"How I treat": a new style of clinical review article
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Journal Article↗
Biomedical subjects
K Kaushansky
Publications and source records attributed to K Kaushansky.
The glycan domain of thrombopoietin enhances its secretion.
Thrombopoietin (TPO) is the major regulator of megakaryocyte development and platelet production. The hormone is structurally characterized by an amino-terminal receptor binding domain (amino acid residues 1-152) predicted to encode a left-handed four-helix bundle structure, and a carboxyl-terminal domain (residues 153-335) that is remarkable for its abundant carbohydrate modification and a lack of homology to other proteins. To investigate the functional role of the carboxyl-terminal glycan domain, we generated truncated forms of murine TPO (TPO1-238, TPO1-174, and TPO1-152) and glycomuteins in which the predicted asparagine (N)-linked sites of glycosylation were sequentially mutated to glutamine (Q), and assayed their secretion and function by comparing them to the native sequence (TPO1-335). Following transient transfection of the corresponding cDNA expression vectors into mammalian cell lines, the secretory efficiencies of the proteins were compared with those of the native hormone. Transfection efficiencies were monitored by cotransfection and reporter gene assay, and TPO secretion was assessed by functional and immunologic assays. We found that full-length TPO was 5-29-fold more efficiently secreted than any of the truncated forms of the hormone in fibroblast and hepatocyte cell lines. Elimination of carboxyl-terminal sites of N-linked glycosylation had a minor impact on secretion of the protein. We conclude that the carboxyl-terminal domain of TPO serves the important role of enhancing secretion of the protein, and in this manner functions as a prosequence.
Amino Acid Sequence↗
Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK).
Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that acts as a stimulator of pre-B lymphocyte cell growth and as a chemoattractant for T cells, monocytes, and hematopoietic stem cells. More recent studies also suggest that megakaryocytes migrate in response to SDF-1. Because genetic elimination of SDF-1 or its receptor lead to marrow aplasia, we investigated the effect of SDF-1 on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-MK]). We report that SDF-1 augments the growth of CFU-MK from whole murine bone marrow cells when combined with thrombopoietin (TPO). The addition of SDF-1 to interleukin-3 (IL-3) or stem cell factor (SCF) had no effect. Specific antagonists for CXCR4 (the sole receptor for SDF-1), T22, and 1-9 (P2G) SDF-1 reduced megakaryocyte colony growth induced by TPO alone, suggesting that many culture systems contain endogenous levels of the chemokine that contributes to the TPO effect. To examine whether SDF-1 has direct effects on CFU-MK, we developed a new protocol to purify megakaryocyte progenitors. CFU-MK were highly enriched in CD41(high) c-kit(high) cells generated from lineage-depleted TPO-primed marrow cells. Because the growth-promoting effects of SDF-1 were also observed when highly purified populations of CFU-MK were tested in serum-free cultures, these results suggest that SDF-1 directly promotes the proliferation of megakaryocytic progenitors in the presence of TPO, and in this way contributes to the favorable effects of the bone marrow microenvironment on megakaryocyte development.
Animals↗
Blood: new designs for a new millennium.
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Animals↗
Tyrosine 462 of the membrane-proximal F'-G' loop of murine Mpl is not essential for high-affinity binding of thrombopoietin.
The ligand binding site of Mpl, the thrombopoietin (Tpo) receptor, has not been determined. Tyr(462)of murine Mpl corresponds to Tyr(421)of the common beta chain of the human IL-3, IL-5 and GM-CSF receptors. Tyr(421)has been identified as essential for high-affinity ligand binding. To determine whether Tyr(462)is similarly required for Tpo binding, wild-type murine Mpl (Mpl-WT) or mutant receptors containing an alanine (Y462A) or lysine (Y462K) in place of Tyr(462)were expressed in BaF3 cells. In proliferation studies, the Y462A mutation had no effect on Tpo-induced growth. In contrast, the Y462K mutation led to an attenuated proliferative response to Tpo. In single-point binding studies, both Mpl-WT and Y462A cells were able to bind [(125)I]Tpo in a specific manner. In contrast, there was a marked reduction in binding of [(125)I]Tpo by Y462K cells. Mpl-WT cells bound Tpo with a K(d)of approximately 330 pM, while Y462A cells bound Tpo with a K(d)of approximately 268 pM. The binding affinity of Y462K cells was below that quantifiable by Scatchard analysis. This study suggests that unlike the corresponding Tyr(421)of the common human beta chain, Tyr(462)of murine Mpl is not required for high-affinity ligand binding, although it may be located in proximity to the ligand binding site.
