Proplatelet Formation Is Regulated by the Rho/Rock Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Yunhua Chang ◽  
Frederic Aurade ◽  
Frederic Larbret ◽  
Jean Pierre Le Couedic ◽  
Laurence Momeux ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Actin Dynamics ◽  
Rock Inhibitor ◽  
Actin Reorganization ◽  
Human Cd34 ◽  
Myosin Light Chain 2 ◽  
Proplatelet Formation ◽  
Cytoskeleton Dynamics ◽  
Mlc Phosphorylation

Abstract Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.

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.

Gene regulation by mechanotransduction in fibroblasts

2007 ◽  
Vol 32 (5) ◽  
pp. 967-973 ◽  
Author(s):  
Matthias Chiquet ◽  
Vildan Tunç-Civelek ◽  
Ana Sarasa-Renedo
Keyword(s):  
Mechanical Stress ◽  
Cyclic Strain ◽  
Actin Dynamics ◽  
Transcriptional Level ◽  
Rock Inhibitor ◽  
Messenger Ribonucleic Acid ◽  
Tenascin C ◽  
Mrna Induction

Mechanical forces are important for connective tissue homeostasis. How do fibroblasts sense mechanical stress and how do they translate this information into an adaptive remodeling of the extracellular matrix (ECM)? Tenascin-C is rapidly induced in vivo by loading muscles and in vitro by stretching fibroblasts. Regulation of tenascin-C expression by mechanical signals occurs at the transcriptional level. Integrin receptors physically link the ECM to the cytoskeleton and act as force transducers: intracellular signals are triggered when integrins engage with ECM, and later when forces are applied. We found that cyclic strain does not induce tenascin-C messenger ribonucleic acid (mRNA) in fibroblasts lacking the β1-integrin chain. An important link in integrin-dependent mechanotransduction is the small guanosine 5′-triphosphatase. RhoA and its target kinase, ROCK. In fibroblasts, cyclic strain activates RhoA and thereby induces ROCK-dependent actin assembly. Interestingly, tenascin-C mRNA induction by cyclic strain was suppressed by relaxing the cytoskeleton with a ROCK inhibitor or by actin depolymerization. Conversely, chemical activators of RhoA enhanced the effect of strain both on actin dynamics and on tenascin-C expression. Thus, RhoA/ROCK-controlled actin dynamics are required for the induction of specific ECM genes by mechanical stress. These findings have implications for the understanding of regeneration and for tissue engineering.

scholarly journals Enhancing Functional Platelet Release In Vivo from in Vitro-Grown Megakaryocytes Using the Protein Kinase Inhibitor SU6656

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1029-1029
Author(s):  
Danuta Jadwiga Jarocha ◽  
Karen K Vo ◽  
Randolph B Lyde ◽  
Vincent M Hayes ◽  
Mortimer Poncz
Keyword(s):  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Rock Inhibitor ◽  
Medicine Research ◽  
Nsg Mice ◽  
Platelet Release

Abstract The clinical demand for platelet transfusions is increasing, threatening the ability to obtain sufficient healthy donors to provide these platelets. Advances in regenerative medicine research have opened the possibility of generating sufficient in vitro-grown megakaryocytes and consequent platelets to supply a portion of the clinical platelet transfusion demand. We have shown that infusing megakaryocytes for obtaining released, functional platelets is a viable alternative strategy than trying to release platelets in vitro. However, for both approaches, in vitro-cultured megakaryocytes have lower ploidy and release fewer platelets than likely occurs in vivo by primary cells. SU6656 inhibitor, a Src kinase inhibitor, has been shown to influence ploidization in several megakaryocyte-like line with purported increase in proplatelets release. However, in our hands, other agents - such as the ROCK inhibitor Y27632 - while increasing polyploidization markedly, inhibited platelet release per infused megakaryocyte in vivo. We grew megakaryocytes from CD34+ cells for 12 days with or without SU6656 (2.5 µM) supplementation during the last 4 days. We found that the SU6656 inhibitor only increased the number of CD34+-derived megakaryocytes by ~15% at the end of the 12 day growth, but more markedly increase the percent of large megakaryocytes measured by FSC parameter in flow cytometry evaluation from 28 up to 41% and percent of high granular megakaryocytes from 27 to 45%. These changes were accompanied with a shift in average ploidy from 4.9 to 6.9 (p<0.0003, N=6). Notably, SU6656-treated megakaryocytes released ~4-fold more platelets per infused megakaryocytes in immunocompromized NSG mice than untreated similarly in vitro-grown megakaryocytes. By 24 hrs, there were 6.5-fold platelets from the infused SU6656-treated megakaryocytes than control untreated (p<0.037, N=6). Released platelets from the drug-treated and untreated megakaryocytes had similar levels of percent thiazole orange positivity as an indication that they were young platelets. Importantly, baseline annexin V, CD62p and PAC1 binding prior to agonist exposure were also similarly and increased to the same extent after thrombin (1U/ml) stimulation. Additionally, incorporation into a growing cremaster laser injury-induced thrombus in vivo was similar further indicating retained function by the platelets released from the drug-treated megakaryocytes. A number of strategies such as modifying the level of transcription factors have been proposed to increase the size, ploidy or proplatelets release from in vitro-grown megakaryocytes. In none of these cases have these released platelets in vivo biology been examined and demonstrated to replicate high release number per megakaryocyte and retained functionality. We show that terminal exposure of in vitro-grown megakaryocytes to the non-specific inhibitor SU6656 significantly increases in vivo yield while leaving in vivo half-life and functionality intact. The exact pathway affected by SU6656 that leads to these results is now being pursued. Disclosures No relevant conflicts of interest to declare.

