c-Mpl and TPO expression in the human central nervous system neurons inhibits neuronal apoptosis.

Thrombopoietin (TPO) is a growth factor for the megakaryocytic/platelet lineage. In this study, we investigated the expression of TPO and its receptor, c-Mpl, in the human central nervous system (CNS) and their roles after a neural insult. Our results demonstrate that both TPO and c-Mpl are expressed in the neurons of the human CNS. TPO was also detected in human cerebrospinal fluid. TPO was found to be neuroprotective in hypoxic-ischemic neonatal rat brain models. In these rat models, treatment with TPO reduced brain damage and improved sensorimotor functions. In addition, TPO promoted C17.2 cell proliferation through activation of the PI3K/Akt signaling pathway. Via the Bcl-2/BAX signaling pathway, TPO exerted an antiapoptotic effect by suppressing mitochondrial membrane potentials. Taken together, our results indicate that TPO is neuroprotective in the CNS.

MPL expression on AML blasts predicts peripheral blood neutropenia and thrombocytopenia.

Although the molecular pathways that cause acute myeloid leukemia (AML) are increasingly well understood, the pathogenesis of peripheral blood cytopenia, a major cause of AML mortality, remains obscure. A prevailing assumption states that AML spatially displaces nonleukemic hematopoiesis from the bone marrow. However, examining an initial cohort of 223 AML patients, we found no correlation between bone marrow blast content and cytopenia, questioning the displacement theory. Measuring serum concentration of thrombopoietin (TPO), a key regulator of hematopoietic stem cells and megakaryocytes, revealed loss of physiologic negative correlation with platelet count in AML cases with blasts expressing MPL, the thrombopoietin (scavenging) receptor. Mechanistic studies demonstrated that MPLhi blasts could indeed clear TPO, likely therefore leading to insufficient cytokine levels for nonleukemic hematopoiesis. Microarray analysis in an independent multicenter study cohort of 437 AML cases validated MPL expression as a central predictor of thrombocytopenia and neutropenia in AML. Moreover, t(8;21) AML cases demonstrated the highest average MPL expression and lowest average platelet and absolute neutrophil counts among subgroups. Our work thus explains the pathophysiology of peripheral blood cytopenia in a relevant number of AML cases.

MPL W515L expression induces TGFß secretion and leads to an increase in chemokinesis via phosphorylation of THOC5.

The thrombopoietin receptor (MPL) has been shown to be mutated (MPL W515L) in myelofibrosis and thrombocytosis yet new approaches to treat this disorder are still required. We have previously shown that transcriptome and proteomic effects do not correlate well in oncogene-mediated leukemogenesis. We therefore investigated the effects of MPL W515L using proteomics. The consequences of MPL W515L expression on over 3300 nuclear and 3500 cytoplasmic proteins were assessed using relative quantification mass spectrometry. We demonstrate that MPL W515L expression markedly modulates the CXCL12/CXCR4/CD45 pathway associated with stem and progenitor cell chemotactic movement. We also demonstrated that MPL W515L expressing cells displayed increased chemokinesis which required the MPL W515L-mediated dysregulation of MYC expression via phosphorylation of the RNA transport protein THOC5 on tyrosine 225. In addition MPL W515L expression induced TGFß secretion which is linked to sphingosine 1-phosphate production and the increased chemokinesis. These studies identify several pathways which offer potential targets for therapeutic intervention in the treatment of MPL W515L-driven malignancy. We validate our approach by showing that CD34+ cells from MPL W515L positive patients display increased chemokinesis and that treatment with a combination of MYC and sphingosine kinase inhibitors leads to the preferential killing of MPL W515L expressing cells.

The Eltrombopag antitumor effect on hepatocellular carcinoma.

Currently, sorafenib is the only available chemotherapeutic agent for advanced hepatocellular carcinoma (HCC), but it cannot be used in patients with liver cirrhosis (LC) or thrombocytopenia. In these cases, sorafenib is likely effective if given in combination with treatments that increase the number of platelets, such as thrombopoietin (TPO) receptor agonists. Increasing the platelet count via TPO treatment resulted in reduction of LC. Eltrombopag (EP), a TPO receptor agonist, has been reported to have antitumor effects against certain cancers, despite their lack of TPO receptor expression. We hypothesized that EP may possess antitumor activity against HCC in addition to its ability to suppress hepatic fibrosis by increasing the platelet count. In the present study, the antitumor activity of EP was examined by assessing the inhibition of cell proliferation and then ascertaining the ability of iron supplementation to reverse these effects in HepG2, Hep3B and Huh7 cells. In addition, a cell cycle assay was performed using flow cytometry, and signal transduction was evaluated by analyzing cell cycle-related protein expression. The results of EP were compared with those of the most common iron chelator, deferoxamine (DFO). The combined effect of EP and sorafenib was also assessed. The results revealed that EP exerts antitumor activity in HCC that is mediated by the modulation of intracellular iron content. EP suppressed the expression of the cell cycle-related protein cyclin D1 and elicited cell cycle arrest in the G0/G1 phase. The activity of EP was comparable to that of DFO in HCC, and EP did not compete with sorafenib at low concentrations. In conclusion, our findings suggest that EP is a good candidate chemotherapeutic agent for the treatment of HCC in patients with LC and thrombocytopenia.

