Sarah M. Jacobs-Helber

Distinct Signaling from Stem Cell Factor and Erythropoietin in HCD57 Cells

A recent report (Wu, H., Klingmuller, U., Besmer, P., and Lodish, H. F. (1995) Nature 377, 242-246) documents the interaction of the erythropoietin (EPO) receptor (EPOR) with the stem cell factor (SCF) receptor (c-KIT) and suggests that SCF acts through the EPOR. To elucidate the ability of SCF to affect the erythropoietin signaling pathway, we studied the effect of SCF on EPOR phosphorylation, SHC/ERK-1 activity, and cell proliferation and apoptosis in EPO-dependent HCD57 cells. Treatment of these cells with SCF resulted in phosphorylation of the EPOR. However, SCF-dependent phosphorylation of the EPOR did not initiate an EPO-like intracellular signal. SCF induced proliferation, SHC phosphorylation, and activation of ERK-1 but did not activate the JAK/STAT pathway. SCF stimulated SHC phosphorylation and ERK-1 activation independent of the EPOR in cells where the EPOR was down-regulated; the presence of the EPOR appeared to facilitate SCF activation of SHC and ERK-1. Furthermore, treatment of HCD57 cells with SCF increased cell number over a 3-day treatment, but apoptosis was observed in these cells. These data may illustrate two distinct pathways for erythroid cell proliferation and prevention of apoptosis in response to EPO, thereby providing a system to discriminate these intracellular signals.

AP1 Regulation of Proliferation and Initiation of Apoptosis in Erythropoietin-Dependent Erythroid Cells

ABSTRACT The transcription factor AP1 has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal. We report here that AP1 is necessary for the induction of apoptosis following hormone withdrawal in the erythropoietin (EPO)-dependent erythroid cell line HCD57. AP1 DNA binding activity increased upon withdrawal of HCD57 cells from EPO. A dominant negative AP1 mutant rendered these cells resistant to apoptosis induced by EPO withdrawal and blocked the downregulation of Bcl-XL. JunB is a major binding protein in the AP1 complex observed upon EPO withdrawal; JunB but not c-Jun was present in the AP1 complex 3 h after EPO withdrawal in HCD57 cells, with a concurrent increase injunB message and protein. Furthermore, analysis of AP1 DNA binding activity in an apoptosis-resistant subclone of HCD57 revealed a lack of induction in AP1 DNA binding activity and no change injunB mRNA levels upon EPO withdrawal. In addition, we determined that c-Jun and AP1 activities correlated with EPO-induced proliferation and/or protection from apoptosis. AP1 DNA binding activity increased over the first 3 h following EPO stimulation of HCD57 cells, and suppression of AP1 activity partially inhibited EPO-induced proliferation. c-Jun but not JunB was present in the AP1 complex 3 h after EPO addition. These results implicate AP1 in the regulation of proliferation and survival of erythroid cells and suggest that different AP1 factors may play distinct roles in both triggering apoptosis (JunB) and protecting erythroid cells from apoptosis (c-Jun).

State-of-the-Art Review: Unraveling Distinct Intracellular Signals That Promote Survival and Proliferation: Study of Erythropoietin, Stem Cell Factor, and Constitutive Signaling in Leukemic Cells

This review summarizes selected recent studies of the intracellular signals that allow erythroid cells to survive and proliferate under the control of erythropoietin (EPO) and alteration in signals that contribute to EPO-independent survival and proliferation. The hypothesis explored is that the proliferation and survival signals are distinct and can be separately studied with the proper cell lines and growth factor stimulation. The anti- and pro-apoptotic proteins Bcl-XL and BAD are highly implicated in EPO-dependent survival of erythroid cells. Stat5 activity appears to be upstream of Bcl-XL expression such that pathologic, constitutive activation of Stat5 may be a common event in leukemic cells that become resistant to apoptosis by constitutive expression of Bcl-XL. Other signals apparently also control the expression of Bcl-XL, such as the expression of JunB which seem to be required to suppress Bcl-XL expression when EPO is withdrawn. Apoptosis may also be triggered by inactivation of Bcl-XL by BAD. Dephosphorylation of BAD as a result of withdrawal of survival factors converts prosurvival BAD to proapoptotic BAD. Phosphorylation of BAD at the serine 112 residue seems critical to promoting survival. Constitutive activation of a kinase that phosphorylates BAD serine 112 may, therefore, contribute to resistance to apoptosis in leukemic cells. We describe the resistance of erythroleukemic cells to apoptosis induced by EPO withdrawal apparently caused by constitutive BAD phosphorylation. The resistance to apoptosis in these cells is reversed by treatment with the PI3-kinase inhibitor, LY294002, suggesting that resistance to apoptosis in these cells likely results from constitutive P13-kinase that is an upstream activator of an S-112 BAD kinase. The MAP kinase cascade is apparently active in EPO-dependent and stem cell factor (SCF)-dependent proliferation but not survival. In addition, autocrine tumor necrosis factor-a! (TNF-alpha) may also be a proliferation factor not affecting survival. P13-kinase seems to be required for full EPO-dependent proliferation but is not required for EPO-dependent survival (but it can promote survival when activated).

Role of JunB in Erythroid Differentiation

The role of junB as a regulator of erythroid cell survival, proliferation, and differentiation was tested by controlled expression of JunB in the erythropoietin (EPO)-dependent erythroleukemia cell line HCD57. JunB induced erythroid differentiation as evidenced by increased expression of the erythroid-specific proteins β-globin, spectrin-α, and TER-119. Expression of JunB for at least 48 h was required for the differentiated phenotype to emerge. Differentiation was accompanied by a slower rate of proliferation and an increase in the expression of the cell cycle inhibitory protein p27. p27 protein expression increased due to reduced turnover without changes in transcription, indicating global changes in cell physiology following JunB induction. JunB expression was also studied in mouse and human primary erythroid cells. JunB expression increased immediately in both primary mouse cells and HCD57 cells treated with EPO and quickly returned to base-line levels, followed by a secondary rise in JunB in primary erythroid cells, but not in HCD57 cells, 36–48 h later. This result suggested that the initial EPO-dependent JunB induction was not sufficient to induce differentiation, but that the late EPO-independent JunB expression in primary erythroid cells was necessary for differentiation. This study suggests that JunB is an important regulator of erythroid differentiation.