Different levels of p38 MAP kinase activity mediate distinct biological effects in primary human erythroid progenitors Tim C. P. Somervaille, David C. Linch and Asim Khwaja Department of Haematology, Royal Free and University College Medical School, London, UK Received 25 July 2002; accepted for publication 3 October 2002 Summary. There have been conflicting reports regarding the role of p38 mitogen-activated protein kinase (MAPK) in the regulation of differentiation, proliferation and apoptosis in erythroid cell lines. We have, therefore, examined the functions of this kinase in primary human erythroid pro- genitors. Cells in steady-state culture showed low-level p38 MAPK activity, which decreased further within 1 h of growth factor withdrawal and increased over a limited range within minutes of re-exposure of cells to erythro- poietin or stem cell factor, demonstrating the link between low-level p38 MAPK activity and the prevailing growth factor milieu. Use of the p38 MAPK-specific inhibitor SB203580 demonstrated that this level of activity was necessary for (1) optimal proliferation, (2) erythroid burst- forming unit migration and (3) full upregulation of E-cadherin and CD36 expression, but not haemoglobin A or glycophorin A expression, during human erythroid differ- entiation. In contrast, cells deprived of growth factors for an 8-h period, following a transient decrease in p38 MAPK activity, demonstrated sustained, substantial and caspase- independent increases in p38 MAPK activity, and its blockade using SB203580 reduced the proportion of erythroblasts undergoing apoptosis by 40 ± 7%, demon- strating a role for p38 MAPK in apoptosis induction in human erythroblasts. Thus, in primary human erythro- blasts, different environmental conditions induce different levels of p38 MAPK activity, which have distinct functions. Keywords: erythroblasts, p38 MAP kinase, apoptosis, differentiation, E-cadherin. The conserved p38 mitogen-activated protein kinase (p38 MAPK) pathway (Han et al, 1994; Lee et al, 1994) is activated by many factors and has a variety of biological functions, depending on the cellular context. These func- tions include regulation of inflammatory cytokine produc- tion, apoptosis, cell cycle entry, embryogenesis, placental development and cellular differentiation (reviewed in Tib- bles & Woodgett, 1999; Ihle, 2000; Lee et al, 2000; Martin- Blanco, 2000; Nebreda & Porras, 2000; Ono & Han, 2000; Harper & LoGrasso, 2001). Several investigators have examined the role of p38 MAPK in erythroid cell biology using various cell lines. These have yielded conflicting results, depending on the line studied (Nagata et al, 1997, 1998; Birkenkamp et al, 1999; Nagata & Todokoro, 1999; Jacobs-Helber et al, 2000; Petitfrere et al, 2000; Witt et al, 2000). Activation of p38 MAPK in response to erythro- poietin (Epo) is seen in the mouse cell lines FD-Epo, SKT6 and HCD57 (Nagata et al, 1997, 1998; Jacobs-Helber et al, 2000). Proliferation of FD-Epo cells and HCD57 cells, but not SKT6 cells or human UT-7 cells, is p38 MAPK dependent. Blockade of p38 MAPK activity reduced 72-h proliferation of both FD-Epo cells and HCD57 cells by approximately 50% (Nagata et al, 1998; Jacobs-Helber et al, 2000; Petitfrere et al, 2000). Apoptosis of SKT6 cells following prolonged hyperosmolar shock or heat shock (Nagata & Todokoro, 1999), and of human TF-1 cells deprived of serum and growth factors (Birkencamp et al, 1999), but not of Epo-deprived HCD57 cells (Jacobs-Helber et al, 2000), is p38 MAPK dependent. p38 MAPK blockade in SKT6 cells treated with hyperosmolar shock reduced the proportion of apoptotic cells observed 24 h later by approximately 50% (Nagata & Todokoro, 1999). p38 MAPK blockade in serum- and growth factor-deprived TF-1 cells reduced the proportion of apoptotic cells observed 48 h later by 33% (Birkenkamp et al, 1999). With regards to differentiation, p38 MAPK activity is required for the haemoglobinization of SKT6 cells in response to either Epo or transient hyperosmolar or heat shock (Nagata et al, 1998; Nagata & Todokoro, 1999) of K562 cells in response to butyrate (Witt et al, 2000) and of UT-7 cells in response to tissue inhibitor of metalloproteinases-1 (TIMP-1) (Petitf- rere et al, 2000). Blockade of p38 MAPK reduced 5-d Epo- induced haemoglobinization of SKT6 cells by approximately Correspondence: Tim C. P. Somervaille, Department of Haematol- ogy, Royal Free and University College Medical School, 98 Chenies Mews, London, WC1E 6HX, UK. E-mail: timsomervaille@aol.com British Journal of Haematology, 2003, 120, 876–886 876 Ó 2003 Blackwell Publishing Ltd