25 research outputs found
An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor
Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (Î5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, Î5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. Î5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the Î5TpoR mutant appears to enhance erythroid differentiation when compared with the Tpo-activated wild-type TpoR. Strikingly, individual substitution of K507 or W508 to alanine also induces constitutive TpoR activation, indicating that the K and W residues within the amphipathic KWQFP motif are crucial for maintaining the unliganded receptor inactive. These residues may be targets for activating mutations in humans. Such a motif may exist in other receptors to prevent ligand-independent activation and to allow signaling via multiple flexible interfaces
Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*
The activating W515L mutation in the thrombopoietin receptor (MPL) has been
identified in primary myelofibrosis and essential thrombocythemia. MPL belongs
to a subset of the cytokine receptor superfamily that requires the JAK2 kinase
for signaling. We examined whether the ligand-independent MPLW515L
mutant could signal intracellularly. Addition of the endoplasmic reticulum
(ER) retention KDEL sequence to the receptor C terminus efficiently locked
MPLW515L within its natural ER/Golgi maturation pathway. In
contrast to cells expressing the parental MPLW515L,
MPLW515L-KDEL-expressing FDC-P1 cells were unable to grow
autonomously and to produce tumors in nude mice. When observed, tumor nodules
resulted from in vivo selection of cells leaking the receptor at
their surface. JAK2 co-immunoprecipitated with MPLW515L-KDEL but
was not phosphorylated. We generated disulfide-bonded MPLW515L
homodimers by the S402C substitution, both in the normal and KDEL context.
Unlike MPLW515L-KDEL, MPLW515L-S402C-KDEL signaled
constitutively and exhibited cell surface localization. These data establish
that MPLW515L with appended JAK2 matures through the ER/Golgi
system in an inactive conformation and suggest that the
MPLW515L/JAK2 complex requires membrane localization for JAK2
phosphorylation, resulting in autonomous receptor signaling
Gfi-1B plays a critical role in terminal differentiation of normal and transformed erythroid progenitor cells
Multistage hematopoietic stem cell regulation in the mouse: A combined biological and mathematical approach
International audienceWe have reconciled steady-state and stress hematopoiesis in a single mathematicalmodel based on murine in vivo experiments and with a focus on hematopoieticstem and progenitor cells. A phenylhydrazine stress was first applied tomice. A reduced cell number in each progenitor compartment was evidenced duringthe next 7 days through a drastic level of differentiation without proliferation,followed by a huge proliferative response in all compartments including longtermhematopoietic stem cells, before a return to normal levels. Data analysisled to the addition to the 6-compartment model, of time-dependent regulationthat depended indirectly on the compartment sizes. The resulting model wasfinely calibrated using a stochastic optimization algorithm and could reproducebiological data in silico when applied to different stress conditions (bleeding,chemotherapy, HSC depletion). In conclusion, our multi-step and time-dependentmodel of immature hematopoiesis provides new avenues to a better understandingof both normal and pathological hematopoiesis
Combined Biological and Modeling Approach of Hematopoiesis: From Native to Stressed Erythropoiesis
International audienc
A major role of TGF-ÎČ1 in the homing capacities of murine hematopoietic stem cell/progenitors
International audienceAbstract Transforming growth factor-ÎČ1 (TGF-ÎČ1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-ÎČ1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-ÎČ1â/â pups, making these studies difficult. Here, we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-ÎČ1â/â neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion, the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-ÎČ1â/â neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Linâ) cells. In addition an in vitroâreduced survival of immature progenitors (Linâ Kit+ Sca+) was observed. Similar defects were found in liver cells from TGF-ÎČ1â/â embryos on day 14 after vaginal plug. These data indicate that TGF-ÎČ1 is a critical regulator for in vivo homeostasis of the HSCs, especially for their homing potential
JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation.
Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses