Thrombopoietin and its receptor.

F. WENDLING; W. VAINCHENKER

Discovery. A portion of the thrombopoietin receptor, the c-mpl gene, was initially discovered fused to viral sequences encoding the envelope protein of the mutant murine myeloproliferative leukemia virus (MPLV). The ability of the oncogene v-mpl to transform a variety of hematopoietic progenitor cells [1-3] and its homology to members of the cytokine receptor superfamily prompted speculation that its cellular homolog was a novel cytokine receptor [3]. Isolation of human and mouse c-mpl cDNAs in 1992 and 1993 confirmed that Mpl belongs to the hematopoietic growth factor receptor superfamily [4-6].

The c-mpl gene is located on human chromosome 1p34 and on murine chromosome 4 [5-8]. In both human and mouse, the c-mpl gene contains 12 exons distributed over 15 kb. Its organisation conforms closely to the pattern observed for the genes of other hematopoietic receptor family members [8-10]. The promoter region lacks conventional TATA and CAAT motifs, but contains consensus binding sequences for several transcriptional regulators including GATA and Ets factors that are implicated in the transcription control of megakaryocyte-specific genes. Site-directed mutagenesis experiments identify one GATA-1 and two Ets motifs which play a crucial role in c-mpl expression. Transactivation assays demonstrate that GATA-1, Ets-1 and Fli-1 efficiently transactivate the c-mpl promoter in heterologous cells [11].

In human, three mRNAs are generated by alternative splicing. Mpl-P encodes a 635 amino acids protein corresponding to the functional receptor; Mpl-K encodes a smaller protein of 572 amino acids produced by premature termination of the transcript within intron 10, and a mRNA lacking exons 9 and 10 encodes a putative soluble form of Mpl [10]. On Northern blot, a c-mpl probe recognizes a 3.7 kb and more faintly a 2.6 kb mRNAs corresponding to Mpl-P and Mpl-K, respectively [4]. The murine c-mpl gene codes for a protein of 625 amino acids, but transcripts of various lengths generated by alternative splicing are also detected: Mpl-2 contains a 8 amino acids in-frame insertion in exon 4; Mpl type II has a 60 amino acids deletion in exon 4 [12] and a soluble Mpl (Mpl-S) may be generated by a mRNA lacking exon 10 [5, 6, 8]. Murine c-mpl is expressed as a 3 kb mRNA in spleen, bone marrow and fetal liver [8]. The physiological role of the different variants are not elucidated. The general structure of the entire molecule is presented in Figure 1.

The extracellular domains of the human and murine Mpl proteins are composed of 463 amino acids and 465 amino acids, respectively, containing several potential N-linked glycosylation sites. Both contain a duplication of the canonical hematopoietin receptor domain (HRD) [9, 13], as found in a minority of cytokine receptors, the common ß chain of the IL-3, GM-CSF and IL-5 receptors [14], and the leukemia inhibitory factor (LIF) alpha chain [15]. The transmembrane domain contains 22 amino acids. The cytoplasmic domain contains 122 amino acids in human and 121 amino acids in mouse. Sequence comparison shows more that 80% amino acids identity in the extracellular domains. The most conserved region is found in the cytoplasmic domain (91% amino acid identity) with an identical stretch of 37 amino acids close to the transmembrane. Like other members of the hematopoietic receptor superfamily, no consensus sequence for kinase activity is found in the Mpl cytoplasmic domain.

Expression of the Mpl gene is restricted to hematopoietic tissues (bone marrow, fetal liver and spleen). No expression is seen in lymphoid organs. Among normal hematopoietic cells, fluorescence-activated cell sorter analysis reveals a strong cell surface expression in differentiating cells belonging to the megakaryocytic (MK) lineage, from the CFU-MK (colony forming unit-MK) stage to platelet [16]. A weaker expression is found on bipotent erythro/megakaryocyte progenitor cells [17]. A recent study demonstrates Mpl expression on approximately 50% of the murine fetal stem cell-enriched population AA4⁺ Sca⁺ c-kit⁺, as well as on 70% of the marrow Lin^lo Sca⁺ c-kit⁺ stem cell subpopulation [18]. Among the human CD34⁺ CD38 stem cell progenitor pool, Mpl expression is detected on about 70% of the cells and CD34⁺ CD38 Mpl⁺ cells engraft SCID mice far more efficiently than CD34⁺ CD38 Mpl cells [18]. Platelets and MK display a single class of high affinity Mpl receptors (approximately 30 receptors/platelet and 1,933 to 12,140 receptors/MK with a kd of 200 to 749 pM) [19-21].

