Thrombopoietin

It has been estimated that an adult human produces nearly 2 × 10¹¹ platelets per day, and this number can increase fourfold to eightfold during times of increased demand. The regulation of this process has been the subject of intense investigation. Kelemen first used the term thrombopoietin in 1958 to describe a humoral substance responsible for enhancing platelet production following the onset of thrombocytopenia. However, it was not until 1994 that five independent groups succeeded in purifying and cloning the responsible cytokine, now known as TPO (previously referred to as cMpl ligand, megakaryocyte growth and development factor [MGDF], and megapoietin). The gene for TPO is located on chromosome 3q27. It encodes a 30 kDa glycoprotein of 353 amino acids that can be divided into two structural domains: an amino-terminal region with homology to human erythropoietin (EPO), and a carboxyl-terminal region that contains multiple N- and O-linked oligosaccharides. The amino terminal 155 residues of human TPO share 21% sequence identity and 46% overall sequence similarity to human EPO. This region mediates binding to the TPO receptor (c-Mpl). The carboxyl region does not share sequence homology with any known protein. TPO is reported to enhance multiple stages of megakaryocyte maturation, including cell size, cell ploidy, and platelet production. The predominant sites of TPO production are the liver and kidney, which secrete it in a generally constitutive fashion. Expression of TPO has been detected by more sensitive methods in BM stroma and spleen in the setting of thrombocytopenia, although this likely accounts for only a minor fraction of total TPO production. A low-level expression has also been reported in the amygdala and hippocam pus of the brain.

Thrombopoietin Receptor (c-Mpl)

The receptor for TPO (TPO receptor; c-Mpl) is the normal homologue of the oncogene v-Mpl, the transforming gene of murine myeloproliferative leukemia virus. It is a 635 amino acid protein that contains a number of distinct functional domains: a 25-amino acid signal peptide, a 465 amino acid extracellular domain, a 22-residue transmembrane domain, and an intracellular domain that contains two conserved motifs, termed Box 1 and Box 2 (Fig. 1). The extracellular domain contains a distal region that negatively influences TPO signaling. It is a member of the type I cytokine receptor superfamily. Like the EPO receptor, it is thought to function as a homodimer. The TPO receptor is expressed on MkPs, as well as ear lier multipotential progenitors, including MEPs, CMPs, and HSCs. TPO receptors are present on the surface of platelets at an estimated density of 20 to 200 receptors per platelet and bind TPO with an affinity of 200 to 560 pM. The binding of TPO to platelets plays an important role in the regulation of total body platelet mass by the TPO–TPO receptor system. Both TPO receptor−/− (c-Mpl−/−) and TPO (TPO−/−) knock-out mice contain ≈85% to 90% lower plate let and megakaryocyte numbers as compared with WT mice. The structure of the megakaryocytes and platelets in these animals is nor mal, reinforcing the notion that TPO signaling plays an important role in the expansion and development of MkPs, but not in terminal maturation and proplatelet release. In addition, the residual platelet production in these mice suggests alternate cytokine, or possibly cytokine-independent, pathways for thrombocytopoiesis. Interbreeding experiments of TPO receptor−/− mice with knock-out mice for IL-3, IL-6, IL-11, or leukemia inhibitory factor (LIF) or their receptors, show that these other cytokines are not responsible for the residual platelet production.

Fig1. THE THROMBOPOIETIN RECEPTOR. Schematic diagram of the thrombopoietin (TPO) receptor depicted as a homodimer with TPO bound. The binding of Janus-kinase 2 (JAK2) at Box 1 of the cytoplasmic tail is shown. Conformational changes in the TPO receptor upon TPO binding result in a juxtaposition of the two cytoplasmic tails, as well as JAK2 autophosphorylation and JAK2-mediated phosphorylation of the c-Mpl cytoplasmic tail (Tyr591, Tyr625, and Tyr630). Activation of signal transducers and activators of transcription, ERK, phosphoinositol-3 kinase (PI-3K)-Akt, and PI3K-mTOR signaling pathways then occurs. mTOR, mammalian target of rapamycin; STAT, signal transducers and activators of transcription. (Reproduced with permission from Geddis AE. Megakaryopoiesis. Semin Hematol. 2010;47:212.)

