Ian S. Hitchcock

Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K(553) and K(573)). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes.

Thrombopoietin from beginning to end.

In the two decades since its cloning, thrombopoietin (TPO) has emerged not only as a critical haematopoietic cytokine, but also serves as a great example of bench‐to‐bedside research. Thrombopoietin, produced by the liver, is the primary regulator of megakaryocyte progenitor expansion and differentiation. Additionally, as TPO is vital for the maintenance of haematopoietic stem cells, it can truly be described as a pan‐haematopoietic cytokine. Since recombinant TPO became available, the molecular mechanisms of TPO function have been the subject of extensive research. Via its receptor, c‐Mpl (also termed MPL), TPO activates a wide array of downstream signalling pathways, promoting cellular survival and proliferation. Due to its central, non‐redundant role in haematopoiesis, alterations of both the hormone and its receptor contribute to human disease; congenital and acquired states of thrombocytosis and thrombocytopenia and aplastic anaemia as a result from dysregulated TPO expression or functional alterations of c‐Mpl. With TPO mimetics now in clinical use, the story of this haematopoietic cytokine represents a great success for biomedical research.

YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation

Thrombopoietin (Tpo), acting through the c-Mpl receptor, promotes the survival and proliferation of hematopoietic stem and progenitor cells and drives megakaryocyte differentiation. The proproliferation and survival signals activated by Tpo must therefore be tightly regulated to prevent uncontrolled cell growth. In this work, we determined the mechanisms that control Tpo-stimulated c-Mpl internalization and defined the processes leading to its degradation. Stimulation of BaF-Mpl cells with Tpo leads to rapid, clathrin-dependent endocytosis of the receptor. Using small interfering RNA (siRNA), we found that inhibition of adaptor protein 2 (AP2), which mediates endocytosis of transmembrane proteins, strongly attenuates Tpo-stimulated c-Mpl internalization. AP2 interacts with YXXPhi motifs and we identified 2 such motifs in c-Mpl (Y(8)RRL and Y(78)RRL) and investigated Tpo-stimulated internalization of receptors bearing point mutations at these sites. After Tpo stimulation, internalization was greatly reduced in c-Mpl Y(78)F and c-Mpl Y(8+78)F, and these cell lines also exhibited increased proliferation and increased strength and duration of Jak2, STAT5, AKT, and ERK1/2 activation in response to Tpo. We also found that the Y(8)RRL motif regulates Tpo-stimulated lysosomal degradation of c-Mpl. Our data establishes that c-Mpl cytoplasmic YRRL motifs are responsible for both Tpo-mediated internalization via interactions with AP2 and lysosomal targeting after endocytosis.

Essential components for a glutamatergic synapse between Merkel cell and nerve terminal in rats.

The exact role of Merkel cells and their possible involvement in mechanosensation is unclear. The aim of this study was to determine, in the adult rat sinus hair follicle, the expression pattern of a number of vesicular proteins involved in neurotransmitter release to provide a clearer understanding of Merkel cell signalling mechanisms. We identified prominent expression and co-localization of the glutamatergic vesicle loading proteins VGLUT1 and VGLUT2 at the site of the sinus hair follicle known to be densely populated with Merkel cells. We also found expression of the vesicle recycling proteins synaptogyrin and syntaxin-6 in the same region of the hair follicle. Our data suggest that glutamate signalling is involved in Merkel cell mechanosensation and that vesicular trafficking is commonplace in the Merkel cell-neurite complex.

A novel activating, germline JAK2 mutation, JAK2R564Q, causes familial essential thrombocytosis

Along with the most common mutation, JAK2V617F, several other acquired JAK2 mutations have now been shown to contribute to the pathogenesis of myeloproliferative neoplasms (MPNs). However, here we describe for the first time a germline mutation that leads to familial thrombocytosis that involves a residue other than Val617. The novel mutation JAK2R564Q, identified in a family with autosomal dominant essential thrombocythemia, increased cell growth resulting from suppression of apoptosis in Ba/F3-MPL cells. Although JAK2R564Q and JAK2V617F have similar levels of increased kinase activity, the growth-promoting effects of JAK2R564Q are much milder than those of JAK2V617F because of at least 2 counterregulatory mechanisms. Whereas JAK2V617F can escape regulation by the suppressor of cytokine signaling 3 and p27/Kip1, JAK2R564Q-expressing cells cannot. Moreover, JAK2R564Q-expressing cells are much more sensitive to the JAK inhibitor, ruxolitinib, than JAK2V617F-expressers, suggesting that lower doses of this drug may be effective in treating patients with MPNs associated with alternative JAK2 mutations, allowing many undesirable adverse effects to be avoided. This work provides a greater understanding of the cellular effects of a non-JAK2V617F, MPN-associated JAK2 mutation; provides insights into new treatment strategies for such patients; and describes the first case of familial thrombosis caused by a JAK2 residue other than Val617.

The thrombopoietin receptor, MPL, is critical for development of a JAK2V617F-induced myeloproliferative neoplasm.

The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F(+) transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F(+) mice, JAK2V617F(+)Mpl(-/-) mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl(+/-) mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F(+)Tpo(-/-) mice, although thrombocytosis was reduced compared with JAK2V617F(+) mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F(+) MPNs, highlighting an entirely novel target for therapeutic intervention.

