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Biomedical subjects

Kenneth Kaushansky

Publications and source records attributed to Kenneth Kaushansky.

At least 19 recordsLinked to original sources
PubMed2006

Hematopoietic growth factors, signaling and the chronic myeloproliferative disorders.

The chronic myeloproliferative diseases (CMDs) are a group of conditions characterized by unregulated blood cell production, that due either to excessive numbers of erythrocytes, leukocytes or platelets, or their defective function cause symptoms and signs of fatigue, headache, ruddy cyanosis, hemorrhage, abdominal distension, and the complications of vascular thrombosis. In the late 19th century Vaquez provided the first description of polycythemia vera (PV) and Hueck defined idiopathic myelofibrosis (IMF). In 1920, di Guglielmo established criteria for patients with essential thrombocythemia (ET). In 1951, Dameshek argued that these disorders, along with chronic myelogenous leukemia (CML) display many similar clinical and laboratory features [Dameshek W. Some speculations on the myeloproliferative syndromes. Blood 1951;6:372-5], and grouped them. In 2002, the World Health Organization expanded the definition of CMDs to also include chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia/hypereosinophilic syndrome (CEL/HES) and systemic mast cell disorder (SMCD) [Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 2002;100:2292-302]. While the molecular pathogenesis of CML is well known [Melo JV, Deininger MW. Biology of chronic myelogenous leukemia-signaling pathways of initiation and transformation. Hematol Oncol Clin North Am 2004;18:545-68], and the causes of CEL/HES and SMCD have been identified in about half of all cases [Gotlib J, Cools J, Malone III JM, Schrier SL, Gilliland DG, Coutre SE. The FIP1L1-PDGFRalpha fusion tyrosine kinase in hypereosinophilic syndrome and chronic eosinophilic leukemia: implications for diagnosis, classification, and management. Blood 2004; 103:2879-91; Valent P, Akin C, Sperr WR, Horny HP, Metcalfe DD. Mast cell proliferative disorders: current view on variants recognized by the World Health Organization. Hematol Oncol Clin North Am 2003; 17:1227-41], until very recently the etiologies of the three classically defined CMDs, PV, IMF and ET, were poorly understood. Each of these disorders is characterized by excessive hematopoiesis, a process usually dependent on one or more hematopoietic growth factors (HGFs). This review will focus on how our knowledge of the molecular mechanisms by which HGFs are produced, bind cell surface receptors and transduce survival and proliferative signals have provided the platform on which the multiple origins of CMDs can be understood and novel therapeutic interventions designed.

Chronic Disease↗
PubMed2006

Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities.

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However, the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g., the brain) is higher than for treatment of anemia. Notably, a dose-dependent risk of adverse effects has been associated with rhEPO administration, especially in high-risk groups, including polycythemia-hyperviscosity syndrome, hypertension, and vascular thrombosis. Of note, several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity, primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover, we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here, we demonstrate that rhEPO administration in the rat increases systemic blood pressure, reduces regional renal blood flow, and increases platelet counts and procoagulant activities. In contrast, carbamylated rhEPO, a heteromeric receptor-specific ligand that is fully tissue protective, increases renal blood flow, promotes sodium excretion, reduces injury-induced elevation in procoagulant activity, and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands, which appear to elicit fewer adverse effects, may be especially useful in clinical settings for tissue protection.

Animals↗
PubMed2006

Endomitotic megakaryocytes form a midzone in anaphase but have a deficiency in cleavage furrow formation.

Megakaryocyte differentiation is marked by development of progressive polyploidy and accumulation of large nuclear mass and cytoplasmic volume. During differentiation, megakaryocytes undergo repeated incomplete cell cycles in which mitosis is aborted in late anaphase with failure of cytokinesis, termed endomitosis. Recent studies have postulated that failure of Aurora-B kinase to localize to the spindle midzone is responsible for endomitosis in megakaryocytes. In diploid cells, the translocation of Aurora-B kinase is critical for positioning of the cleavage furrow, in part through its phosphorylation of the Rho family GTPase activating protein MgcRacGAP which in turn alters activity of RhoA. However, we have previously demonstrated that Aurora-B kinase localizes to centromeres and is functional in endomitotic megakaryocytes. Here, we show that endomitotic megakaryocytes form midzone structures that recruit Aurora-B kinase and its substrate MgcRacGAP. Although many cells with polyploid anaphases showed cortical localization of Aurora-B kinase, we did not observe accumulation of RhoA in furrows or formation of an actin ring. When mitotic exit was induced by inhibition of cdk1, diploid control cells formed furrows exhibiting cortical RhoA but megakaryocytes exited endomitosis without evidence of furrowing. Therefore, localization of Aurora-B kinase to the midzone is normal in endomitotic megakaryocytes but furrowing is abnormal. These data suggest that endomitotic MKs fail to complete cytokinesis due to aberrant regulation of furrowing at a step subsequent to the localization of Aurora-B kinase, possibly involving the activation or localization of RhoA. This work explores the mechanism of a normally occurring furrowing defect in a nonmalignant primary cell.