scholarly journals Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Xiaolin Chen ◽  
Jianhui Chen ◽  
Xianfan Li ◽  
Zengpu Yu
Keyword(s):  
Fluorescence Probe ◽  
Endothelial Permeability ◽  
Barrier Dysfunction ◽  
Actin Reorganization ◽  
Barrier Disruption ◽  
Rhoa Activity ◽  
Filtration Barrier ◽  
Activation Assay ◽  
Mlc Phosphorylation

Abstract Objective Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro. Materials and methods HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed. Results HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure. Conclusions Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

Effectiveness of a Novel Humanized VB22B Minibody against Human TPO Receptor Compared with Recombinant TPO and Eltrombopag Using Primary Human CD34+ Cells and Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4559-4559
Author(s):  
Eri Matsuki ◽  
Akiko Yamane ◽  
Shinichiro Okamoto ◽  
Yoshitaka Miyakawa
Keyword(s):  
Bone Marrow ◽  
Platelet Aggregation ◽  
Colony Formation ◽  
Human Platelets ◽  
Receptor Agonists ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proplatelet Formation

Abstract Abstract 4559 Thrombopoietin (TPO) is a cytokine produced primarily by the liver and kidney that regulates platelet production by stimulating proliferation and differentiation of hematopoietic stem cells, megakaryocytic progenitor cells and megakaryocytes via activation of its receptor, c-Mpl. Recently, TPO receptor agonists such as eltrombopag and romiplostim have been approved for chronic ITP. huVB22B was created as a novel humanized form of murine sc(Fv) 2VB22B minibody (BLOOD, 2005) which activates human c-Mpl by CDR grafting. The advent of these various TPO receptor agonists prompted us to consider the differences in their mechanisms of action, efficacy or potency. However, to date, there has been no in vivo or in vitro study directly comparing the effects of different TPO receptor agonists. In this study, we compared the efficacy of huVB22B on CFU-GM, CFU-E, CFU-Megakaryocyte (CFU-MK), megakaryocyte maturation (DNA ploidy and proplatelet formation) with those of recombinant human TPO (rhTPO) and eltrombopag. Primary human CD34+ bone marrow cells were cultured with various concentrations of rhTPO, huVB22B and eltrombopag using methylcellulose based media. In serum-free condition, 0.286 nM rhTPO, 0.182 nM huVB22B and 17.7 mcM eltrombopag demonstrated almost equivalent efficacy of megakaryocyte colony formation. At these concentrations, all agents demonstrated similar in vitro efficacy for colony formation of CFU-GM and CFU-E, proplatelet formation and nuclear maturation of megakaryocytes. In preliminary results, huVB22B induced maturation of CFU-MK earlier than rhTPO and eltrombopag, suggesting that huVB22B might have some potential to increase human platelets faster than other agents in vivo. This is compatible with the observation that huVB22B induced tyrosine phosphorylation of STAT3, STAT5 and JAK2 faster and stronger than rhTPO and eltrombopag in human primary platelets. Both rhTPO and huVB22B enhanced low-dose ADP and collagen-induced human platelet aggregation in vitro. In contrast, eltrombopag did not enhance ADP or collagen-induced platelet aggregation, although it induced activation of JAK-STAT pathway in human platelets. Contrary to the fact that huVB22B induces phosphorylation of intracellular signaling molecules faster and stronger than rhTPO in human platelets, the priming effect by huVB22B on platelet aggregation was much weaker than rhTPO. In conclusion, we confirmed that newly created huVB22B minibody induced colony formation of CFU-MK, CFU-E, CFU-GM and maturation of megakaryocytes from human bone marrow-derived CD34+ cells in vitro. The differences among TPO receptor agonists observed in our study would lead to further understanding of the basic biology of megakaryopoiesis and the action of TPO receptor agonists. Disclosures: Okamoto: Alexion: Research Funding. Miyakawa:GlaxoSmithKline: Consultancy.