Amino Acid Sequence↗
Use of thrombopoietic growth factors in acute leukemia.
Several hematopoietic growth factors have been shown to affect megakaryocyte development, and two, interleukin (IL)-11 and thrombopoietin (TPO) are presently being evaluated for use in patients with thrombocytopenia. In two studies patients who required one or more platelet transfusions during their first course of chemotherapy were found to require fewer platelet transfusions if their second cycle was augmented with IL-11. The drug was generally safe, with cardiovascular compromise the only significant complication occurring in a minority of patients. Although these reports included patients with various malignancies, studies of IL-11 in patients with myeloproliferative disorders have not been presented. In several clinical trials in cancer patients treatment with TPO was safe, and when administered early following a moderately aggressive cytotoxic insult was effective in accelerating platelet recovery. In addition, in both pre-clinical and clinical trials, TPO given to stem cell donors during mobilization lead to accelerated hematopoietic recovery. Finally, TPO appears safe when administered to patients with acute myelogenous leukemia (AML), both with respect to acute toxicity and long-term outcome of the leukemia. However, when used following a 7-day course of standard chemotherapy, the agent does not appear to accelerate platelet recovery. As such, additional clinical trials to test different growth factor regimens are ongoing. A number of studies have suggested that megakaryocytic growth factors may play a role in the biology of myeloproliferative disorders. Given the potential for adversely affecting patients with these disorders, the affects of IL-11 or TPO in patients with AML must continue to be carefully studied.
Acute Disease↗
CDP/Cut DNA binding activity is down-modulated in granulocytes, macrophages and erythrocytes but remains elevated in differentiating megakaryocytes.
DNA binding by the CCAAT-displacement protein, the mammalian homologue of the Drosophila melanogaster Cut protein, was previously found to increase sharply in S phase, suggesting a role for CDP/Cut in cell cycle progression. Genetic studies in Drosophila indicated that cut plays an important role in cell-type specification in several tissues. In the present study, we have investigated CDP/Cut expression and activity in a panel of multipotent hematopoietic cell lines that can be induced to differentiate in vitro into distinct cell types. While CDP/Cut DNA binding activity declined in the pathways leading to macrophages, granulocytes and erythrocytes, it remained elevated in megakaryocytes. CDP/Cut was also highly expressed in primary megakaryocytes isolated from mouse, and some DNA binding activity could be detected. Altogether, these results raise the possibility that CDP/Cut may be a determinant of cell type identity downstream of the myelo-erythroid precursor cell. Another possibility, which does not exclude a role in lineage identity, is that CDP/Cut activity in megakaryocytes is linked to endomitosis. Indeed, elevated CDP/Cut activity in differentiating megakaryocytes and during the S phase of the cell cycle suggests that it may be required for DNA replication.
Animals↗
Dysregulated activation of activator protein 1 in keratinocytes of atopic dermatitis patients with enhanced expression of granulocyte/macrophage-colony stimulating factor.
Keratinocytes of patients with atopic dermatitis produce high amounts of granulocyte/macrophage colony-stimulating factor, a factor essential for dendritic cell function and thus for the development of skin immune responses. In contrast to keratinocytes cultured from nonatopic, healthy individuals, granulocyte/macrophage colony-stimulating factor mRNA could be detected in unstimulated cultures of atopic dermatitis keratinocytes, and phorbol myristate acetate induced much greater granulocyte/macrophage colony-stimulating factor mRNA levels in these cells, although the decay kinetics were not altered. Using reporter gene (chloramphenicol acetyl transferase) analysis, a minimal granulocyte/macrophage colony-stimulating factor promoter was shown to confer constitutive and phorbol-myristate-acetate-induced regulation of transcriptional activity in keratinocytes, and significantly higher levels of chloramphenicol acetyl transferase activity were measured in lysates of unstimulated and phorbol-myristate-acetate-treated atopic dermatitis keratinocytes than in control keratinocyte cultures. Electrophoretic mobility shift assays showed that low levels of NF-kappa B binding activity could be induced by phorbol myristate acetate in both normal and atopic dermatitis keratinocytes. By contrast, activator protein 1 complexes were efficiently induced, and they were invariably present at higher levels in nuclear lysates of atopic dermatitis keratinocytes. Atopic dermatitis keratinocyte nuclear lysates had higher constitutive levels of c-Jun, and phorbol myristate acetate promoted an earlier and stronger expression of c-Jun, JunB, and of the phosphorylated forms of c-Fos. A dysregulated activation of activator protein 1 may be implicated in the molecular mechanisms leading to increased granulocyte/macrophage colony-stimulating factor expression in atopic dermatitis keratinocytes. J Invest Dermatol 115:1134-1143 2000
Adolescent↗
Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis.
