fold basal at 30 seconds), whereas both the Triton-soluble and G-actin pools simultaneously decrease (50% decrease at 30 seconds). Concurrent growth of one F-actin pool (Triton-insoluble) and loss of another F-actin pool (Triton-soluble) emphasize the functional uniqueness of the F-actin pools and can occur only if the Triton- soluble F-actin anneals or cross-links filament-to-filament with the Triton-insoluble fraction or if the Triton-insoluble F-actin pool first depolymerizes to monomer, which is then added to the Triton-insoluble pool. Because from 0 to 30 seconds after FMLP activation G-actin never increases, but, like the Triton-soluble F-actin progressively decreases, the results suggest that F-actin growth results from simultaneous new filament growth by monomer addition to the Triton- insoluble F-actin and cytoskeletal remodelling by Triton-soluble F- actin annealing or cross-linking to Triton-insoluble F-actin. These findings offer important new insights into the mechanism(s) of actin polymerization in CTF-activated human PMNs.

scholarly journals Airway smooth muscle tone increases actin filamentogenesis and contractile capacity

2020 ◽  
Vol 318 (2) ◽  
pp. L442-L451
Author(s):  
Morgan Gazzola ◽  
Cyndi Henry ◽  
Katherine Lortie ◽  
Fatemeh Khadangi ◽  
Chan Young Park ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Muscle Tone ◽  
Coiled Coil ◽  
Traction Force

Force adaptation of airway smooth muscle (ASM) is a process whereby the presence of tone (i.e., a sustained contraction) increases the contractile capacity. For example, tone has been shown to increase airway responsiveness in both healthy mice and humans. The goal of the present study is to elucidate the underlying molecular mechanisms. The maximal force generated by mouse tracheas was measured in response to 10−4 M of methacholine following a 30-min period with or without tone elicited by the EC30 of methacholine. To confirm the occurrence of force adaptation at the cellular level, traction force generated by cultured human ASM cells was also measured following a similar protocol. Different pharmacological inhibitors were used to investigate the role of Rho-associated coiled-coil containing protein kinase (ROCK), protein kinase C (PKC), myosin light chain kinase (MLCK), and actin polymerization in force adaptation. The phosphorylation level of the regulatory light chain (RLC) of myosin, the amount of actin filaments, and the activation level of the actin-severing protein cofilin were also quantified. Although ROCK, PKC, MLCK, and RLC phosphorylation was not implicated, force adaptation was prevented by inhibiting actin polymerization. Interestingly, the presence of tone blocked the activation of cofilin in addition to increasing the amount of actin filaments to a maximal level. We conclude that actin filamentogenesis induced by tone, resulting from both actin polymerization and the prevention of cofilin-mediated actin cleavage, is the main molecular mechanism underlying force adaptation.

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