scholarly journals Activation of mtor Mediates Hyperglycemia-Induced Renal Glomerular Endothelial Hyperpermeability via the RhoA/ROCK/pmlc Signaling Pathway

2021 ◽  
Author(s):  
Jianhui Chen ◽  
Xianfan Li ◽  
Xiaolin Chen ◽  
Zengpu Yu
Keyword(s):  
Fluorescence Probe ◽  
Endothelial Permeability ◽  
Barrier Dysfunction ◽  
Actin Reorganization ◽  
Barrier Disruption ◽  
Rhoa Activity ◽  
Filtration Barrier ◽  
Activation Assay ◽  
Mlc Phosphorylation

Abstract OBJECTIVE: Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro.MATERIALS AND METHODS: HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed.RESULTS: HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure.CONCLUSIONS: Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

scholarly journals Development of Poly Lactic/Glycolic Acid (PLGA) Microspheres for Controlled Release of Rho-Associated Kinase Inhibitor

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Sho Koda ◽  
Naoki Okumura ◽  
Junji Kitano ◽  
Noriko Koizumi ◽  
Yasuhiko Tabata
Keyword(s):  
High Performance ◽  
Glycolic Acid ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Double Emulsion ◽  
Plga Microspheres ◽  
Rock Inhibitor ◽  
Drug Delivery Carrier ◽  
Composition Ratios

Purpose. The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA) as a drug delivery carrier of Rho kinase (ROCK) inhibitor for the treatment of corneal endothelial disease. Method. ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1), and a double emulsion [(W1/O)/W2] was formed with dichloromethane (O) and polyvinyl alcohol (W2). Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. Results. Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. Conclusions. ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7–10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases.

Dasatinib Induced Thrombocytopenia Might Be Due To The Inhibition Of Proplatelet Formation Of Megakaryocytes Via The Pathways Including Rho/Rock and Rac

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1071-1071
Author(s):  
Kazunori Murai ◽  
Shugo Kowata ◽  
Yukiteru Fujishima ◽  
Yasuhiko Tsukushi ◽  
Takahiro Mine ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Biological Effects ◽  
Physiological Concentration ◽  
Western Blots ◽  
Minimum Concentration ◽  
Proplatelet Formation ◽  
Molecular Signals

Abstract Background Dasatinib is a highly potent BCR-ABL inhibitor with established efficacy and safety in imatinib-resistant/intolerant patients with chronic myeloid leukemia (CML). The DASISION reported a significantly higher rate of CCyR after 1 year (83%), MMR after 1 year (46%) and CMR4.5 after 3 years (22%). Grade 3/4 thrombocytopenia was more frequent adverse events. Although Mazharian et al (Blood 2011) reported the inhibition of PPF by dasatinib, molecular signals in dasatinib-induced PPF inhibition has remained unclear. In this study, we elucidated the biological effects of dasatinib on proliferation and maturation of megakaryocytic progenitor cells and maturation of megakaryocytes. In addition, the molecular signals of G-proteins were revealed in the process of PPF inhibition by dasatinib. Methods and Materials To evaluate the biological effects of dasatinib, the following experiments were performed in the presence of dasatinib; (1) CFU-Meg assay in vitro culture of murine bone marrow (BM) cells with murine TPO, IL-3 and IL-6, (2) megakaryocyte ploidy analysis in vitro culture of murine BM MNCs with murine TPO and SCF, (3) proplatelet formation (PPF) assay in vitro culture of murine megakaryocytes. To evaluate the molecular signals in inhibited PPF by dasatinib, Western blots were performed. Activated Rho, Rac, phosphorylated myosin light chain (p-MLC), phosphorylated Lyn (p-Lyn) and p-Src in murine platelets were evaluated. Results (1) CFU-Megs (colonies per 1x 105 BM-MNCs) did not decrease significantly at 0.01, 0.1, 1, 10, 100 nM (dasatinib 0 nM; 32.6 ± 13.1, 0.1 nM: 27.6 ± 8.4, 1 nM: 24.8 ± 7.8, 10 nM: 28.8 ± 12.1, 100 nM: 23.0 ± 7.6). (2) There was no difference in DNA ploidy of cultured megakaryocytes in the presence of dasatinib (0, 0.01 to 100 nM). (3) PPF decreased significantly at 1, 10, 100 nM of dasatinib (0 nM: 40.6 ± 2.5%, 1 nM: 29.4 ± 2.4% p<0.01, 10 nM: 23.6 ± 1.7% p<0.01, 100 nM: 24.0± 2.4 % p<0.01). According to the previous reports as to the plasama concentrations of dasatinib, minimum concentration was 2∼5nM. These results indicated that dasatinib did not suppress megakaryocytic proliferation and differentiation but did inhibit PPF of megakaryocytes at the physiological concentration of dasatinib. Activated Rho, Rac, p-MLC and p-Lyn in murine platelets, incubated in the presence of dasatinib, were evaluated by the Western blots. Activated Rho and p-MLC were up-regulated and activated Rac was down-regulated at 50 nM of dasatinib. Dasatinib reduced levels of p-Lyn and p-Src. Therefore we added Y27632, which was the ROCK (Rho kinase) inhibitor, to the megakaryocytes culture in the presence of dasatinib to evaluate the PPF activity. The inhibition of PPF by dasatinib was cancelled significantly by the addition of Y27632 (control; 25.4 ± 0.9%, dasatinib 100 nM; 12.4 ± 1.3% p<0.01, Y27632 10 μM; 34.5 ± 2.0% p<0.01, dasatinib100 nM + Y27632 10μM; 27.5 ± 1.5%). Furthermore, to elucidate the precise role of Rac pathways in PPF, NSC23776, Rac inhibitor, was added to megakaryocytes culture for PPF. PPF decreased significantly in the presence of NSC23776 (0 nM: 29.8 ± 4.7%, 50 µM: 12.1 ± 1.7% p<0.01). Conclusion Dasatinib induced thrombocytopenia might not be due to the inhibition of megakaryocyte colony formations but the inhibition of PPF of megakaryocytes via pathways including Rho/Rock and Rac. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of endothelin type B receptors (EDNRB) on smooth muscle cells is controlled by MKL2, ternary complex factors, and actin dynamics