Thrombopoietin (TPO) plays a critical role in megakaryocyte proliferation and differentiation. Using various cultured cell lines, several recent studies have implicated the mitogen-activated protein kinase (MAPK) pathway in megakaryocyte differentiation. In the study reported here, we examined the role played by thrombopoietin-induced MAPK activity in a cytokine-dependent cell line (BAF3/Mpl) and in primary murine megakaryocytes. In both systems, extracellular signal-regulated protein kinase (ERK) 1 and 2 MAPK phosphorylation was rapidly induced by TPO stimulation. To identify the Mpl domain responsible for MAPK activation, BAF3 cells expressing truncated forms of the Mpl receptor were studied. Phosphorylation of ERKs did not require elements of the cytoplasmic signaling domain distal to Box 2 and was not dependent on phosphorylation of the adapter protein Shc. ERK activation in murine megakaryocytes was maximal at 10 minutes and was markedly decreased over the subsequent 3 hours. Next, the physiologic consequences of MAPK inhibition were studied. Using the MAPK kinase (MEK) inhibitor, PD 98059, blockade of MAPK activity substantially reduced TPO-dependent proliferation in BAF3/Mpl cells and markedly decreased mean megakaryocyte ploidy in cultures. To exclude an indirect effect of MAPK inhibition on stromal cells in whole bone marrow, CD41(+) cells were selected and then cultured in TPO. The number of polyploid megakaryocytes derived from the CD41-selected cells was also significantly reduced by MEK inhibition, as was their geometric mean ploidy. These studies show an important role for MAPK in TPO-induced endomitosis and underscore the value of primary cells when studying the physiologic effects of signaling pathways.
Animals↗
Deletion of the extracellular membrane-distal cytokine receptor homology module of Mpl results in constitutive cell growth and loss of thrombopoietin binding.
The thrombopoietin receptor, Mpl, is a member of the cytokine receptor superfamily. The extracellular domain of Mpl contains two copies of the cytokine receptor homology module (CRM). Mpl is encoded by c-mpl, the cellular homologue of the oncogene v-mpl. The oncogenic potential of v-mpl may arise from deletion of all but the 43 most membrane-proximal amino acids of the extracellular domain of the wild-type receptor. To test the hypothesis that the extracellular domain of Mpl plays a role in controlling receptor activity, we created mutants of murine Mpl in which the membrane-distal CRM was either deleted or replaced by the membrane-proximal CRM. Introduction of these mutant receptors into factor-dependent BaF3 cells led to constitutive cell growth in the absence of growth factor. Both mutant receptors failed to bind 125I-Tpo. These results suggest that the membrane-distal CRM of Mpl acts as a brake on cell proliferation and that this region is required for ligand binding.
Animals↗
Thrombopoietin signal transduction requires functional JAK2, not TYK2.
The Janus family of tyrosine kinases (JAKs) plays a critical role in signal transduction by members of the cytokine receptor superfamily. In response to ligand-receptor interaction, these nonreceptor tyrosine kinases are rapidly phosphorylated and activated, triggering tyrosine phosphorylation and activation of downstream signaling intermediates. Upon binding to its receptor, the product of the proto-oncogene c-mpl, thrombopoietin (TPO) activates both JAK2 and TYK2 in multiple cell lines as well as megakaryocytes and platelets. To study whether one or both of these kinases are essential for TPO signal transduction, we engineered a parental human sarcoma cell line (2C4) as well as sarcoma cell lines that are deficient in JAK2 expression (gamma2A) or TYK2 expression (U1A) to express the wild-type Mpl receptor. The ability of TPO to induce tyrosine phosphorylation of Mpl and multiple intracellular substrates in each cell line was then examined. Our results demonstrate that JAK2-deficient cells (gamma2A-Mpl) are unable to initiate TPO-mediated signaling. In contrast, cells that are TYK2-deficient (U1A-Mpl) are able to induce tyrosine phosphorylation of Mpl, JAK2, STAT3, and Shc as efficiently as parental cells (2C4-Mpl). These data indicate that JAK2 is an essential component of Mpl signaling and that, in the absence of JAK2, TYK2 is incapable of initiating TPO-induced tyrosine phosphorylation.