Actins↗
PubMed2006

Blocking the alpha 4 integrin-paxillin interaction selectively impairs mononuclear leukocyte recruitment to an inflammatory site.

Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice], which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice, mice bearing the alpha4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however, the alpha4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer's patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis.

Amino Acid Substitution↗
PubMed2006

The Mpl receptor expressed on endothelial cells does not contribute significantly to the regulation of circulating thrombopoietin levels.

OBJECTIVE: Blood levels of thrombopoietin (TPO) are regulated in part by cellular degradation following its binding to the cell surface receptor c-mpl. Previous reports have demonstrated that in addition to hematopoietic cells, c-mpl is expressed on and functions in several types of endothelial cells (ECs). We hypothesized that the c-mpl expressed on ECs would contribute to the regulation of circulating TPO levels. METHODS: To test this hypothesis we transplanted c-mpl-null and wild-type (WT) control mice with WT marrow stem cells, resulting in two groups of posttransplant chimeric animals, one expressing c-mpl on megakaryocytes and platelets only and one in which the receptor is expressed on both hematopoietic and ECs. Should EC c-mpl take up TPO and degrade it, we predicted that c-mpl-null mice reconstituted with WT cells would display increased TPO levels and an increased steady state platelet count compared to the WT recipients. RESULTS: Contrary to our prediction, for up to 6 months posttransplantation both platelet counts and TPO levels in both groups of transplanted mice were virtually identical. CONCLUSIONS: Our results indicate that the EC c-mpl receptor does not contribute significantly to the regulation of TPO levels or to steady-state platelet counts. These results also imply that patients with congenital amegakaryocytic thrombocytopenia, lacking the c-mpl receptor, who have successfully been engrafted with normal hematopoietic stem cells should have normal (not elevated) TPO levels and that gene replacement strategies designed to restore c-mpl in these patients do not need to target ECs to establish the normal regulation of TPO.

Animals↗
PubMed2006

Transcriptional regulation of megakaryopoiesis: thrombopoietin signaling and nuclear factors.

PURPOSE OF REVIEW: Thrombopoietin, the primary regulator of megakaryopoiesis, acts by modulating several nuclear transcription factors. This review focuses on recent studies that have provided new insights into the functional roles of these proteins. RECENT FINDINGS: Among the transcription factors responsible for megakaryopoiesis, important roles for the signal transducer and activator of transcription and Forkhead box proteins have been defined. In addition, in the past several years, two new groups of transcription factors have been found to be affected by thrombopoietin signaling, homeodomain-containing HOX proteins and hypoxia-inducible factor. The HOX transcription factors, which play key roles in body pattern development, are also expressed in adult hematopoietic stem cells and play pivotal roles in their proliferation. Thrombopoietin controls the levels of expression and modifies the function of homeodomain proteins in thrombopoietin-responsive leukemic cells and in primary immature hematopoietic cells. Levels of hypoxia-inducible factor, a master transcription factor required for the adaptation to hypoxic conditions, is also affected by thrombopoietin in these cells. SUMMARY: The discovery that a diverse range of transcription factors are downstream effectors of thrombopoietin helps to explain the molecular mechanisms by which the hormone affects hematopoiesis.

Animals↗
PubMed2005

Thrombopoietin (TPO) induces c-myc expression through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR in TPO-dependent cell lines and primary megakaryocytes.

