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

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2222-2231 ◽  
Author(s):  
Ian S. Hitchcock ◽  
Maximus M. Chen ◽  
Jennifer R. King ◽  
Kenneth Kaushansky
Keyword(s):  
Point Mutations ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Lysosomal Degradation ◽  
Hematopoietic Stem ◽  
Thrombopoietin Receptor ◽  
Stem And Progenitor Cells ◽  
Increased Strength ◽  
Interfering Rna ◽  
Stimulation Of

Abstract 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 YXXΦ motifs and we identified 2 such motifs in c-Mpl (Y8RRL and Y78RRL) and investigated Tpo-stimulated internalization of receptors bearing point mutations at these sites. After Tpo stimulation, internalization was greatly reduced in c-Mpl Y78F and c-Mpl Y8+78F, 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 Y8RRL 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.

scholarly journals A thrombopoietin receptor antagonist is capable of depleting myelofibrosis hematopoietic stem and progenitor cells

Blood ◽  
2016 ◽  
Vol 127 (26) ◽  
pp. 3398-3409 ◽  
Author(s):  
Xiaoli Wang ◽  
David Haylock ◽  
Cing Siang Hu ◽  
Wioleta Kowalczyk ◽  
Tianbo Jiang ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
New Approaches ◽  
Thrombopoietin Receptor ◽  
Cd34 Cells ◽  
Key Points ◽  
Stem And Progenitor Cells

Key Points Treatment of MF CD34+ cells with a TPO receptor antagonist selectively depletes MF HSCs and HPCs. Agents that target the TPO receptor represent potentially new approaches for the treatment of MF patients.

scholarly journals Direct Toll-Like Receptor-Mediated Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Macrophages

Stem Cells ◽  
2012 ◽  
Vol 30 (7) ◽  
pp. 1486-1495 ◽  
Author(s):  
Javier Megías ◽  
Alberto Yáñez ◽  
Silvia Moriano ◽  
José-Enrique O'Connor ◽  
Daniel Gozalbo ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Toll Like Receptor ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Stimulation Of

scholarly journals LNK (SH2B3) Inhibition Expands Healthy and Fanconi Anemia Human Hematopoietic Stem and Progenitor Cells

2021 ◽  
Author(s):  
Nicholas Holdreith ◽  
Grace Y Lee ◽  
Vemika Chandra ◽  
Carlo Salas Salinas ◽  
Peter Nicholas ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Adaptor Protein ◽  
Negative Regulator ◽  
Hematopoietic Stem ◽  
Monogenic Diseases ◽  
Stem And Progenitor Cells ◽  
Human Hscs

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for a variety of hematological diseases. Allogenic HSCT requires hematopoietic stem cells (HSCs) from matched donors and comes with cytotoxicity and mortality. Recent advances in genome modification of HSCs have demonstrated the possibility of using autologous HSCT-based gene therapy to cure monogenic diseases, such as the inherited bone marrow failure (BMF) syndrome Fanconi Anemia (FA). However, for FA and other BMF syndromes insufficient HSC numbers with functional defects results in delayed hematopoietic recovery and increased risk of graft failure. We and others previously identified the adaptor protein Lnk (Sh2b3) as a critical negative regulator of murine HSC homeostasis. However, whether LNK (SH2B3) controls human HSCs has not been studied. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs (shRNAs) resulted in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Importantly, LNK depletion enhanced cytokine mediated JAK/STAT activation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA. In xenotransplant, engraftment defects of FANCD2-depleted FA-like HSCs were markedly improved by LNK inhibition. Finally, targeting LNK in primary bone marrow HSPCs from FA patients enhanced their colony forming potential in vitro. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT and HSCT-based gene therapy.

scholarly journals Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar K. Azhagiri ◽  
Prathibha Babu ◽  
Vigneshwaran Venkatesan ◽  
Saravanabhavan Thangavel
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Target Cells ◽  
Gene Manipulation ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Base Editing ◽  
Stem And Progenitor Cells

AbstractThe advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.