Thrombopoietin Receptor Downstream Signaling Pathways

The TPO receptor lacks intrinsic tyrosine kinase activity. Instead, ligand binding is thought to induce a conformational change in the homodimeric receptor and stimulates the cytoplasmic tyrosine kinase Janus-kinase 2 (JAK2), which binds to Box 1 of the cytoplasmic tail. This results in tyrosine phosphorylation of multiple targets, including signal transducers and activators of transcription (STATs), Shc adaptor protein, and the TPO receptor itself (Tyr591, Tyr625, and Tyr630). Additional signaling pathways activated upon TPO receptor engagement include the mitogen-activated protein kinase (MAPK) p38, p42/p44 extracellular signal-regulated kinase 1 (ERK1/ERK2), phosphoinositol-3-kinase-AKT (PI3K-AKT), and PI3K-Mammalian target of rapamycin (mTOR) signaling pathways.

Several of these downstream signaling pathways have been shown to be functionally important in TPO-mediated effects on mega karyocytopoiesis. Double STAT5a/STAT5b–deficient mice have impaired platelet production as well as defects in early multipotent progenitor cells. Moreover, megakaryocyte-selective overexpression of a dominant-negative mutant STAT3 in transgenic mice reduces platelet recovery following 5-fluorouracil–induced myelosuppression. These findings suggest a functional role for STAT family members in thrombocytopoiesis.

Studies in primary megakaryocytes show a requirement for PI3 AKT signaling in TPO-induced cell cycling. This involves the silencing of the Forkhead O family of transcription factors. Activation of the p42/p44-MAPK plays an important role in TPO-induced maturation and endomitosis. The mTOR signaling pathway is involved in TPO-mediated megakaryocytic progenitor proliferation and possibly terminal megakaryocyte size determination, ploidy, and cellular maturation.

Negative Regulation of Thrombopoietin Signaling

As with other receptor-mediated signaling processes, feedback mechanisms exist to limit or turn off the signal once initiated to avoid uncontrolled growth. Lnk, an adaptor protein implicated in immunoreceptor and cytokine receptor signaling negatively modulates TPO signaling in megakaryocytes. Overexpression of Lnk decreases TPO-dependent megakaryocyte growth and polyploidization in BM-derived cultures. Conversely, loss of Lnk expression by gene targeting results in increased numbers of megakaryocytes, accentuated megakaryocyte polyploidization, and a myeloproliferative disorder in mice. This correlates with enhanced and prolonged TPO-mediated induction of STAT3, STAT5, AKT, and MAPK signaling pathways.

Following TPO binding, the TPO receptor is internalized and subsequently degraded. This process depends on dileucine repeats, and Tyr⁵⁹¹ and Tyr⁶²⁵ within the TPO receptor cytoplasmic tail, and involves ubiquitinylation via the E3 ubiquitin ligase c-Cbl.

Regulation of Platelet Mass by Thrombopoietin

 Platelet counts are typically held at a relatively fixed level in humans, ranging from 150,000 to 400,000/mm3. The maintenance of plate let numbers by the TPO-TPO receptor system involves an unusual homeostatic mechanism among hematopoietic cytokine-mediated regulation. This is sometimes referred to as the “sponge” model (Fig. 2). Unlike other cytokines, TPO is secreted predominantly in a constitutive manner, mostly from the liver and kidney. High-affinity TPO receptors present on the platelet surface bind free TPO and internalize it, where it is degraded. Therefore, when platelet counts are low, less TPO is removed, and more are available to stimulate megakaryocytopoiesis in the BM. Conversely, when platelet counts rise above a given set point, they act as a “sink” for TPO, binding and destroying it before it can stimulate megakaryocytopoiesis in the BM. Thus total platelet mass is preserved, rather than absolute plate let numbers. This may explain the mild to moderate thrombocytopenia seen in certain disorders associated with large platelets, such as Bernard–Soulier syndrome.

Fig2. REGULATION OF PLATELET COUNT BY THROMBOPOIETIN (TPO): THE “SPONGE” MODEL. TPO is secreted at a constitutive rate primarily in the liver, and perhaps other sources such as the kidney, into the circulation. There it binds with high affinity to TPO receptors (c-Mpl) present on the surface of platelets. The TPO is then internalized by the platelets and degraded. Free TPO (i.e., TPO not bound to platelets) enters the bone marrow and stimulates megakaryocytopoiesis. Thus in the presence of high platelet counts, little free TPO is available to stimulate megakaryocytopoiesis. Conversely, low platelet numbers lead to increased free TPO and active megakaryocytopoiesis. The net result is the preservation of total platelet mass.

