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 in junB 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 in junB 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).

FIG. 1

Withdrawal of EPO from HCD57 cells induces AP1 activity. For each panel, bracket indicates AP1 complex, lower arrow indicates unbound [γ-³²P]ATP-labeled TRE, and asterisk indicates shifted band which is not completely competed by unlabeled TRE. Nuclear extracts were incubated with 10⁶ cpm of labeled TRE in the absence (−) or presence (+) of 10-fold excess of unlabeled TRE (cold comp.). (a) EMSA of nuclear extracts incubated in the presence of [γ-³²P]ATP-labeled TRE. Cells were washed three times in serum-free medium and incubated in EPO-free medium containing 25% fetal calf serum; 10⁷ cells were harvested 0, 1, 3, 6, and 24 h after the withdrawal of EPO (hrs-EPO). (b) Supershift analysis of nuclear extracts from HCD57 cells deprived of EPO for 3 h and incubated with specific antibodies to the Fos and Jun family members. Upper arrows indicate the presence of JunB (lane 2), JunD (lane 3), and FosB (lane 8) in the AP1 complex. nrs, normal rabbit serum; JB, JunB; JD, JunD; Fp, Fos pan antibody; F1, Fra-1; F2, Fra-2; FB, FosB. (c and d) Supershift analysis of nuclear extracts (nuc. ext) from HCD57 cells cultured in the presence (+) or absence (−) of EPO overnight (o/n) or for 3 h and incubated with an antibody (Ab) specific for cJun. (c) Overnight exposure of the assay; (d) 10-day exposure of the same experiment. Upper arrow indicates presence of cJun in the AP1 complex 3 h following treatment with EPO (c, lane H) but not when the cells are deprived of EPO for 3 h (d, lane D). c-Jun is still present in the complex after overnight treatment with EPO (d, lane B). All lanes in panels c and d are normalized for approximately equal radioactivities shifted into the AP1 complex. Thus, while the radioactivities in lanes C and D and lanes G and H are comparable, the films for lanes A and B and lanes E and F were exposed seven times longer to compensate for very low AP1 binding.

FIG. 2

JunB mRNA and protein expression in EPO-deprived cells. (a) Northern blot analysis of junB expression in HCD57 and HCD57-SREI cells upon EPO withdrawal. Upper arrow indicates position of junB message; lower arrow indicates 28S rRNA to show equal loading of samples. Positions of migration of 4.4-, 2.37-, and 1.35-kb molecular size markers are indicated at the left. (b) Western blot analysis of 40 μg of nuclear extracts from HCD57 cells deprived of EPO for 0, 1, 3, 6, and 24 h (hrs-EPO) probed with an anti-JunB antibody. Arrow indicates presence of JunB protein 1 and 3 h after EPO deprivation. (c) EMSA of HCD57 (lanes A to F) and HCD57-SREI (lanes G to L) cells deprived of EPO and incubated in the presence (+) or absence (−) of unlabeled TRE. AP1 complex, noncompeting complex, and unbound [γ-³²P]ATP-labeled TRE are labeled as indicated for Fig. 1. Protein isolation and EMSA were conducted as indicated for Fig. 1a. A loss in AP1 activity in HCD57-SREI cells is observed (lanes H to J) compared to that in HCD57 cells (lanes B to D).

FIG. 3

Stimulation of EPO-induced proliferation of HCD57 cells is accompanied by a rapid increase in AP1 activity. AP1 complex, noncompeting complex, and unbound [γ-³²P]ATP-labeled TRE are labeled as indicated for Fig. 1. (a) EMSA of nuclear extracts isolated from HCD57 cells which have been deprived of EPO for 18 h and then stimulated with EPO for 0, 1, 3, 6, and 24 h. An increase in AP1 activity is observed over the first 3 h following treatment with EPO (lanes B to D). cc, cold competitor. (b) Supershift analysis of nuclear extracts isolated from HCD57 cells cultured in EPO for 3 h, using the Jun and Fos antibodies indicated in Fig. 1b. Arrows indicate the presence of all Fos family members (upper right arrow) and JunD (upper left arrow) in the AP1 complex.

FIG. 4

The presence of a dominant negative AP1 mutant blocks apoptosis in HCD57 cells. (A) Western blot analysis of 40 μg of nuclear protein from HCD57, HCD57-TAM-67, and HCD57 cells transfected with vector alone (HCD57-NEO^R), using an anti-c-Jun antibody. Upper and lower arrows indicate the presence of the c-Jun and TAM-67 proteins, respectively. (b and c) Growth and survival properties of HCD57 and HCD57-TAM-67 cells exposed to EPO. (b) Proliferative response of HCD57 and HCD57-TAM-67 cells to EPO. Cells were cultured in the absence or presence of 1 U of EPO/ml. Data are indicated as percentages of the starting number of cells. ⧫, HCD57 cells cultured in EPO; ▴, HCD57-TAM-67 cells cultured in EPO; ▪, HCD57 cells cultured in the absence of EPO; ×, HCD57-TAM-67 cells cultured in the absence of EPO for 0, 24, 48, 72, and 96 h. Data are indicated as percentages of the starting number of cells. (d) Prevention of apoptosis by TAM-67 in HCD57 cells. Cells were cultured in the presence of EPO (lanes A to C and G to I) or in the absence of cytokine (lanes D to F and J to L) for the number of hours indicated. Ten micrograms of genomic DNA from HCD57 (lanes A to F) and HCD57-TAM-67 (lanes G to L) cells was resolved on a 2.25% agarose gel. Arrow indicates 100-bp marker.

FIG. 5

JNK levels decrease following EPO withdrawal from HCD57 cells. An in vitro kinase assay of JNK activity in HCD57 cells cultured in the presence of an anti-EPO antibody for the hours indicated was done. Cell lysates were immunoprecipitated with an anti-JNK antibody and tested for the ability to phosphorylate a GST-Jun fusion protein. Phosphorylated proteins were resolved on an SDS–12% polyacrylamide gel. Arrow indicates position of the [γ-³²P]ATP-labeled GST-Jun fusion protein.

FIG. 6

Bcl-X_L levels are maintained in EPO-deprived HCD57-TAM-67 cells. Total cellular proteins were isolated from HCD57 and HCD57-TAM-67 cells and were subjected to Western blot analysis with an anti-Bcl-X_L antibody. Positions of molecular weight markers are indicated at the left. Arrows indicate the presence of the Bcl-X_L protein. Bcl-X_L levels decreased 72 h following EPO withdrawal in HCD57 cells (top) but were unchanged in HCD57-TAM-67 cells deprived of EPO for 96 h (bottom).

FIG. 7

Normal human erythroid cells demonstrate an increase in AP1 activity when deprived of EPO. AP1 complex, noncompeting complex, and unbound [γ-³²P]ATP-labeled TRE are labeled as indicated for Fig. 1. Nuclear extracts from a culture highly enriched for CFU-Es cultured in the presence of EPO (lane A) or deprived of EPO for 1 and 3 h (lanes B and C) were incubated in the presence of a [γ-³²P]ATP-labeled TRE as indicated in Fig. 1 and in the presence (+) or absence (−) of unlabeled TRE. An increase in AP1 activity is observed 1 and 3 h after withdrawal of EPO (lanes B and C).