Robust hematopoietic progenitor cell commitment in the presence of a conflicting cue.

Hematopoietic lineage commitment is regulated by cytokines and master transcription factors, but it remains unclear how a progenitor cell chooses a lineage in the face of conflicting cues. Through transcript counting in megakaryocyte-erythroid progenitors undergoing erythropoiesis, we show that the expression levels of the pro-erythropoiesis transcription factor EKLF (also known as KLF1) and receptor EpoR are inversely correlated with their pro-megakaryopoiesis counterparts, FLI-1 and TpoR (also known as MPL). Notably, as progenitors commit to the erythrocyte lineage, EpoR is upregulated and TpoR is strongly downregulated, thus boosting the potency of the pro-erythropoiesis cue erythropoietin and effectively eliminating the activity of the pro-megakaryopoiesis cue thrombopoietin. Based on these findings, we propose a new model for exclusive decision making that explicitly incorporates signals from extrinsic cues, and we experimentally confirm a model prediction of temporal changes in transcript noise levels in committing progenitors. Our study suggests that lineage-specific receptor levels can modulate potencies of cues to achieve robust commitment decisions.

Itga2b regulation at the onset of definitive hematopoiesis and commitment to differentiation.

Product of the Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression.

A case of acute stent thrombosis during treatment with the thrombopoietin receptor agonist peptide--romiplostim.

Romiplostim is a thrombopoietin receptor agonist that increases platelet counts and restores platelet function in patients with chronic immune thrombocytopenia (ITP). Increase in platelet count and platelet activation has been associated with increased thromboembolic risk. The present case report describes an interesting case of acute stent thrombosis in a patient with chronic immune thrombocytopenic purpura (ITP) being treated with romiplostim.

Inflammation stimulates thrombopoietin (Tpo) expression in rat brain-derived microvascular endothelial cells, but suppresses Tpo in astrocytes and microglia.

Thrombopoietin (Tpo) and its receptor (c-Mpl; TpoR), which primary regulate megakaryopoiesis and platelet production, are also expressed in the central nervous system (CNS). Increased Tpo concentrations are present in the cerebrospinal fluid (CSF) of some patients with bacterial or viral meningitis. Since previous data implicated a proapoptotic role of Tpo on newly generated neuronal cells, we herein elucidated the regulation of Tpo in primary rat neurons (e17), astrocytes, and microglia (p0-p3), as well as in brain-derived vascular endothelial cells of 3-week-old rats after exposure to bacterial lipopolysaccharide (LPS). LPS inhibited Tpo gene expression in astrocytes and microglia, but not in neurons, most likely due to absence of Toll-like receptor 4 in neurons. While Tpo mRNA expression recovered in astrocytes after 24 h, it remained suppressed in microglia. Furthermore, we detected Tpo mRNA expression in primary brain-derived vascular endothelial cells, which also express the TpoR. In these cells, LPS significantly up-regulated Tpo mRNA expression. TpoR mRNA and protein expression remained constitutive in all cell types. Thus, our data provide evidence for a cell-type-specific modulation of Tpo mRNA expression by inflammation in brain-derived cells. Transient down-regulation of Tpo expression in astrocytes and microglia may limit Tpo-induced neuronal cell death in inflammatory brain disorders.

HIV-1 determinants of thrombocytopenia at the stage of CD34+ progenitor cell differentiation in vivo lie in the viral envelope gp120 V3 loop region.

HIV-1 V3 loop clones of virus isolates derived from patients suffering from thrombocytopenia were used for infection of the human thymus/liver conjoint hematopoietic organ that developed in the severe combined immunodeficient mouse (SCID-hu Thy/Liv). The V3 loop clones showed a significantly greater degree of inhibition of megakaryopoiesis than myelopoiesis and erythropoiesis of the human CD34+ progenitor cells, in vivo. Inhibition of megakaryopoiesis occurs through reduction in c-Mpl expression and consequent decrease in STAT5 activation. Therefore HIV-1 V3 loop sequences of thrombocytopenic patients exhibit preferential inhibition of megakaryocyte lineage-specific differentiation of CD34+ progenitor cells, thus reflecting the patients' clinical condition.

CD90 and CD110 correlate with cancer stem cell potentials in human T-acute lymphoblastic leukemia cells.