Several pieces of evidence support this model. First, it has been known for over 40 years that the peripheral blood platelet count varies inversely with plasma TPO activity. Second, TPO receptor-deficient mice (c-Mpl−/−) have elevated levels of circulating TPO, and this is reduced when the mice are transfused with washed platelets from normal mice. Third, in contrast to platelets from TPO receptor deficient mice, platelets from normal mice bind purified radiolabeled TPO and degrade it. Fourth, TPO levels are low to intermediate in normal individuals and in those with idiopathic thrombocytopenic purpura (where the bound TPO is destroyed along with the platelets). However, following chemotherapy, or in individuals with aplastic anemia, levels are markedly elevated.

Although the model described above likely explains the predominant basal regulation of platelet number by the TPO–TPO receptor signaling system, overlying inducible mechanisms also probably exist. It has been shown that the TPO gene is transcriptionally activated in BM stroma and spleen during times of thrombocytopenia, although the degree to which this may contribute to total TPO levels is uncertain. In addition, IL-6 mediates upregulation of hepatic TPO mRNA transcripts in inflammation-related thrombocytosis. Recent work shows that binding of desialylated platelets to the hepatic Ashwell Morell receptor triggers TPO gene transcription and protein production via a JAK 2/STAT3 pathway, linking platelet turnover directly to TPO production.

Thrombopoietin Signaling in Disease Conditions

Congenital Amegakaryocytic Thrombocytopenia

 Biallelic mutations in the TPO receptor gene cause CAMT (OMIM 604498). In this disorder, megakaryocytes are absent or greatly diminished in number in the BM. Patients typically present shortly after birth with petechiae, bruising, or bleeding. Patients with severe CAMT are at high risk for developing progressive BM failure, typically within the first few years of life. Recently, biallelic loss-of-function mutations in the TPO gene (THPO) have also been linked to BM failure. These observations are consistent with the role of TPO signaling in maintaining HSCs and/or multipotential progenitor cells. It should also be noted that in contrast to humans, TPO receptor−/− (as well as TPO−/−) mice do not develop BM failure states. The reason for this discrepancy is not known, but it highlights important differences between human and mouse hematopoiesis.

Essential Thrombocythemia

 Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm (MPN) associated with sustained excessive megakaryocyte hyperproliferation, thrombocytosis, and abnormal platelet function leading to either hemorrhage or thrombosis. Mutation in c-MPL, Janus activated kinase (JAK2V617F) and calreticulin (CALR) are major drivers of transformation in ET and its related MPNs, polycythemia vera (PV) and MF. While c-MPL mutations are relatively rare, V617F JAK2 mutations are found in ∼50% of patients with ET, ∼95% of patients with PV, and ∼50% of patients with MF. Mutations involving exon 9 of CALR gene were identified in 67% to 88% of cases of MPN which do not carry V617F JAK2 or MPL mutations, particularly ET and MF. Recently, several lines of evidence have established that all three driver mutations activate TPO-c-mpl signaling axis and its downstream pathways.

Mutations leading to constitutive activation of the TPO receptor or enhanced translation efficiency of the TPO gene have also been reported in rare cases of familial thrombocytosis. These two classes of disorders can be distinguished by measuring circulating TPO levels, which are elevated with mutations enhancing TPO mRNA translation efficiency, and decreased with mutations leading to constitutive TPO receptor activation.

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مواضيع ذات صلة

Cellular and Molecular Mechanisms in Development: Morphogens and Cell-to-Cell Signaling

Mechanisms of Endomitosis in Megakaryocytes

Megakaryocytopoiesis

Ontogeny of Erythropoiesis

Cellular Dynamics in Erythropoiesis: Steady State and Stress Erythropoiesis

Erythropoiesis and Iron Regulation

Hematopoietic Microenvironment

Intrinsic Control of Erythropoiesis:Erythropoietin

Nuclear Organization

Unfolded Protein Response

Autophagy

Nonapoptotic forms of cell death