Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin.

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage colony-stimulating factor (GM-CSF)- or erythropoietin (EPO)-dependent human cell line UT7. TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by decreased proliferation, changes in cell morphology, and increased surface expression and mRNA levels of megakaryocytic markers CD41, CD61, and CD42b. When UT7-mpl cells were cultured long-term in EPO instead of GM-CSF, the TPO effect was dominant over that of EPO. Moreover, the differentiation induced by TPO was more pronounced for cells shifted from EPO to TPO than for cells shifted from GM-CSF to TPO, as shown by the appearance of polyploid cells. Mutational analysis of the cytoplasmic domain of mpl showed that proliferation and maturation functions of mpl can be uncoupled. Two functional regions were identified: (i) the first 69 amino acids comprising the cytokine receptor motifs, box I and box 2, which are necessary for both TPO-induced mitogenesis and maturation; and (ii) amino acids 71 to 94, which are dispensable for proliferation but required for differentiation. Surprisingly, however, EPO could complement this latter domain for TPO-induced differentiation, suggesting a close relationship between EPO and TPO signaling

Thrombopoietin activates a STAT5-like factor in hematopoietic cells.

International audienceThrombopoietin (TPO) is a newly cloned cytokine which is the major regulator of circulating platelet levels, acting on both proliferation and differentiation of megakaryocytes. We have investigated the ability of TPO to activate the JAK/STAT pathway in megakaryocytic cell lines. We used either the granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or erythropoietin (EPO)-dependent UT7 cell line in which the murine TPO receptor (mumpl) had been transfected (mumpl-UT7 transfectants) or the MO7E and DAMI cells which express endogenous human TPO receptors. We demonstrated that TPO activates the kinase JAK2 and a STAT5-like transcriptional factor but not STAT1, STAT2, STAT3 or STAT4, in a very rapid and transient manner. In order to better ascertain the specificity of the activation of STAT5-related factor by TPO, we investigated the effect of other cytokines/growth factors. Both GM-CSF and EPO activated the STAT5-like factor. In contrast, neither interferon (IFN)-gamma nor the mitogenic stem cell factor (SCF) activated STAT5, although IFN-gamma did activate STAT1 in those cells. The hematopoietic DNA binding activity related to STAT5 was identified as a p97 tyrosine-phosphorylated protein band which exhibited identical gel mobility to the mammary STAT5. Because v-mpl, a truncated form of the TPO receptor c-mpl, was shown to be oncogenic, we tested the activity of v-mpl on STAT5 and found STAT5 constitutively activated in two different v-mpl-expressing cells, the transiently transfected Cos7 cells and the stable v-mpl-UT7 transfectants. Overall, our data indicate that STAT5 is widely expressed in hematopoietic cells and activated by a number of cytokines, including TPO, GM-CSF and EPO, but not by IFN-gamma or SCF

IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line

International audienceActivation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival

Concomitant multipotent and unipotent dental pulp progenitors and their respective contribution to mineralised tissue formation

Upon in vitro induction or in vivo implantation, the stem cells of the dental pulp display hallmarks of odontoblastic, osteogenic, adipogenic or neuronal cells. However, whether these phenotypes result from genuine multipotent cells or from coexistence of distinct progenitors is still an open question. Furthermore, determining whether a single cell-derived progenitor is capable of undergoing a differentiation cascade leading to tissue repair in situ is important for the development of cell therapy strategies. Three clonal pulp precursor cell lines (A4, C5, H8), established from embryonic ED18 first molars of mouse transgenic for a recombinant plasmid adeno-SV40, were induced to differentiate towards the odonto/osteogenic, chondrogenic or adipogenic programme. Expression of phenotypic markers of each lineage was evaluated by RT-PCR, histochemistry or immunocytochemistry. The clones were implanted into mandibular incisors or calvaria of adult mice. The A4 clone was capable of being recruited towards at least 3 mesodermal lineages in vitro and of contributing to dentin-like or bone formation, in vivo, thus behaving as a multipotent cell. In contrast, the C5 and H8 clones displayed a more restricted potential. Flow cytometric analysis revealed that isolated monopotent and multipotent clones could be distinguished by a differential expression of CD90. Altogether, isolation of these clonal lines allowed demonstrating the coexistence of multipotential and restricted-lineage progenitors in the mouse pulp. These cells may further permit unravelling specificities of the different types of pulp progenitors, hence facilitating the development of cell-based therapies of the dental pulp or other cranio-facial tissues