The presence of c-mpl mRNA or cell surface protein is found on pluripotent, megakaryoblastic, some erythroleukemic and one monocytic permanent cell lines [22-24]. No expression is detected in pre-B, B, T, NK and plasmocytic cell lines, or in blast cells from acute lymphoblastic leukemia [25]. However, in about 50% of the cases, c-mpl mRNA is detected in blast cells from primary acute myeloid leukemia (AML, French-American-British subtypes M0, M2, M4 and M7), in a substantial fraction of refractory anemia with excess of blast (RAEB) and chronic myelomonocytic leukemia cells [26]. In addition, blast cells expressing Mpl are able to proliferate in response to TPO in about 60% of the cases [25-28]. In patient with secondary AML developing during the progression of myeloproliferative or myelodysplastic syndromes, a correlation between the level of Mpl expression and poor prognosis was established [26].

No c-mpl transcripts have been detected by RT-PCR on a large panel of primary solid tumors of various origin [24, 29].

Mutant murine Mpl receptors have been generated by amino acid substitution within a critical dimer interface conserved between the erythropoietin (EPO) and the growth hormone receptors. Substitution by cysteine residues induces homodimerization and constitutive activation of Mpl. Moreover, transfected factor-dependent cell lines expressing these mutants become tumorigenic [30]. These data imply that ligand-induced Mpl activation involves receptor homodimerization. Using retrovirus-mediated gene transfer, an activating point mutation (Ser⁴⁹⁸ to Asn⁴⁹⁸) located in the transmembrane domain of the human Mpl has been identified [31]. Expression of the mutant in factor-dependent cell lines abrogates factor dependency and leads to tumorigenicity [31].

Detailed studies of the hematopoietic growth factor receptors indicate that ligand/receptor interaction leads to tyrosine phosphorylation of intracellular signaling molecules including various members of the Janus tyrosine kinase (JAKs), signal transducer and activator of transcription (STATs), mitogen-activated protein kinase (MAPKs) families, the adaptor protein Shc and the receptors themselves [for a review see 32]. A number of studies have documented the molecular mechanism involved in Mpl signal transduction in a variety of cellular contexts. In human megakaryoblastic UT-7, MO7e and DAMI cell lines, Mpl ligand induces a rapid and transient tyrosine phosphorylation of JAK2 and TYK2, and three STAT members (STAT1, STAT3 and STAT5). No detectable activation of the other members of the JAK- and STAT families is observed [33-35]. A time-course study shows that upon ligand binding JAK2, Shc and the Mpl receptor itself become phosphorylated within 1 min. However, direct interaction between JAK2 and Mpl is only detectable after 20 min of ligand stimulation and increases after 60 min suggesting that an other tyrosine kinase might be involved in receptor phosphorylation [36]. Nevertheless, JAK2 is essential since hematopoietic progenitors from JAK2-deficient mice are unresponsive to TPO. In human platelets and megakaryocytes, Mpl ligand induces tyrosine phosphorylation of JAK2, TYK2, Shc and Grb2 confirming the studies performed with cell lines [37-39]. Studies were performed to dissect the signaling events that are more specifically involved in megakaryocytic differentiation. Chimeric receptors containing deletions within the intracytoplasmic domain have identified the regions required for the generation of mitogenic versus differentiation signals [40-47]. Recently, it has been reported that Mpl-induced MK differentiation is tightly dependent on a strong and long-lasting activation of the MAPK signaling pathway [47, 48].

In 1994, 5 independent groups published the purification and cloning of the ligand for Mpl (Mpl-L), also named thrombopoietin (TPO), megakaryocyte growth and development factor (MGDF) or megapoietin. Researchers at Genentech and Amgen used purification schemes centered around Mpl-affinity columns to isolate the ligand from the plasma of thrombocytopenic pigs and dogs, respectively. Active proteins with identical amino-terminal sequences but various molecular weights (70, 30, 28 and 18 kDa) were isolated. Degenerate oligonucleotides were designed to obtain a probe which was used to screen a human fetal liver cDNA library [49, 50]. The group at ZymoGenetics treated IL-3-dependent murine Ba/F3 cells expressing Mpl with the mutagen 2-ethylmethane-sulfonate and selected mutants for their autonomous growth. Among several autonomous mutant clones secreting IL-3, one of the clone produced an activity that could be neutralized by a soluble form of Mpl. cDNA library pools were prepared from this clone and expressed in mammal cells. Using the pool-breakdown technique, a positive clone was identified from which a full-length cDNA encoding murine TPO was isolated [51]. Two other groups, one at Kirin's Pharmaceutical Research Laboratory [52] and the other at MIT [53] isolated TPO directly from the plasma of thrombocytopenic animals using standard purification methods.