Although cancer stem cells (CSCs) have been recently identified in myeloid leukemia, published data on lymphoid malignancy have been sparse. T-acute lymphoblastic leukemia (T-ALL) is characterized by the abnormal proliferation of T-cell precursors and is generally aggressive. As CD34 is the only positive-selection marker for CSCs in T-ALL, we performed extensive analysis of CD markers in T-ALL cell lines. We found that some of the tested lines consisted of heterogeneous populations of cells with various levels of surface marker expression. In particular, a small subpopulation of CD90 (Thy-1) and CD110 (c-Mpl) were shown to correlate with stem cell properties both in vitro and in transplantation experiments. As these markers are expressed on hematopoietic stem cells, our results suggest that stem cell-like population are enriched in CD90+/CD110+ fraction and they are useful positive-selection markers for the isolation of CSCs in some cases of T-ALL.

Rescue of multi-lineage hematopoiesis during HIV-1 infection by human c-mpl gene transfer and reconstitution of CD34+ progenitor cells in vivo.

Cytopenias arising from hematopoietic abnormalities are a severe common complication contributing to early mortality in HIV/AIDS patients. The proto-oncogene c-mpl, identified as the thrombopoietin receptor is involved in multilineage differentiation of CD34+ hematopoietic progenitor cells. We have introduced the c-mpl gene into CD34+ cells via transduction of the lentivirus p156RRLsinPPTmPGK-CMPL-PRE. The lentiviral construct expresses c-mpl on approximately 90% of purified CD34+ cells. These transduced cells have then been reconstituted into human fetal thymus/liver implants in severe combined immunodeficient mice (SCID-hu Thy/Liv). The c-mpl expression on transduced CD34+ cells is not susceptible to downregulation due to the effects of HIV-1 infection. Reconstituted CD34+ cells transduced with control lentivirus, p156RRLsinPPTmPGK-EGFP-PRE, express EGFP at > 90%. Reconstituted c-mpl expressing SCID-hu implants show almost maximum rescue (approximately 90%) of myelopoiesis, erythropoiesis and megakaryopoiesis, during HIV-1 infection in vivo, at 6 weeks post-infection. We also show that the differentiated multi-lineage progeny colonies and thymocytes in mice reconstituted with the c-mpl transduced CD34+ cells, carry the HLA Class I loci phenotypes of these donor cells, in the implants of the recipient SCID-hu animals. We propose a gene therapeutic strategy, with c-mpl as the major genetic component, to address the morbidity and mortality resulting from cytopenias in HIV infected patients.

Angiogenesis, tumor necrosis factor-alpha and procoagulant factors in coronary artery giant aneurysm of a fatal infantile Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is an infantile febrile illness of unknown origin characterized by clinical, laboratory and histopathologic features of systemic vasculitis. METHODS AND RESULTS: We report a 3-month-old female infant with incomplete KD who suddenly died despite intravenous immunoglobulin, aspirin, steroid and heparin treatment. Postmortem examination confirmed the echocardiographically detected giant coronary aneurysms and showed occlusive thrombosis in the giant aneurysm of the left anterior descending coronary artery, associated with neoangiogenesis, macrophage infiltration and immunostaining for tissue factor (a strong initiator of the coagulation cascade), thrombopoietin receptor and tumour necrosis factor-alpha. CONCLUSIONS: These findings show the association of angiogenesis, tumor necrosis factor-alpha and procoagulant factors, with macrophage infiltration in coronary artery aneurysms of a fatal infantile KD.

Impact of chicken thrombopoietin and its receptor c-Mpl on hematopoietic cell development.

OBJECTIVE: The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chicken TPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. MATERIALS AND METHODS: Chicken TPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. RESULTS: Both chicken TPO and its receptor c-Mpl were identified and cloned. Expression of chicken TPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chicken TPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. CONCLUSION: The characterized chicken thrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.

Molecular mimicry in the chronic myeloproliferative disorders: reciprocity between quantitative JAK2 V617F and Mpl expression.

An activating JAK2 mutation (JAK2 V617F) is present in the chronic myeloproliferative disorders (MPDs), polycythemia vera (PV), idiopathic myelofibrosis (IMF), and essential thrombocytosis (ET). JAK2 is also a chaperone for Mpl and responsible for its cell-surface expression. We observed a reciprocal relationship between neutrophil JAK2 V617F allele percentage and platelet Mpl expression in JAK2 V617F-positive PV, IMF, and ET patients. However, severely impaired platelet Mpl expression was present in JAK2 V617F-negative MPD patients. While JAK2 V617F allele status did not necessarily correlate with the clinical MPD phenotype, the degree of impaired platelet Mpl expression did. We conclude that multiple molecular abnormalities are involved in the pathogenesis of the MPDs and that aberrant Mpl expression may be a common denominator of aberrant signaling in both the JAK2 V617F-positive and JAK2 V617F-negative MPDs.