2018 ◽  
Vol 315 (6) ◽  
pp. C873-C884 ◽  
Author(s):  
Katarzyna K. Krawczyk ◽  
Gry Freja Skovsted ◽  
Ljubica Perisic ◽  
Rasmus Dreier ◽  
Jais Oliver Berg ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ternary Complex ◽  
Transcriptional Control ◽  
Muscle Cells ◽  
Actin Dynamics ◽  
Type B ◽  
Human Coronary Artery ◽  
Rock Inhibitor

The endothelin type B receptor (ETB or EDNRB) is highly plastic and is upregulated in smooth muscle cells (SMCs) by arterial injury and following organ culture in vitro. We hypothesized that this transcriptional plasticity may arise, in part, because EDNRB is controlled by a balance of transcriptional inputs from myocardin-related transcription factors (MRTFs) and ternary complex factors (TCFs). We found significant positive correlations between the TCFs ELK3 and FLI1 versus EDNRB in human arteries. The MRTF MKL2 also correlated with EDNRB. Overexpression of ELK3, FLI1, and MKL2 in human coronary artery SMCs promoted expression of EDNRB, and the effect of MKL2 was antagonized by myocardin (MYOCD), which also correlated negatively with EDNRB at the tissue level. Silencing of MKL2 reduced basal EDNRB expression, but depolymerization of actin using latrunculin B (LatB) or overexpression of constitutively active cofilin, as well as treatment with the Rho-associated kinase (ROCK) inhibitor Y27632, increased EDNRB in a MEK/ERK-dependent fashion. Transcript-specific primers indicated that the second EDNRB transcript (EDNRB_2) was targeted, but this promoter was largely unresponsive to LatB and was inhibited rather than stimulated by MKL2 and FLI1, suggesting distant control elements or an indirect effect. LatB also reduced expression of endothelin-1, but supplementation experiments argued that this was not the cause of EDNRB induction. EDNRB finally changed in parallel with ELK3 and FLI1 in rat and human carotid artery lesions. These studies implicate the actin cytoskeleton and ELK3, FLI1, and MKL2 in the transcriptional control of EDNRB and increase our understanding of the plasticity of this receptor.

Proplatelet formation is regulated by the Rho/ROCK pathway

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4229-4236 ◽  
Author(s):  
Yunhua Chang ◽  
Frédéric Auradé ◽  
Frédéric Larbret ◽  
Yanyan Zhang ◽  
Jean-Pierre Le Couedic ◽  
...  
Keyword(s):  
Rho Kinase ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Actin Organization ◽  
Human Cd34 ◽  
Myosin Light Chain 2 ◽  
Cd34 Cells ◽  
Proplatelet Formation ◽  
High Level

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 dominant-negative 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.

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