Carl W. Jackson

Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development

Despite the importance of blood platelets in health and disease, the mechanisms regulating their formation within megakaryocytes are unknown. We generated mice lacking the hematopoietic subunit (p45) of the heterodimeric erythroid transcription factor NF-E2. Unexpectedly, NF-E2-/- mice lack circulating platelets and die of hemorrhage; their megakaryocytes show no cytoplasmic platelet formation. Though platelets are absent, serum levels of the growth factor thrombopoietin/MGDF are not elevated above controls. Nonetheless, NF-E2-/- megakaryocytes proliferate in vivo in response to thrombopoietin administration. Thus, as an essential factor for megakaryocyte maturation and platelet production, NF-E2 must regulate critical target genes independent of the action of thrombopoietin. These findings provide insight into the genetic analysis of megakaryocyte maturation and thrombopoiesis.

Phospholipase Cγ2 Is Essential in the Functions of B Cell and Several Fc Receptors

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

A transcription-factor-binding surface of coactivator p300 is required for haematopoiesis

The coactivators CBP (Cre-element binding protein (CREB)-binding protein) and its paralogue p300 are thought to supply adaptor molecule and protein acetyltransferase functions to many transcription factors that regulate gene expression. Normal development requires CBP and p300, and mutations in these genes are found in haematopoietic and epithelial tumours. It is unclear, however, which functions of CBP and p300 are essential in vivo. Here we show that the protein-binding KIX domains of CBP and p300 have nonredundant functions in mice. In mice homozygous for point mutations in the KIX domain of p300 designed to disrupt the binding surface for the transcription factors c-Myb and CREB, multilineage defects occur in haematopoiesis, including anaemia, B-cell deficiency, thymic hypoplasia, megakaryocytosis and thrombocytosis. By contrast, age-matched mice homozygous for identical mutations in the KIX domain of CBP are essentially normal. There is a synergistic genetic interaction between mutations in c-Myb and mutations in the KIX domain of p300, which suggests that the binding of c-Myb to this domain of p300 is crucial for the development and function of megakaryocytes. Thus, conserved domains in two highly related coactivators have contrasting roles in haematopoiesis.

Slug, a highly conserved zinc finger transcriptional repressor, protects hematopoietic progenitor cells from radiation-induced apoptosis in vivo

We show here that a zinc finger transcriptional repressor, Slug, which is aberrantly upregulated by the E2A-HLF oncoprotein in pro-B cell acute leukemia, functions as an antiapoptotic factor in normal hematopoietic progenitor cells. Slug(-/-) mice were much more radiosensitive than wild-type mice, dying earlier and showing accentuated decreases in peripheral blood cell counts, as well as abundant microhemorrhages and widely disseminated bacterial microabscesses throughout the body. Slug expression was detected in diverse subsets of hematopoietic progenitors, but not in more differentiated B and T lymphoid cells, and there was a significant increase in apoptotic (TUNEL-positive) bone marrow progenitor cells in irradiated Slug(-/-) mice compared to wild-type controls. These results implicate Slug in a novel survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage.

A role for cyclin D3 in the endomitotic cell cycle.

Platelets, essential for thrombosis and hemostasis, develop from polyploid megakaryocytes which undergo endomitosis. During this cell cycle, cells experience abrogated mitosis and reenter a phase of DNA synthesis, thus leading to endomitosis. In the search for regulators of the endomitotic cell cycle, we have identified cyclin D3 as an important regulatory factor. Of the D-type cyclins, cyclin D3 is present at high levels in megakaryocytes undergoing endomitosis and is markedly upregulated following exposure to the proliferation-, maturation-, and ploidy-promoting factor, Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the platelet lineage display a striking increase in endomitosis, similar to changes seen following Mpl ligand administration to normal mice. Electron microscopy analysis revealed that unlike such treated mice, however, D3 transgenic mice show a poor development of demarcation membranes, from which platelets are believed to fragment, and no increase in platelets. Thus, while our model supports a key role for cyclin D3 in the endomitotic cell cycle, it also points to the unique role of Mpl ligand in priming megakaryocytes towards platelet fragmentation. The role of cyclin D3 in promoting endomitosis in other lineages programmed to abrogate mitosis will need further exploration.