Thrombopoietin (TPO) and its receptor (c-Mpl) are the major regulators of megakaryocyte and platelet production and serve a critical and non-redundant role in hematopoietic stem cell (HSC) biology. TPO signals through the Jak-STAT, Ras-Raf-MAPK, and PI3K pathways, and promotes survival, proliferation, and polyploidization in megakaryocytes. The proto-oncogene c-myc also plays an important role in many of these same processes. In this work we studied the regulated expression of c-myc in megakaryocytic cell lines and primary cells by quantitative real-time RT-PCR. We found that TPO induced expression of c-myc in 1 h in both hematopoietic cell lines (UT-7 and BaF3/Mpl) and mature murine megakaryocytes. The TPO-induced expression of c-myc was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting that TPO stimulated c-myc expression through a PI3K-dependent pathway. Of interest, our study showed that overexpression of active Akt did not rescue the effect of PI3K blockade on c-myc expression, rather, enhanced it. In addition, inhibitors of protein kinase C (PKC)zeta and the target of rapamycin (mTOR) also failed to affect c-myc mRNA expression, while c-myc mRNA expression was reduced by inhibition of the mitogen activated protein kinase (MAPK) pathway. Therefore, we conclude that TPO stimulates c-myc expression in primary megakaryocytes through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR.

Animals↗
PubMed2005

Thrombopoietin stimulates vascular endothelial cell growth factor (VEGF) production in hematopoietic stem cells.

Thrombopoietin (TPO) is a pivotal and nonredundant hematopoietic cytokine, supporting the survival, self-renewal activity and proliferation of hematopoietic stem and progenitor cells, the growth and differentiation of megakaryocytes, and the functional activation of their progeny, blood platelets. TPO exerts these effects through regulating the abundance or subcellular localization of several transcription factors, including the homeodomain proteins HOXB4 and HOXA9. In addition to these effects, TPO helps orchestrate a cytokine-network in the bone marrow microenvironment that supports hematopoietic stem cell (HSC) function. In recent studies we have shown that TPO stimulates production of vascular endothelial cell growth factor (VEGF), another cytokine vital for HSC physiology, promoting their survival and expansion into committed hematopoietic progenitors. Like several other effects of the cytokine, the effect of TPO on VEGF expression is mediated by stabilization and activation of the primary transcription factor responsible for VEGF expression, the oxygen tension responsive hypoxia inducible factor-1 (HIF-1). Together with the observation that bone marrow microenvironment is hypoxic and hypoxia simulates the repopulating activity of HSCs, our observations suggest that TPO mimics hypoxia and controls important genes required for HSC cycling, including VEGF, adding to our understanding of how the hormone contributes to HSC function.

Animals↗
PubMed2005

Thrombopoietin enhances expression of vascular endothelial growth factor (VEGF) in primitive hematopoietic cells through induction of HIF-1alpha.

Thrombopoietin (TPO), the primary regulator of thrombopoiesis, is also an important, nonredundant mediator of hematopoietic stem cell (HSC) development. For example, following transplantation, HSC expansion is approximately 15-fold more robust in normal than in Tpo(-/-) mice. Vascular endothelial growth factor (VEGF) also plays an important role in HSC development, where it acts in an intracellular autocrine fashion to promote cell survival. Thus, we tested the hypothesis that TPO affects the autocrine production of VEGF to account for its favorable effects on HSCs. We found that VEGF transcripts are reduced in purified sca-1(+)/c-kit(+)/Gr-1(-) marrow cells derived from Tpo(-/-) mice and that TPO induces VEGF transcripts in these primitive hematopoietic cells. Additional studies determined that TPO induces VEGF expression by increasing the level of its primary transcription factor, hypoxia-inducible factor 1alpha (HIF-1alpha), by enhancing its protein stability. Moreover, VEGF expression is important for the TPO effect on primitive hematopoietic cells because blockade of the VEGF receptor with a specific inhibitor substantially blunts TPO-induced growth of single sca-1(+)/c-kit(+)/Gr-1(-) marrow cells in serum-free cultures. Along with previous findings that TPO affects Hox transcription factors that regulate HSC proliferation, these data contribute to our growing understanding of the mechanisms by which a hormone can influence stem cell development.

Animals↗
PubMed2005

Engagement of integrin alpha4beta1 enhances thrombopoietin-induced megakaryopoiesis.

