Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.

Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation.     

Disparate effects of interleukin 11 and thrombopoietin on megakaryocytopoiesis in vitro

The effects of interleukin-11 (IL-11) and thrombopoietin (TPO) on murine megakaryocytopoiesis were studied using a serum-free culture system. Acting alone, both IL-11 and TPO increased the number of acetylcholinesterase (AchE)(+)cells (megakaryocytes), the latter being more potent than the former. TPO, but not IL-11, increased the mean AchE activity per megakaryocyte (AchE activity/megakaryocyte). TPO increased both the number of megakaryocytes with high ploidy, and of those with low ploidy. In contrast, IL-11 increased only the number of megakaryocytes with high ploidy. The effect of TPO on megakaryocyte ploidy was stronger than that of IL-11. Both IL-11 and TPO increased the proportion of large megakaryocytes, but the latter was more potent than the former. While the stimulatory effects of IL-11 and TPO on the number of megakaryocytes were enhanced by IL-3 or stem cell factor (SCF), synergism of IL-11 or TPO with IL-3 or SCF in stimulating AchE activity/megakaryocyte was inconsistent. IL-11 and TPO stimulated the formation of colony-forming units of megakaryocyte in the presence of IL-3, but not alone, with similar maximum colony numbers for both cytokines. Our findings thus demonstrate that IL-11 principally stimulates megakaryocyte maturation rather than the proliferation of megakaryocytes, whereas TPO stimulates both.     

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.     

Thrombopoietin induces phosphoinositol 3-kinase activation through SHP2, Gab, and insulin receptor substrate proteins in BAF3 cells and primary murine megakaryocytes

Thrombopoietin (TPO) is a recently characterized member of the hematopoietic growth factor family that serves as the primary regulator of megakaryocyte (MK) and platelet production. The hormone acts by binding to the Mpl receptor, the product of the cellular proto-oncogene c-mpl. Although many downstream signaling targets of TPO have been identified in cell lines, primary MKs, and platelets, the molecular mechanism(s) by which many of these molecules are activated remains uncertain. In this report we demonstrate that the TPO-induced activation of phosphoinositol 3-kinase (PI3K), a signaling intermediate vital for cellular survival and proliferation, occurs through its association with inducible signaling complexes in both BaF3 cells engineered to express Mpl (BaF3/Mpl) and in primary murine MKs. Although a direct association between PI3K and Mpl could not be demonstrated, we found that several proteins, including SHP2, Gab2, and IRS2, undergo phosphorylation and association in BaF3/Mpl cells in response to TPO stimulation, complexes that recruit and enhance the enzymatic activity of PI3K. To verify the physiological relevance of the complex, SHP2-Gab2 association was disrupted by overexpressing a dominant negative SHP2 construct. TPO-induced Akt phosphorylation was significantly decreased in transfected cells suggesting an important role of SHP2 in the complex to enhance PI3K activity. In primary murine MKs, TPO also induced phosphorylation of SHP2, its association with p85 and enhanced PI3K activity, but in contrast to the results in cell lines, neither Gab2 nor IRS2 are phosphorylated in MKs. Instead, a 100-kDa tyrosine-phosphorylated protein (pp100) co-immunoprecipitated with the regulatory subunit of PI3K. These findings support a model where PI3K activity is dependent on its recruitment into TPO-induced multiphosphoprotein complexes, implicate the existence of a scaffolding protein in primary MKs distinct from the known Gab and IRS proteins, and suggest that, in contrast to erythroid progenitor cells that employ Gab1 in PI3K signaling complexes, utilization of an alternate member of the Gab/IRS family could be responsible for specificity in TPO signaling.     