Stimulation of Human Hematopoiesis: The Androstene Link.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4166-4166
Author(s):  
Jessie R. Groothuis ◽  
Dwight R. Stickney ◽  
Ajay Malik ◽  
Armando Garsd ◽  
Christopher Reading ◽  
...  
Keyword(s):  
Plasminogen Activator Inhibitor ◽  
Angiogenic Factors ◽  
Elderly Subjects ◽  
Plasminogen Activator Inhibitor 1 ◽  
Maximum Increase ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Complete Blood Counts ◽  
Stimulation Of

Abstract 5-Androstene-3β, 17β-diol (AED) is a naturally occurring member of the androstene series of steroids. In models of chemotherapy and radiation-induced mylosuppression, AED expanded hematopoietic stem and progenitor cells (HSPC), decreased need for clinical support and increased survival. We studied hematopoietic activity of AED vs. Placebo in healthy adult (20–64 years, n=9) and elderly (65–72 years, n=9) subjects. AED was safe and well tolerated. Complete blood counts were serially obtained between study days 1–56. Bone marrow (BM) samples were obtained on days -1 and 7. AED produced significant increases in average neutrophil counts (NT) and platelet counts (PLT) as compared to placebo. Adult NT significantly increased between days 2–7 (40–73%, p=0.05) and persisted through day 28 (41.5%); adult PLT increased (19.9%) by day 14, and achieved significance by day 21 (15.6%, p=0.01) and increases persisted through day 28 (19.4%, p=0.02). In elderly subjects, NT significantly increased by 56–96% (days 4–6, p=0.05) which persisted through day 28 (30.7%). Fewer elderly subjects demonstrated robust PLT responses; maximum increase in PLT was seen on day 14 (31.5%) and persisted through day 28. BM samples at day 7 showed increased cellularity in AED but not placebo-treated subjects which was consequently reflected as an increase in peripheral blood NT and PT in AE-treated subjects. BM phenotype changes and maturation potential were estimated from immunophenotyping and CFU assay in vitro. BM precursor populations were elevated in most treated subjects on day 7; this reflected an activity of AED on precursor cells that resulted in a decrease in the CD34+CD38− and CFU-F populations and an increase in erythrocyte (BFU-E, CFU-E), and granulocyte (CFU-GM) precursors and megakaryocytes. Elderly patients (but not adults) had increased levels of two angiogenic factors; vascular endothelial growth factor and plasminogen activator inhibitor-1 in plasma (day 5) and in BM (day 7). These findings suggest that AED has a regenerative role in elderly BM and that the stimulation of angiogenic factors precedes an increase in BM cellularity which in turn leads to an increase in circulating terminally differentiated blood elements.

scholarly journals Endocytosis of the Thrombopoietin Receptor Mpl Regulates Both Megakaryocyte and Erythroid Maturation in Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2477-2477
Author(s):  
Nathan Eaton ◽  
Melissa M. Lee-Sundlov ◽  
Theresa A. Dlugi ◽  
Jon Wieser ◽  
Haley E. Ramsey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Critical Role ◽  
Surface Expression ◽  
Extramedullary Hematopoiesis ◽  
Cell Surface Expression ◽  
Elevated Plasma ◽  
Laboratory Equipment ◽  
Hematopoietic Stem ◽  
Thrombopoietin Receptor ◽  
Stem And Progenitor Cells