Base Sequence↗
Thrombopoietin and hematopoietic stem cell development.
Thrombopoietin, the long sought primary regulator of thrombopoiesis, was cloned four years ago. In addition to its fulfilling most, if not all, of the expected biological activities relating to megakaryocyte and platelet development, the availability of the recombinant hormone and reagents to characterize its receptor have allowed detailed investigation of additional biological activities. In cultures of purified populations of candidate stem cells, thrombopoietin supports the survival, and augments the proliferation of hematopoietic stem cells when present together with interleukin-3 or steel factor. The progeny of such cultures are not skewed in their developmental potential; colony-forming cells of all lineages arise from thrombopoietin-stimulated stem cells. Evidence for an important effect of thrombopoietin on stem cell physiology in vivo are equally compelling. Genetic elimination of thrombopoietin or its receptor leads to a profound reduction not only of megakaryocytes and platelets, but also of committed myeloid progenitors of all types, primitive progenitors and hematopoietic stem cells. When administered to animals, thrombopoietin profoundly stimulates thrombopoiesis and enhances the number of hematopoietic progenitor cells of all lineages, and when used in most animal models of myelosuppressive therapy, accelerates the recovery of platelet, erythrocyte and leukocyte production. Thus, thrombopoietin appears to be more than a lineage-restricted growth factor.
Animals↗
The enigmatic megakaryocyte gradually reveals its secrets.
The recent cloning of thrombopoietin has brought many insights into the cellular and molecular mechanisms of megakaryocyte and platelet development. Thrombopoietin was cloned based on its binding to the product of the proto-oncogene c-mpl and was found to affect all aspects of thrombopoiesis. Many of the molecular pathways that mediate thrombopoietin action have been discerned. Upon hormone binding, the megakaryocyte thrombopoietin receptor homodimerizes, activating members of the JAK family of kinases, which, in turn, phosphorylate the receptor, generating docking sites for second messengers that affect multiple signalling pathways. Ultimately, cellular proliferative and anti-apoptotic mechanisms are initiated, increasing megakaryocyte numbers, as are processes that uncouple DNA synthesis from nuclear and cytoplasmic division, generating polyploid cells. As the net result of thrombopoietin action is an expansion of cells that give rise to mature platelets, the availability of the recombinant hormone has provided new opportunities to manipulate blood cell development for therapeutic benefit.
Animals↗
Thrombopoietin signal transduction: studies from cell lines and primary cells.
Thrombopoietin (TPO) and its receptor Mpl support all of the developmental step necessary for megakaryocytopoiesis. In the past few years, the signaling pathways utilized by this member of the cytokine receptor family have been extensively studied, especially JAK/STAT, Ras/MAP kinase, Shc, and other adapter molecules. Many if not most of the secondary signaling pathways activated by thrombopoietin have also been identified upon binding of other hematopoietic growth factors to their cognate receptors, making the study of Mpl signaling representative of the field in general. However, identifying unique molecules or combinations of signals that direct megakaryocyte development has been an elusive goal and has led some investigators to conclude that there is little specificity during Mpl signal transduction. In this article we review the data regarding Mpl signaling with particular attention to the methods employed and critical interpretation of the data generated. Future studies will have to focus on primary bone marrow cells and intact animal models rather than transformed cell lines. Furthermore, it is likely that a comprehensive, integrative analysis of the many pathways activated by ligand binding will be necessary to understand the physiology of cytokine signaling.
Animals↗
Megakaryocyte growth factors and acute myeloid leukemia.
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Acute Disease↗
Thrombopoietin: from theory to reality.
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Animals↗