The human TPO cDNA has an open reading frame encoding 353 amino acids consisting of a 21 amino acids secretory signal and a mature polypeptide of 332 amino acids with a predicted molecular mass of 35 kDa. The protein is composed of two domains. The amino-terminal domain of 153 amino acids (EPO-like domain) displays considerable sequence similarity to EPO (23% identity and 50% similarity when conservative substitutions are taken into account). This domain contains two disulfide bonds and no N-linked glycosylation sites. Analysis of truncated forms of TPO showed that the amino-terminal region (TPO_1-153) is sufficient to fully activate Mpl. The carboxy-terminal half of the protein (amino acid 154-332) has no homology to any other known protein. It contains six potential N-linked and several O-linked glycosylation sites. Native TPO is heavily glycosylated with carbohydrate increasing as much as 50% the molecular mass of the protein (68-85 kDa). The C-terminus domain, required for efficient secretion and glycosylation, increases the stability and the potency of the molecule in vivo [49, 50, 54-56]. Although a crystal structure of TPO is not yet available, computer modeling predicts that the N-terminal domain forms a four-alpha-helix bundle, a structure similar to that of growth hormone, EPO and other members of the hematopoietic cytokine family [57, 58].

Two natural alternative splice forms of TPO have been identified in human, pig and mouse. TPO-2 [55] or LPPQ variant [59] has a deletion of 4 amino acids within the EPO-like domain at the junction of exons 4 and 5 which maintains the same reading frame. A second variant is produced by an internal splice within the last exon causing a frame shift. Each variant is present in similar frequencies in TPO-producing organs. Transient expression experiments indicate that these proteins are very poorly or not secreted. The biological function and significance of these splice variants are unknown.

The human TPO locus spans over 6 kb. The coding region is organized in 5 exons with intron/exon boundaries precisely corresponding to that of the EPO gene. There is one or two upstream noncoding exons [55, 60]. Exon 1 encodes a 5'-untranslated sequence and the first 4 amino acids of the secretory signal. The entire carboxyl-domain of the protein is encoded by exon 5 with no intron at the junction between the two TPO domains [55, 61] (Figure 2). The 5'-flanking region of the TPO gene does not reveal putative TATA- or CAAT-box motifs. The molecular mechanisms controlling the expression of the TPO gene are still unknown. Multiple sites for initiation of transcription have been identified by S1 nuclease mapping, 5'RACE or RNase protection assays [60, 62, 63]. A region located from -88 to -58 and containing a core motif for Ets family proteins appears to be essential for optimal transcription. Co-expression experiments show that the Ets family member E4TF1/GABP is required for high expression in liver [63].

The TPO gene is located on human chromosome 3q27-q28 [64, 65] and in the in proximal region of mouse chromosome 16 [60]. Since thrombocytosis or dysmegakaryopoiesis is often observed in patients with 3q21 or 3q26 rearrangements, a possible involvement of the TPO gene was examined. No transcriptional activation or chromosomal rearrangement were detected so far excluding an involvement of the TPO gene in these malignancies [64-66].

TPO transcripts are detected in several organs throughout the body, but the main sources are the liver and kidney [49, 51, 67]. In situ hybridization techniques indicate that TPO is produced by parenchymal and in sinusoidal endothelial cells in the liver [68, 69] and in proximal convoluted tubules in the kidney [70]. Biologically active TPO is found in culture supernatants from primary rat hepatocytes, mouse sinusoidal endothelial cells, hepatoma cell lines from various species, a human embryonic kidney cell line and bone marrow-derived stromal cells [69, 71-73]. TPO is a hormone circulating in the blood with a half-life of 20-30 hours [74].

