Somervaille, Tim Charles Plomer; (2002) Survival signal transduction pathways in primary human erythroblasts. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human erythropoiesis is regulated by the haemopoietic growth factors erythropoietin (Epo) and stem cell factor (SCF), which act to prevent the apoptosis of erythroid progenitor cells, and encourage their proliferation, through the binding and activation of their specific surface membrane receptors EpoR and c-kit respectively. However, the relevant intracellular pathways downstream of these receptors which are responsible for the transduction of survival signals in human erythroblasts are not known. In order to investigate this a system for the in vitro culture of primary human erythroblasts was developed. Using the cells generated it was demonstrated that the phosphoinositide-3- kinase (PI3K)/Akt pathway was regulated in response to both Epo and SCF and was centrally implicated in survival signalling in this cellular context. Furthermore Epo and SCF were shown to suppress the activity of the PI3K/Akt target, the constitutively active serine/threonine kinase glycogen synthase kinase-3 (GSK3). Growth factor deprived primary human erythroblasts, which undergo apoptosis rapidly, were protected from apoptosis by pharmacological inhibitors of GSK3, and to a lesser extent by inhibitors of p38 MAP kinase, demonstrating that increases in the activity of GSK3 and p38 MAP kinase following growth factor withdrawal act to induce apoptosis. Apoptosis in human erythroblasts was shown to correlate with a caspase independent conformational change in the Bcl-2 family member Bax, and this was also prevented by inhibition of GSK3 activity. Increasing cellular GSK3 activity in isolation, as achieved with a tamoxifen inducible GSK3?-ER fusion protein transduced into primary human erythroblasts, did not, however, induce apoptosis in these cells, suggesting that although increased GSK3 activity is central to the early stages of apoptosis induction, its effects are dependent on concomitant changes in the activity of other signal transduction pathways. In summary, the data presented in this thesis show for the first time that GSK3 activity is regulated in response to Epo and SCF in human erythroblasts and that the rise in GSK3 activity following growth factor withdrawal is causally implicated in the process of apoptosis induction.

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