Role of a serine/threonine kinase, Mst1, in megakaryocyte differentiation

Platelets, which play a central role in thrombosis and hemostasis, develop from megakaryocytes. Signal transduction originated from the megakaryocyte growth and development factor, the Mpl ligand, which leads to megakaryocyte differentiation, polyploidization, and maturation, has been gradually characterized. In this study, we report the inducibility of Mst1, a recently described serine/threonine kinase, by Mpl ligand and the effect of its induced expression on megakaryocyte differentiation. The steady-state level of mst1 message and Mst1-associated kinase activity increased in response to Mpl ligand. Ectopic expression of human mst1 in a mouse megakaryocytic cell line resulted in a drastic increase in DNA content per cell. Elevated expression of megakaryocyte differentiation markers, such as acetylcholine esterase, PF4, and GPIIb was also observed in hmst1-expressing cells. Activation of p38 MAPK, a known downstream effector of Mst1, was shown to be required for polyploidization, but not for enhanced expression of differentiation markers. Our study thus designates Mst1 as a Mpl ligand-responsive signaling molecule that promotes induction of lineage-specific cellular programming.     

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.     

Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.     

Phosphatidylinositol 3-kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocytic progenitors.

Thrombopoietin and its receptor (Mpl) support survival and proliferation in megakaryocyte progenitors and in BaF3 cells engineered to stably express Mpl (BaF3/Mpl). The binding of thrombopoietin to Mpl activates multiple kinase pathways, including the Jak/STAT, Ras/Raf/MAPK, and phosphatidylinositol 3-kinase pathways, but it is not clear how these kinases promote cell cycling. Here, we show that thrombopoietin induces phosphatidylinositol 3-kinase and that phosphatidylinositol 3-kinase is required for thrombopoietin-induced cell cycling in BaF3/Mpl cells and in primary megakaryocyte progenitors. Treatment of BaF3/Mpl cells and megakaryocytes with the phosphatidylinositol 3-kinase inhibitor LY294002 inhibited mitotic and endomitotic cell cycl-ing. BaF3/Mpl cells treated with thrombopoietin and LY294002 were blocked in G(1), whereas megakaryocyte progenitors treated with thrombopoietin and LY294002 showed both a G(1) and a G(2) cell cycle block. Expression of constitutively active Akt in BaF3/Mpl cells restored the ability of thrombopoietin to promote cell cycling in the presence of LY294002. Constitutively active Akt was not sufficient to drive proliferation of BaF3/Mpl cells in the absence of thrombopoietin. We conclude that in BaF3/Mpl cells and megakaryocyte progenitors, thrombopoietin-induced phosphatidylinositol 3-kinase activity is necessary but not sufficient for thrombopoietin-induced cell cycle progression. Phosphatidylinositol 3-kinase activity is likely to be involved in regulating the G(1)/S transition.     

Thrombopoietin and hematopoietic stem cells

Thrombopoietin (TPO) is the cytokine that is chiefly responsible for megakaryocyte production but increasingly attention has turned to its role in maintaining hematopoietic stem cells (HSCs). HSCs are required to initiate the production of all mature hematopoietic cells, but this differentiation needs to be balanced against self-renewal and quiescence to maintain the stem cell pool throughout life. TPO has been shown to support HSC quiescence during adult hematopoiesis, with the loss of TPO signaling associated with bone marrow failure and thrombocytopenia. Recent studies have shown that constitutive activation mutations in Mpl contribute to myeloproliferative disease. In this review, we will discuss TPO signaling pathways, regulation of TPO levels and the role of TPO in normal hematopoiesis and during myeloproliferative disease.Key words: thrombopoietin, TPO, Mpl, hematopoietic stem cell, hematopoiesis, Jak2, MPLW515K, MPLW515L     

Signaling by the Mpl receptor involves IKK and NF-kappaB

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.     