Circulating levels of TPO are elevated in the plasma of thrombocytopenic animals and inversely correlated to the platelet number [75]. A similar inverse relation exists between EPO and red blood cells, or G-CSF (granulocyte-colony stimulating factor) and neutrophil levels. Two models were proposed to understand how TPO is regulated: TPO production could be upregulated by the platelet demand or, alternatively, TPO level could be directly regulated by the platelet mass. No transcriptional regulation of the TPO gene is seen in the liver or kidney from mice rendered either severely thrombocytopenic by radiation, chemotherapy or antiplatelet antibodies treatment, or markedly thrombocythemic by platelet transfusions [70, 76-78]. In addition, no variation in TPO alternative splice transcripts encoding inactive or non secreted proteins is detected in these organs [55, 60, 76, 77, 79]. Moreover, the gene dosage effect on the peripheral platelet number observed in heterozygous TPO knock out mice strongly suggests that there is no regulation of TPO transcription [80]. Nevertheless, TPO is also produced by marrow stromal cells and it is reported that a transcriptional regulation may occur in the bone marrow in response to the platelet demand [81, 82].

The central role of platelets in the plasmatic clearance of TPO is demonstrated by numerous studies. Elevated TPO levels are seen in c-mpl deficient mice having low platelet and MK numbers [83]. When these mice are transfused with platelets from normal donors, plasma TPO level rapidly decreases [79]. Platelets actively bind TPO, internalize and degradate the protein [19, 79, 84]. Unexpectedly, the predicted elevation of TPO level is not observed in the profoundly thrombocytopenic NF-E2 knock out mice in which the major defect is the inability of MK to produce platelets [85]. This observation indicates that the MK mass also contributes to the regulation of the plasmatic concentration of TPO [86]. The obtention of sensitive ELISA assays [87] show that, in thrombocytopenic patients, serum TPO levels are more correlated with the combined MK and platelet mass than with the platelet counts [78]. High TPO levels are found in patients with aplastic anemia where thrombocytopenia is associated with MK hypoplasia [88]. In contrast, patients with immune thrombocytopenic purpura (ITP) exhibit normal or only mildly elevated TPO levels [89-91]. Together, these data are consistent with a model in which TPO is constitutively synthesized and the level in circulation is regulated by sequestration through binding to Mpl present on the surface of both platelets and MKs.

The TPO/Mpl system has been examined in myeloproliferative disorders to understand whether abnormalities were involved in the pathogenesis of these diseases. In essential thrombocythemia (ET), it was shown that serum TPO levels are either normal or slightly elevated compared to normal subjects. Given the regulation of TPO serum level by the platelet and MK masses, this observation was quite unexpected and suggested that an impaired Mpl expression or a TPO overexpression or both could be implicated in this disorder. Two studies show that Mpl expression is markedly reduced in platelets from ET patients as compared to platelets from normal subjects, but Mpl-mediated signaling is normal [92, 93]. Similar findings have been reported in patients with polycythemia vera and agnostic myelofibrosis (PMF), but not in ET patients [94]. Because CD34⁺ cells isolated from ET and PMF patients produce autonomously developing MK colonies at a single cell level, activation of Mpl by missense mutation was investigated. So far, no mutation in the c-mpl coding region and intron/exon junctions is reported [95, 96]. A study was performed on a family with hereditary thrombocythemia in which affected members have elevated serum TPO concentrations. It is demonstrated that a splice donor mutation in the TPO gene is responsible for an overexpression of TPO due to an increased translational efficiency [97]. These data provide new insights into the physiopathology of myeloproliferative disorders.

The potent and lineage dominant action of the Mpl/TPO system on the regulation of platelet production is demonstrated by the generation of Mpl- and TPO- knock out mice [80, 83]. Homozygous animals display an identical phenotype with a 80%-90% reduction in platelet counts and a markedly low number of MK with a reduced ploidy. Although red blood cell and leukocyte counts of homozygous animals are similar to wild-type littermates, Mpl- and TPO-deficient mice show a 50 to 60% reduction in the absolute numbers of all myeloid committed progenitors, including blast cell-colony forming cell [98, 99]. In addition, Mpl-deficient mice have a reduction in the number of colony-forming unit-spleen (both day 8 and day 12 CFU-S) and their bone marrow stem cells have reduced self-renewal potential [100]. Administration of TPO to TPO-deficient mice restores their progenitors to normal or above normal levels [80]. Together, these data demonstrate the essential physiological role of the Mpl/TPO system in the regulation of megakaryopoiesis and platelet production. They also clearly indicate that Mpl/TPO has a broad action in hematopoiesis being required to maintain and/or expand early and committed progenitor cells (Figure 3).