Acute Myeloid Leukemia-Associated Mkl1 (Mrtf-a) Is a Key Regulator of Mammary Gland Function

ABSTRACT Transcription of immediate-early genes—as well as multiple genes affecting muscle function, cytoskeletal integrity, apoptosis control, and wound healing/angiogenesis—is regulated by serum response factor (Srf). Extracellular signals regulate Srf in part via a pathway involving megakaryoblastic leukemia 1 (Mkl1, also known as myocardin-related transcription factor A [Mrtf-a]), which coactivates Srf-responsive genes downstream of Rho GTPases. Here we investigate Mkl1 function using gene targeting and show the protein to be essential for the physiologic preparation of the mammary gland during pregnancy and the maintenance of lactation. Lack of Mkl1 causes premature involution and impairs expression of Srf-dependent genes in the mammary myoepithelial cells, which control milk ejection following oxytocin-induced contraction. Despite the importance of Srf in multiple transcriptional pathways and widespread Mkl1 expression, the spectrum of abnormalities associated with Mkl1 absence appears surprisingly restricted.

Acute megakaryoblastic leukaemia associated with intrinsic platelet dysfunction and constitutional ring 21 chromosome in a young boy

A 3‐year‐old boy with pancytopenia and a paucity of circulating blast cells was found to have acute megakaryoblastic leukaemia. Histopathologic investigation of the bone marrow biopsy disclosed replacement by megakaryoblasts and mild‐to‐moderate reticulin fibrosis. the megakaryocytic origin of these cells was confirmed by their staining properties and by cross‐reactivity with rabbit anti‐rat platelet serum. Treatment with adriamycin and cytosine arabinoside induced a complete remission of this otherwise rapidly fatal disease. Before chemotherapy, the patients platelets showed decreased aggregation in response to thrombin and adenosine diphosphate, as well as a defective thrombin‐induced serotonin release reaction. Neither functional defect resolved after remission induction, indicating that the platelets were intrinsically abnormal. Most striking was the finding of a constitutional chromosomal defect, a ring No. 21 chromosome, in addition to an abnormal malignant stem line. This appears to be the first reported instance of a constitutional r(21) chromosome associated with acute leukaemia.

The roles of ADP and TXA2 in botrocetin/VWF‐induced aggregation of washed platelets

Summary.  Background: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib‐IX‐V complex initiates a cascade of events leading to αIIbβ3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination‐induced GPIbα‐mediated platelet activation have not been fully described. Methods: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Gαq, or αIIbβ3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, αIIbβ3 and Ca2+ in botrocetin/VWF‐induced signaling. Results: Our data demonstrate that botrocetin/VWF/GPIbα‐mediated agglutination results in calcium‐independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbα‐elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2‐induced αIIbβ3 activation and ADP signaling. TXA2 or ADP can activate αIIbβ3, but both are required for α‐granule secretion and aggregation. Botrocetin/VWF‐induced dense granule secretion is Gαq‐dependent. α‐Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an αIIbβ3‐dependent manner. Conclusions: In contrast to adhesion or shear stress‐induced GPIb‐elicited signaling, agglutination‐elicited GPIb signaling that activates αIIbβ3 requires TXA2. Agglutination‐elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination‐elicited GPIb signaling causes αIIbβ3 activation by a mechanism that is distinct from those used by adhesion, or shear stress‐induced GPIb signaling.

Identification of the Src Family Kinases, Lck and Fgr in Platelets Their Tyrosine Phosphorylation Status and Subcellular Distribution Compared With Other Src Family Members

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.

Pronounced thrombocytosis in transgenic mice expressing reduced levels of Mpl in platelets and terminally differentiated megakaryocytes

We generated mice expressing a full-length Mpl transgene under the control of a 2-kb Mpl promoter in an Mpl(-/-) background, effectively obtaining mice that express full-length Mpl in the absence of other Mpl isoforms. These mice developed thrombocytosis with platelet levels approximately 5-fold higher than wild-type controls and markedly increased megakaryocyte numbers. The reintroduction of one wild-type Mpl allele restored normal platelet counts. We excluded the deletion of Mpl-tr, a dominant-negative isoform, as the underlying molecular cause for thrombocytosis. Instead, we found that transgene expression driven by the 2-kb Mpl promoter fragment was decreased during late megakaryocyte maturation, resulting in strongly diminished Mpl protein expression in platelets. Because platelets exert a negative feedback on thrombopoiesis by binding and consuming Tpo in the circulation through Mpl, we propose that the severe reduction of Mpl protein in platelets in Mpl-transgenic Mpl(-/-) mice shifts the equilibrium of this feedback loop, resulting in markedly elevated levels of megakaryocytes and platelets at steady state. Although the mechanism causing decreased expression of Mpl protein in platelets from patients with myeloproliferative disorders differs from this transgenic model, our results suggest that lowering Mpl protein in platelets could contribute to raising the platelet count.