Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression

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Abstract

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.

Authors

Konstantin Levay, Vladlen Z. Slepak

FTY720, a new alternative for treating blast crisis chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphocytic leukemia

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Abstract

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome–positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL⁺ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190^BCR/ABL myeloid and lymphoid cell lines and CML-BC^CD34+ and Ph1 ALL^CD34+/CD19+ progenitors but not of normal CD34⁺ and CD34⁺/CD19⁺ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190^BCR/ABL-driven [including p210/p190^BCR/ABL (T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.

Authors

Paolo Neviani, Ramasamy Santhanam, Joshua J. Oaks, Anna M. Eiring, Mario Notari, Bradley W. Blaser, Shujun Liu, Rossana Trotta, Natarajan Muthusamy, Carlo Gambacorti-Passerini, Brian J. Druker, Jorge Cortes, Guido Marcucci, Ching-Shih Chen, Nicole M. Verrills, Denis C. Roy, Michael A. Caligiuri, Clara D. Bloomfield, John C. Byrd, Danilo Perrotti

The antithrombotic potential of selective blockade of talin-dependent integrin α_IIbβ₃ (platelet GPIIb–IIIa) activation

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Abstract

In vitro studies indicate that binding of talin to the β3 integrin cytoplasmic domain (tail) results in integrin αIIbβ3 (GPIIb–IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the β3 tail. We introduced a β3(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a β3(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate αIIbβ3 require binding of talin to the β3 tail. β3(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride–induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of β3(Y747A) and virtually all β3-null animals examined. Remarkably, less than 5% of β3(L746A) animals exhibited this form of bleeding. These results establish that αIIbβ3 activation in vivo is dependent on the interaction of talin with the β3 integrin cytoplasmic domain. Furthermore, they suggest that modulation of β3 integrin–talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.

Authors

Brian G. Petrich, Per Fogelstrand, Anthony W. Partridge, Nima Yousefi, Ararat J. Ablooglu, Sanford J. Shattil, Mark H. Ginsberg

The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo

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Abstract

NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.

Authors

Dmitriy N. Atochin, Annie Wang, Victor W.T. Liu, Jeffrey D. Critchlow, Ana Paula V. Dantas, Robin Looft-Wilson, Takahisa Murata, Salvatore Salomone, Hwa Kyoung Shin, Cenk Ayata, Michael A. Moskowitz, Thomas Michel, William C. Sessa, Paul L. Huang

Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III

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Abstract

Single gene mutations in β integrins can account for functional defects of individual cells of the hematopoietic system. In humans, mutations in β2 integrin lead to leukocyte adhesion deficiency (LAD) syndrome and mutations in β3 integrin cause the bleeding disorder Glanzmann thrombasthenia. However, multiple defects in blood cells involving various β integrins (β1, β2, and β3) occur simultaneously in patients with the recently described LAD type III (LAD-III). Here we show that the product of a single gene, Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), controlled the activation of all 3 integrins in the hematopoietic system. Neutrophils from CalDAG-GEFI–/– mice exhibited strong defects in Rap1 and β1 and β2 integrin activation while maintaining normal calcium flux, degranulation, and ROS generation. Neutrophils from CalDAG-GEFI–deficient mice failed to adhere firmly to stimulated venules and to migrate into sites of inflammation. Furthermore, CalDAG-GEFI regulated the activation of β1 and β3 integrins in platelets, and CalDAG-GEFI deficiency caused complete inhibition of arterial thrombus formation in mice. Thus, mice engineered to lack CalDAG-GEFI have a combination of defects in leukocyte and platelet functions similar to that of LAD-III patients.

Authors

Wolfgang Bergmeier, Tobias Goerge, Hong-Wei Wang, Jill R. Crittenden, Andrew C.W. Baldwin, Stephen M. Cifuni, David E. Housman, Ann M. Graybiel, Denisa D. Wagner

Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

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Abstract

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1–/– mice, decreased in Kcc3–/– mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1–/– or Kcc3–/– mice were of normal density, rbc of Kcc1–/–Kcc3–/– mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1–/–Kcc3–/– SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K+ content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

Authors

Marco B. Rust, Seth L. Alper, York Rudhard, Boris E. Shmukler, Rubén Vicente, Carlo Brugnara, Marie Trudel, Thomas J. Jentsch, Christian A. Hübner

Deficiency of Src homology 2 domain–containing inositol 5-phosphatase 1 affects platelet responses and thrombus growth