In vitro, TPO alone has a potent and lineage-specific action on late CFU-MK progenitor cells inducing their proliferation, ploidization and maturation [101-104]. MK and platelets produced in culture are functionally and morphologically identical to bone marrow MK and blood-derived platelets [105-108]. Several studies provide evidences that TPO acts synergistically with early acting cytokines in stimulating the proliferation of primitive hematopoietic progenitors [109-118]. Experiments performed with highly enriched population of hematopoietic stem cells show that TPO added to c-kit ligand (SCF) or IL-3 speeds entry into the cell cycle of quiescent stem cells and greatly augments the production of committed progenitors [110, 111]. Addition of TPO to SCF or Flk2/Flt3 ligand greatly enhances the expansion of CD34⁺ CD38 progenitors, maintains their primitive phenotype and their capacity for multilineage differentiation [116-118]. Thus, TPO is an important cytokine for ex vivo expansion of CFU-MK and primitive progenitor cells.

To date, two companies produce recombinant human Mpl ligand for therapeutic use. Amgen-Kirin-ZymoGenetics develops PEG-rHuMGDF (pegylated recombinant human megakaryocyte growth and development factor), a truncated nonglycosylated form of TPO produced in Escheri-chia coli and covalently coupled to polyethylene glycol to increase the in vivo life span of the protein [119]. Genentech-Pharmacia-Upjohn produces a full length glycosylated TPO form in CHO cells (rHuTPO). In normal animals, TPO/PEG-rHuMGDF increases several fold platelet and MK numbers, but does not affect the number of peripheral red blood cells and mononuclear cells [120-124]. In various models of thrombocytopenia induced by myelosuppressive or myeloablative therapies, TPO/rHuMGDF reduces the severity of the platelet nadir, accelerates platelet recovery and reduces mortality [125-127]. After bone marrow transplantation, TPO/PEG-rHuMGDF treatment has either no effect [128] or accelerates platelet recovery [129]. Interestingly, both group of investigators reported that the rate of platelet reconstitution is highly hastened when donors are pretreated with TPO/PEG-rHuMGDF prior to graft harvest [128, 130]. In addition to the major effect on platelet recovery, TPO/PEG-rHuMGDF also dramatically accelerates the recovery of all progenitor classes, improves neutrophil and reticulocyte recovery and mobilized stem/progenitor cells [122, 125, 126, 128, 131-133].

Clinical evaluation of rHuTPO/PEG-rHuMGDF are ongoing [for reviews see 134, 135]. As predicted from preclinical studies, rHuTPO/PEG-rHuMGDF given to cancer patients before receiving chemotherapy resulted in a dose-dependent increase in platelet counts, expansion of marrow myeloid, erythroid and multipotential progenitor cells and a marked mobilization of progenitor cells in peripheral blood [74, 119, 133, 136]. There was no change in white blood cell count and hematocrit. Platelets harvested at peak response have normal appearance and function normally in assays of aggregation and ATP-release in response to various agonists. No major drug-related side effects and no evidence of ischaemia or thrombo-embolism is reported, even in one patient who experienced a very high number of platelets (1,876 x 10⁹/L on day 17) [119, 136]. After chemotherapy, injection of PEG-rHuMGDF accelerated the time for platelet recovery and reduced the nadir platelet count [137, 138]. Basser et al. reported the combined effects of increasing doses of PEG-rHuMGDF and G-CSF (Filgrastim, 5 µg/kg/day)
given to patients treated with carboplatin and cyclophosphamide [139]. No difference in the depth of the platelet nadir count was noted between PEG-rHuMGDF-treated patients and the placebo group. However, platelet recovery to baseline levels occurred earlier (day 17 versus day 22). Notably, when PEG-rHuMGDF was administered both before and after chemotherapy, platelet recovery was hastened as it was during the second cycle of chemotherapy. In severe thrombocytopenia induced by high chemotherapy regimens, the molecule has only a modest influence on the platelet nadir. This may be explained by the high levels of plasmatic endogenous TPO found in these patients. In the phase I trials, the route and frequency of TPO administration were different. PEG-rHuMGDF was given subcutaneously during 10 days [119, 136], while rhuTPO was administrated intravenously as a single dose [74]. Nevertheless, platelet responses were similar. These data indicate that rHuTPO/PEG-rHuMGDF (i) is well tolerated, (ii) is a powerful agent to increase platelet counts in normal or mildly thrombocytopenic patients and (iii) that more studies are needed to define the optimal dosage, route delivery and schedule at which TPO should be administrated in order to reduce the need of platelet transfusions.

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