Zhengyu Wang

Thymocyte emigration is mediated by active movement away from stroma-derived factors

T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage-restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell-derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond.

Ubiquitin-dependent Degradation of Cyclin B Is Accelerated in Polyploid Megakaryocytes

During the endomitotic cell cycle of megakaryocytic cell lines, the levels of cyclin B1 and the activity of cyclin B1-dependent Cdc2 kinase, although detectable, are reduced as compared with megakaryocytes undergoing a mitotic cell cycle. The levels of cyclin A, however, are comparable during both cell cycles. The expression of cyclin B1 mRNA is also equivalent in proliferating and polyploidizing cells. In the current study, we found that the rate of cyclin B1 protein degradation is enhanced in polyploidizing megakaryocytes. This finding has led us to further investigate whether the ubiquitin-proteosome pathway responsible for cyclin B degradation is accelerated in these cells. Our data indicate that polyploidizing megakaryocytic cell lines and primary bone marrow cells treated with the megakaryocyte proliferation- and ploidy-promoting factor, the c-Mpl ligand, display increased activities of the ubiquitin-proteosome pathway, which degrades cyclin B, as compared with proliferating megakaryocytic cell lines or diploid bone marrow cells, respectively. This degradation has all the hallmarks of a ubiquitin pathway, including the dependence on ATP, the appearance of high molecular weight conjugated forms of cyclin B, and inhibition of the proteolytic process by a mutated form of the ubiquitin-conjugating enzyme Ubc4. Our studies also indicate that the ability to degrade cyclin A is equivalent in both the mitotic and endomitotic cell cycles. The increased potential of polyploid megakaryocytes to degrade cyclin B may be part of the cellular programming that leads to aborted mitosis.

Signaling by the Mpl receptor involves IKK and NF‐κB*

Binding of tumor necrosis factor‐α (TNF‐α) to its receptor activates IKK complex, which leads to inducement of NF‐κB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF‐κB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand‐responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKα and IKKβ. Proliferating megakaryocytes displayed a constitutive DNA‐binding activity of NF‐κB p50 homodimers and of NF‐κB p50–p65 heterodimers. As expected, reduced IKK activity in Mpl ligand‐treated cells was associated with a significant reduction in NF‐κB DNA binding activity and in the activity of a NF‐κB‐dependent promoter. Our study is thus the first to identify a constitutive NF‐κB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF‐κB activities. Since a variety of proliferation‐promoting genes and anti‐apoptotic mechanisms are activated by NF‐κB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis. J. Cell. Biochem. 85: 523–535, 2002.