OBJECTIVE: Studies in numerous adherent cell systems have indicated that engagement of integrins is required for cell survival and proliferation. Although not classically thought of as an adherent cell type, megakaryocytes in the marrow develop in juxtaposition to endothelial cells which display a number of integrin counter-receptors. Moreover, a number of other hematopoietic cell types, including stem cells and erythroid progenitors, have been shown to engage and be affected by integrin ligands. METHODS: The role of beta1 integrins in thrombopoietin-mediated megakaryopoiesis was studied using both gain-of-function and loss-of-function strategies. RESULTS: We found that pan-blockade of integrins with a relatively nonspecific disintegrin blocked TPO-induced MK growth, but that an alpha5beta1 disintegrin, and a function-blocking monoclonal antibody, failed to affect megakaryopoiesis in vitro. In contrast, a neutralizing alpha4beta1 monoclonal antibody blocked TPO-induced MK growth, and an integrin alpha4beta1 ligand, the H296 fragment of fibronectin, enhanced MK growth at all concentrations of TPO. CONCLUSIONS: These findings have important implications for thrombopoiesis in general, and potentially for the enhanced platelet production found in states of systemic inflammation and following the use of therapeutic strategies designed to block alpha4beta1 integrin engagement in states of chronic inflammation and autoimmunity.

Animals↗
PubMed2005

The molecular mechanisms that control thrombopoiesis.

Our understanding of thrombopoiesis--the formation of blood platelets--has improved greatly in the last decade, with the cloning and characterization of thrombopoietin, the primary regulator of this process. Thrombopoietin affects nearly all aspects of platelet production, from self-renewal and expansion of HSCs, through stimulation of the proliferation of megakaryocyte progenitor cells, to support of the maturation of these cells into platelet-producing cells. The molecular and cellular mechanisms through which thrombopoietin affects platelet production provide new insights into the interplay between intrinsic and extrinsic influences on hematopoiesis and highlight new opportunities to translate basic biology into clinical advances.

Animals↗
PubMed2005

Thrombopoietin and the hematopoietic stem cell.

The molecular mechanisms that underlie the favorable effects of thrombopoietin on stem cell survival, self-renewal, and expansion are unknown. On the basis of known effects of HoxB4, HoxA9, and vascular endothelial cell growth factor on stem cells, we explored whether TPO might affect these pathways. We found that TPO enhances the transcription of HoxB4, induces the nuclear localization of HoxA9, and upregulates VEGF expression by reducing the ubiquination of hypoxia inducible factor.

Animals↗
PubMed2004

Megakaryocytes express functional Aurora-B kinase in endomitosis.

Endomitosis (EnM) in megakaryocytes (MKs) is characterized by abortion of mitosis in late anaphase and failure of cytokinesis; subsequent reinitiation of DNA synthesis results in polyploidy. Ablation of chromosomal passenger proteins including Aurora-B kinase causes defects in late anaphase and cytokinesis in diploid cells; thus one hypothesis is that the expression or function of these proteins in polyploid MKs is abnormal. It has been reported that Aurora-B kinase mRNA is decreased in polyploid megakaryocytic cells, suggesting that deficiency of Aurora-B kinase is responsible for EnM. We examined the localization of Aurora-B kinase and additional members of the chromosomal passenger protein and aurora kinase families in MKs. We found that in EnM MKs (1) Aurora-B kinase is present and appropriately localized to centromeres in early EnM; (2) in low-ploidy human MKs, centromeric localization of survivin and inner centromere protein (INCENP) can also be demonstrated; (3) the function of Aurora-B kinase, as measured by Ser10 phosphorylation of histone H3, is intact; and (4) aurora-A kinase localizes appropriately to centrosomes in EnM. These results suggest that EnM MKs appropriately express functional Aurora-B kinase and related proteins in early anaphase, making a simple deficiency of this protein an unlikely explanation for polyploidy in this cell type.

Anaphase↗
PubMed2004

Inherited thrombocytopenias: toward a molecular understanding of disorders of platelet production.

PURPOSE OF REVIEW: To review the defined syndromes of inherited thrombocytopenia and discuss new genetic data for several disorders that shed light on the process of megakaryopoiesis. RECENT FINDINGS: The genes responsible for several inherited thrombocytopenias have been recently discovered, including congenital amegakaryocytic leukemia, amegakaryocytic thrombocytopenia with radio-ulnar synostosis, familial platelet syndrome with predisposition to acute myelogenous leukemia, Paris-Trousseau, Wiskott-Aldrich syndrome, and the May-Hegglin, Sebastian, Epstein, and Fechner syndromes. These clinical syndromes, combined with studies in mouse and in vitro models, reveal the importance of these genes for normal hematopoiesis. SUMMARY: Although inherited syndromes of thrombocytopenia are rare, characterization of mutations in these disorders has contributed greatly to our understanding of megakaryocyte and platelet development. A systematic registry of congenitally thrombocytopenic individuals would almost certainly lead to new genetic discoveries.

Animals↗