Emerging neuroskeletal signalling pathways: a review

Recent work has demonstrated that neurotransmitters, signalling molecules primarily associated with the nervous system, can have profound effects on the skeleton. Bone cells express a broad range of neurotransmitter receptors and transporters, and respond to receptor activation by initiating diverse intracellular signalling pathways, which modulate cellular function. Evidence of neuronal innervation in skeletal tissues, neurotransmitter release directly from bone cells and functional effects of pharmacological manipulation support the existence of a complex and functionally significant neurotransmitter‐mediated signalling network in bone. This review aims to concisely summarise our current understanding of how neurotransmitters affect the skeletal system, focusing on their origin, cellular targets and functional effects in bone.

ARE UNCONVENTIONAL NMDA RECEPTORS INVOLVED IN SLOWLY ADAPTING TYPE I MECHANORECEPTOR RESPONSES

Specific immunohistochemical staining for NMDA receptor NR2A/B subunits was found in the outer root sheath layer of rat sinus hair (whisker) follicle. Co-localization with CK 20 confirmed that Merkel cells were stained. The NR2A/B staining seen on Merkel cells was pericellular. In addition it appeared that NF70-positive staining was in close proximity to, but did not colocalise with NR2A/B immunoreactivity, indicating that NR2A/B was only expressed by Merkel cells and not their adjacent nerve terminals. Merkel cells and the nerve terminals have previously been associated with electrophysiological recordings from slowly adapting type I (St I) mechanoreceptor unit activity. Pharmacological experiments with isolated sinus hairs using a wide range of ionotropic glutamate receptor antagonists found that only certain NMDA receptor blockers depressed St I unit responses to mechanical stimuli. AMPA/kainate receptor antagonists (CNQX and NBQX, 100 microM) had no effect, nor did classical competitive NMDA receptor antagonists, D-AP5 (600 microM) and R-CPP (100 microM), nor the NMDA glycine site antagonist 5,7-dichlorokynurenic acid (100 microM). The only effective NMDA receptor blockers were those selective for the polyamine site: ifenprodil (IC50 20 microM) and Ro 25-6981 (IC50 approximately 50 microM), and the associated ion channel: MK 801, ketamine and (+/-)-1-(1,2-diphenylethyl)piperidine (IC50 < 100 microM). The two enantiomers of MK 801 were equipotent. All effects were long lasting, consistent with their non-/uncompetitive actions. The most potent drug tested, ifenprodil, at an effective dose of 30 microM, had a mean recovery time of 74 min. A three-fold increase in drug concentration was required to depress St II units (associated with non-synaptic lanceolate endings). Changes in Zn2+ did not affect St I unit responses. These data suggest that unconventional NMDA receptors are involved in St I unit responses, but question the notion of a glutamatergic synapse between the Merkel cell and nerve terminal.

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms

Posttranscriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biologic processes. We dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and controls, and integrated data with transcriptomic and proteomic platforms. We validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a 3-member subset defines essential thrombocythemia (ET). The genetic signature includes functional guide and passenger strands of the previously uncharacterized miR 490 (5p and 3p), which displayed restricted, low-level expression in megakaryocytes/platelets (compared with leukocytes), and aberrant expression during thrombocytosis, most profound in ET. Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic colony differentiation and/or lineage specification. Integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis and support a developmentally restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation.

JAK2V617F-positive endothelial cells contribute to clotting abnormalities in myeloproliferative neoplasms

Significance The myeloproliferative neoplasms (MPNs) are a group of hematological malignancies characterized by increased numbers of myeloid blood cells, such as platelets, erythrocytes, and neutrophils. The main causes of illness and death in patients with MPNs are arterial and venous clotting and also, conversely, bleeding complications. However, the causes of these conditions are poorly understood. In this paper, we use a mouse model of MPNs to determine the cell types responsible for abnormal clotting in MPNs. We demonstrate that endothelial cells, the cell type that lines all blood vessels, have a significant role to play in MPN bleeding complications, potentially identifying a new cellular target for MPN therapies. The Janus kinase 2 (JAK2) V617F mutation is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombohemorrhagic incidents are the most common causes of morbidity and mortality in patients with MPNs, the events causing these clotting abnormalities remain unclear. To identify the cells responsible for the dysfunctional hemostasis, we used transgenic mice expressing JAK2V617F in specific lineages involved in thrombosis and hemostasis. When JAK2V617F was expressed in both hematopoietic and endothelial cells (ECs), the mice developed a significant MPN, characterized by thrombocytosis, neutrophilia, and splenomegaly. However, despite having significantly higher platelet counts than controls, these mice showed severely attenuated thrombosis following injury. Interestingly, platelet activation and aggregation in response to agonists was unaltered by JAK2V617F expression. Subsequent bone marrow transplants revealed the contribution of both endothelial and hematopoietic compartments to the attenuated thrombosis. Furthermore, we identified a potential mechanism for this phenotype through JAK2V617F-regulated inhibition of von Willebrand factor (VWF) function and/or secretion. JAK2V617F+ mice display a condition similar to acquired von Willebrand syndrome, exhibiting significantly less high molecular weight VWF and reduced agglutination to ristocetin. These findings greatly advance our understanding of thrombohemorrhagic events in MPNs and highlight the critical role of ECs in the pathology of hematopoietic malignancies.