Thrombopoietin and hematopoietic stem cells

Thrombopoietin (TPO) is the cytokine that is chiefly responsible for megakaryocyte production but increasingly attention has turned to its role in maintaining hematopoietic stem cells (HSCs). HSCs are required to initiate the production of all mature hematopoietic cells, but this differentiation needs to be balanced against self-renewal and quiescence to maintain the stem cell pool throughout life. TPO has been shown to support HSC quiescence during adult hematopoiesis, with the loss of TPO signaling associated with bone marrow failure and thrombocytopenia. Recent studies have shown that constitutive activation mutations in Mpl contribute to myeloproliferative disease. In this review, we will discuss TPO signaling pathways, regulation of TPO levels and the role of TPO in normal hematopoiesis and during myeloproliferative disease.     

Differential signalling of NH2-terminal flag-labelled thrombopoietin receptor activated by TPO or anti-FLAG antibodies

In this report, we compared activation of NH2-terminal FLAG-labelled thrombopoietin receptor (Mpl) by anti-FLAG antibodies and by thrombopoietin (TPO). We found that anti-FLAG monoclonal antibodies M1 dimerize FLAG-labelled receptor and trigger proliferation of BaF3/FLAG-Mpl cells. In UT7/FLAG-Mpl cells, activation of the FLAG-Mpl receptor by low TPO concentrations triggered proliferation, while high concentrations triggered differentiation. Activation of FLAG-Mpl receptors in these cells by all tested concentrations of M1 antibodies induced proliferation but not differentiation. Low TPO concentrations induced similar to M1 antibodies level of Jak2, Stat3, Stat5 and Akt phosphorylation. In contrast, only TPO and not M1 antibodies activated Erks phosphorylation. Since the anti-FLAG antibodies do not react with the TPO binding site of the receptor, we hypothesize that they can trigger a distinct signal by dimerizing Mpl in a manner different from that induced by TPO.     

Regulation of cell differentiation by hNUDC via a Mpl-dependent mechanism in NIH 3T3 cells

Thrombopoietin receptor (Mpl) belongs to the cytokine receptor surperfamily with a large extracellular N-terminal portion responsible for cytokine recognition and binding. Thrombopoietin (TPO) has so far been the only widely studied cytokine for Mpl. However we have recently identified human NUDC (hNUDC), previously described as a human homolog of a fungal nuclear migration protein, as another putative binding partner of Mpl. The purpose of this study is to test the extent of the functioning of hNUDC by identifying protein-protein interactions with Mpl in mammalian cells. The full-length cDNAs encoding Mpl and hNUDC were cloned into pEGFP-N1 and pDsRed2-N1 respectively which were subsequently expressed as Mpl-EGFP (green) and hNUDC-DsRed (red) fusion proteins. Using ELISA and immunofluorescence studies, we have demonstrated the direct binding of hNUDC to cell surface-captured Mpl. We also observed that hNUDC induced significant changes in cellular morphology in NIH 3T3 cells stably transfected with pMpl-EGFP. Interestingly, these morphological changes were characteristic of cells undergoing megakaryocyte differentiation. Extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) have been shown to mediate such megakaryocyte-like differentiation. In addition, co-expression of Mpl-EGFP and hNUDC-DsRed led to the release of hNUDC-DsRed into the culture medium.     

Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)

Thrombopoietin (TPO) and its receptor (Mpl) have long been associated with megakaryocyte proliferation, differentiation, and platelet formation. However, studies have also shown that the extracellular domain of Mpl (Mpl-EC) interacts with human (h) NUDC, a protein previously characterized as a human homolog of a fungal nuclear migration protein. This study was undertaken to further delineate the putative binding domain on the Mpl receptor. Using the yeast two-hybrid system assay and co-immunoprecipitation, we identified that within the Mpl-EC domain 1 (Mpl-EC-D1), amino acids 102–251 were strongly involved in ligand binding. We subsequently expressed five subdomains within this region with T7 phage display. Enzyme-linked immunosorbent binding assays identified a short stretch of peptide located between residues 206 and 251 as the minimum binding domain for both TPO and hNUDC. A series of sequential Ala replacement mutations in the region were subsequently used to identify the specific residues most involved in ligand binding. Our results point to two hydrophobic residues, Leu²²⁸ and Leu²³⁰, as having substantial effects on hNUDC binding. For TPO binding, mutations in residues Asp²³⁵ and Leu²³⁹ had the largest effect on binding efficacy. In addition, deletion of the conservative motif WGSWS reduced binding capacity for hNUDC but not for TPO. These separate binding sites on the Mpl receptor for TPO and hNUDC raise interesting implications for the cytokine-receptor interactions.     

AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl

Thrombopoietin (TPO) plays a pivotal role in megakaryopoiesis. TPO initiates its biological effects by binding to its receptor Mpl. A recombinant protein consisting of a carrier Fc domain linked to multiple Mpl-binding domains was constructed, and is called AMG531. To define the biological activity of AMG531, we examined the ability of AMG531 to support CFU-Meg growth and to promote megakaryocyte maturation in vitro. AMG531 stimulates CFU-Meg growth in a dose-dependent manner, and acts in concert with erythropoietin, stem cell factor, interleukin-3, and interleukin-6 to enhance CFU-Meg growth, similar to parallel experiments with TPO. AMG531-stimulated serum-free liquid cultures support the development of mature polyploid megakaryocytes with a predominant DNA content of 32 N and 64 N, identical to that of parallel TPO-stimulated cultures. Competitive binding experiments show that AMG531 effectively competes with 125I-TPO for binding to BaF3-Mpl cells or normal platelets. Treatment of BaF3-Mpl cells with AMG531 or with TPO resulted in rapid tyrosine phosphorylation of Mpl, JAK2, and STAT5. These results indicate that AMG531 is a potent stimulant of megakarypoiesis in vitro, and provide support for its further characterization in vivo.     

Role of a serine/threonine kinase,Mst1, in megakaryocyte differentiation

Platelets, which play a central role in thrombosis and hemostasis, develop from megakaryocytes. Signal transduction originated from the megakaryocyte growth and development factor, the Mpl ligand, which leads to megakaryocyte differentiation, polyploidization, and maturation, has been gradually characterized. In this study, we report the inducibility of Mst1, a recently described serine/threonine kinase, by Mpl ligand and the effect of its induced expression on megakaryocyte differentiation. The steady‐state level of mst1 message and Mst1‐associated kinase activity increased in response to Mpl ligand. Ectopic expression of human mst1 in a mouse megakaryocytic cell line resulted in a drastic increase in DNA content per cell. Elevated expression of megakaryocyte differentiation markers, such as acetylcholine esterase, PF4, and GPIIb was also observed in hmst1‐expressing cells. Activation of p38 MAPK, a known downstream effector of Mst1, was shown to be required for polyploidization, but not for enhanced expression of differentiation markers. Our study thus designates Mst1 as a Mpl ligand‐responsive signaling molecule that promotes induction of lineage‐specific cellular programming. J. Cell. Biochem. 76:44–60, 1999. © 1999 Wiley‐Liss, Inc.     

Repression of A TAF(II)32 isoform as part of a program of genes regulated during mpl ligand-induced megakaryocyte differentiation.

Circulating platelets, essential for thrombosis and hemostasis, originate from megakaryocytes. Megakaryocyte growth, differentiation and survival processes are regulated by the c-Mpl receptor ligand. In the current study we used differential display to identify part of the program of genes regulated during Mpl ligand-induced murine megakaryocyte differentiation. Several of the genes, including the retinoblastoma binding protein p84, were found to be induced, while others were repressed. One such repressed gene was identified as a TATA-binding protein (TBP)-Associated Factor (TAF) family member, TAF(II)32, previously reported to be upregulated during apoptosis. Our analysis of various cell types suggested that the previously identified species homologs, human TAF(II)32 and murine TAF(II)32, are in fact different isoforms, which we propose to re-name TAF(II)32alpha and TAF(II)32beta, respectively. Only the TAF(II)32beta isoform is regulated during Mpl ligand-induced megakaryocyte differentiation, which suggests individual roles for the two forms.