The thrombopoietin receptor Mpl plays a critical role in thrombopoiesis, as it is the primary signaling constituent in megakaryocyte (MK) differentiation from hematopoietic stem and progenitor cells (HSPCs) and maintains circulating blood thrombopoietin levels via clathrin-mediated endocytosis. Thus, thrombopoiesis is tightly controlled by Mpl cell-surface expression on HSPCs, MKs, and platelets. Previous work by our group has shown that Dnm2fl/flPF4-Cre (Dnm2Plt-/-) mice, lacking the large endocytic GTPase dynamin 2 (DNM2) specifically within MKs and platelets, develop a variety of clinical phenotypes that closely resemble myelofibrosis, such as bone marrow fibrosis, marked HSPC expansion, MK hyperplasia, extramedullary hematopoiesis, and severe splenomegaly (Bender, Giannini, et al. Blood. 2015;125(6):1014-1024). Dnm2Plt-/- mice also displayed elevated plasma TPO levels and constitutive JAK2 activation in platelets, due to defective Mpl endocytosis. Here, the role of Mpl endocytosis in the maintenance of normal hematopoiesis was assessed by generating Dnm2Plt-/- mice in the Mpl-/- background. At 3 weeks of age, Mpl-/- Dnm2Plt-/- mice displayed significantly reduced HSPCs, a near complete depletion of bone marrow MKs, similar to Mpl-/- mice, indicating that Mpl is the primary receptor contributing to the aberrant hyperproliferative phenotype of Dnm2Plt-/- mice. However, Mpl-/- Dnm2Plt-/- mice also showed severe anemia, defects in erythroblast maturation, grossly elevated plasma erythropoietin (EPO) levels, and splenomegaly, resulting in early fatality by 3 to 4 weeks of age, suggesting that Mpl contributes to normal erythropoiesis in young mice. Mpl-/- and Mpl+/- Dnm2Plt-/- mice displayed reduced erythroblast development at 3 weeks of age, which returned to normal with adulthood. Taken together, the data shows that DNM2-dependent Mpl endocytosis is required for steady-state hematopoiesis and provides novel insights into a developmentally-controlled role for Mpl in normal erythropoiesis. Disclosures Sola-Visner: Sysmex America, Inc.: Other: Laboratory equipment on loan, Research Funding.

scholarly journals Inferring the dynamic of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms

Blood ◽  
2021 ◽  
Author(s):  
Matthieu Mosca ◽  
Gurvan Hermange ◽  
Amandine Tisserand ◽  
Robert John Noble ◽  
Christophe Marzac ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Driver Mutations ◽  
Molecular Response ◽  
Hematopoietic Stem ◽  
Thrombopoietin Receptor ◽  
Recurrent Mutations ◽  
Genes Encoding ◽  
Stem And Progenitor Cells ◽  
Hierarchical Bayesian Inference

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells (HSC) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon alpha (IFNα) has demonstrated some efficacy in inducing molecular remission in MPN. In order to determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in MPN patients by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured several times per year the clonal architecture of early and late hematopoietic progenitors (84,845 measurements) and the global variant allele frequency in mature cells (409 measurements). Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSC. Our data support the hypothesis that IFNα targets JAK2V617F HSC by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSC and increases with high IFNα dosage in heterozygous JAK2V617F HSC. Besides, we found that the molecular responses of CALRm HSC to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and high dosage of IFNα correlates with worse outcomes. Together, our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dosage.

scholarly journals Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Blood ◽  
2010 ◽  
Vol 115 (6) ◽  
pp. 1254-1263 ◽  
Author(s):  
Sebastian J. Saur ◽  
Veena Sangkhae ◽  
Amy E. Geddis ◽  
Kenneth Kaushansky ◽  
Ian S. Hitchcock
Keyword(s):  
Negative Regulation ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Cytokine Signaling ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Negative Regulatory Pathway ◽  
Thrombopoietin Receptor ◽  
Interfering Rna ◽  
Regulation Of Growth

Abstract 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 (K553 and K573). 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.

Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells

2001 ◽  
Vol 38 (2) ◽  
pp. 139-147
Author(s):  
Jan W. Gratama ◽  
D. Robert Sutherland ◽  
Michael Keeney
Keyword(s):  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Stem And Progenitor Cells
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