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Abstract

Platelets are critical for normal hemostasis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Src homology 2 domain–containing inositol 5-phosphatase 1 (SHIP1) is a 5-phosphatase capable of dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate second messenger into phosphatidylinositol 3,4-bisphosphate. SHIP1 plays a critical role in regulating the level of these 2 lipids in platelets. Using SHIP1-deficient mice, we found that its loss affects platelet aggregation in response to several agonists with minor effects on fibrinogen binding and β3 integrin tyrosine phosphorylation. Accordingly, SHIP1-null mice showed defects in arterial thrombus formation in response to a localized laser-induced injury. Moreover, these mice had a prolonged tail bleeding time. Upon stimulation, SHIP1-deficient platelets showed large membrane extensions, abnormalities in the open canalicular system, and a dramatic decrease in close cell-cell contacts. Interestingly, SHIP1 appeared to be required for platelet contractility, thrombus organization, and fibrin clot retraction. These data indicate that SHIP1 is an important element of the platelet signaling machinery to support normal hemostasis. To our knowledge, this is the first report unraveling an important function of SHIP1 in the activation of hematopoietic cells, in contrast to its well-documented role in the negative regulation of lymphocytes.

Authors

Sonia Séverin, Marie-Pierre Gratacap, Nadège Lenain, Laetitia Alvarez, Etienne Hollande, Josef M. Penninger, Christian Gachet, Monique Plantavid, Bernard Payrastre

Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model

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Abstract

Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and vWF are known to induce in vitro calcium mobilization in platelets. Using a calcium-sensitive fluorochrome and digital multichannel intravital microscopy to image unstimulated and stimulated platelets, calcium mobilization was monitored as a reporter of platelet activation (as distinct from platelet accumulation) during thrombus formation in live mice. In the absence of vWF, platelet activation was normal, but platelet adherence and aggregation were attenuated during thrombus formation following laser-induced injury in the cremaster muscle microcirculation. In WT mice treated with lepirudin, platelet activation was blocked, and platelet adherence and aggregation were inhibited. The kinetics of platelet activation and platelet accumulation were similar in FcRγ–/– mice lacking glycoprotein VI (GPVI), GPVI-depleted mice, and WT mice. Our results indicate that the tissue factor–mediated pathway of thrombin generation, but not the collagen-induced GPVI-mediated pathway, is the major pathway leading to platelet activation after laser-induced injury under the conditions employed. In the tissue factor–mediated pathway, vWF plays a role in platelet accumulation during thrombus formation but is not required for platelet activation in vivo.

Authors

Christophe Dubois, Laurence Panicot-Dubois, Justin F. Gainor, Barbara C. Furie, Bruce Furie

Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect

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Abstract

The regulation of HSC proliferation and engraftment of the BM is an important but poorly understood process, particularly during ontogeny. Here we show that in mice, all HSCs are cycling until 3 weeks after birth. Then, within 1 week, most became quiescent. Prior to 4 weeks of age, the proliferating HSCs with long-term multilineage repopulating activity displayed an engraftment defect when transiting S/G2/M. During these cell cycle phases, their expression of CXC chemokine ligand 12 (CXCL12; also referred to as stromal cell–derived factor 1 [SDF-1]) transiently increased. The defective engrafting activity of HSCs in S/G2/M was reversed when cells were allowed to progress into G1 prior to injection or when the hosts (but not the cells) were pretreated with a CXCL12 antagonist. Interestingly, the enhancing effect of CXCL12 antagonist pretreatment was exclusive to transplants of long-term multilineage repopulating HSCs in S/G2/M. These results demonstrate what we believe to be a new HSC regulatory checkpoint during development. They also suggest an ability of HSCs to express CXCL12 in a fashion that changes with cell cycle progression and is associated with a defective engraftment that can be overcome by in vivo administration of a CXCL12 antagonist.

Authors

Michelle B. Bowie, Kristen D. McKnight, David G. Kent, Lindsay McCaffrey, Pamela A. Hoodless, Connie J. Eaves

Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies

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Abstract

Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A. We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain–deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific αIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8trans mice, the bleeding phenotype of FVIIInull mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII–immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII.

Authors

Qizhen Shi, David A. Wilcox, Scot A. Fahs, Hartmut Weiler, Clive W. Wells, Brian C. Cooley, Drashti Desai, Patricia A. Morateck, Jack Gorski, Robert R. Montgomery