Involvement of the Jak/Stat Signalling Pathway in the Erythropoietin-Induced Prevention of Apoptosis in Erythroid Cell Lines

Abstract

The highly conserved cysteine proteases of the ICE/CED-3 family are thought to be key components of the apoptotic machinery. However, the targets of these proteases, and the consequences of their cleavage, are unknown. Drosophila melanogaster not only shows widespread cell death during development but also can be used for powerful genetic screens, making it an ideal system to identify such targets.I have cloned a D. melanogaster ICE / CED-3 homologue, drICE, by degenerate PCR. drICE has 39% identity with human CPP32 and 31% identity with CED-3, and is expressed at all stages of Drosophila development. This is the first potential component of the conserved apoptotic machinery to be identified in Drosophila. I intend to use drICE as the basis for genetic screens to identify both its upstream regulators and downstream targets.Proliferation and differentiation of erythroid progenitor cells is promoted by the glycoprotein hormone erythropoietin (EPO). In addition, EPO plays a basic role for the suppression of apoptosis during certain stages of erythroid maturation [I]. There is increasing experimental evidence for the involvement of specific regions within the cytoplasmic domain of the EPO-receptor in the different biological activities of EPO [2]. We have previously shown, that distinct signalling events may be linked to differentiation, proliferation and maintenance of viability [3]. EPO induces multiple signalling pathways including the raslMAPK and the JAKISTAT-cascades. Both pathways were shown to be involved in the induction of proliferation and maturation of erythroid cells [3], but their role for the suppression of apoptosis remains to be elucidated. Therefore our recent studies aimed at defining the contribution of the JAKlSTAT pathway for the EPO-induced survival.Here we present data, demonstrating that EPO stimulates a transient induction of STATS DNA-binding activity in the factor-independent cell line J2E and the two EPO-dependent cell lines HCD57 and TFI. The cells become commited to apoptosis after withdrawal of fetal calf serum and EPO suppresses cell death under these circumstances. We show here that this inhibition of cell death goes along with a long term activation of the transcription factor STATS. Our results suggest a crucial role of the JAKlSTAT signalling cascade in the EPO-induced prevention of apoptosis in erythroleukemic cells. Ref.: [ I ] M. The anthracycline anti-neoplastic agent, daunorubicin, can induce apoptosis in cells [I].Ceramide synthase activity is enhanced following treatment of cells with this drug, giving rise to increased ceramide, an established inducer of apoptotic events. Furthermore, inhibition of this enzyme is sufficient to block ceramide elevation and apoposis. We now demonstrate that Watment of hemampoetic cells with daunorubicin results in the activation of the transcription factor NFKB, an event also induced by ceramide. Da-bicin also potentiated the induction of an NFrB-linked reporter gene by TNF. This was in contrast to results with C2-ceramide. 'where no effect ...