Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells

Abstract

p38alpha, p38beta, p38gamma, and p38delta are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38alpha and p38gamma transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38delta mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38beta is minimally expressed in early CD34(+) hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38alpha, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38alpha, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38alpha does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38alpha and p38delta isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor alpha, an inflammatory cytokine that is modulated by p38alpha, is expressed by differentiating erythroblasts and inhibition of p38alpha or tumor necrosis factor alpha results in reduction in differentiation. Taken together, our data demonstrate that both p38alpha and delta isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

Fig. 1.

Expression pattern of p38 MAPK α, β, δ, and γ mRNA in CD34⁺ early hematopoietic cells and differentiating primary erythroid progenitors. RT-PCR analysis was performed by using p38 MAPK isoform-specific primers using early-uncommitted CD34⁺ cells and differentiating erythroid progenitors as indicated. As positive controls, 18S ribosomal RNA gene was also amplified by RT-PCR.

Fig. 2.

Growth factor withdrawal induces phosphorylation/activation of MKK3/6, p38α, and MAPKAP-2 in primary erythroid progenitors. Primary erythroid progenitors on day 8 of culture were deprived of Epo and SCF for indicated periods or stimulated with Epo or SCF after growth factor starvation. Cell lysates were collected and analyzed by immunoblotting with anti-phospho MKK3/6 antibodies (A) or anti-phospho p38α antibodies (B). Each of the blots were then stripped and reprobed with either anti-tubulin (A) or anti-p38α (B) antibodies to confirm equal protein loading. (C) Day 8 cells were growth factor starved as indicated and stimulated with Epo or SCF. Total cell lysates were immunoprecipitated with antibodies against MAPKAP-2. The immunocomplexes were then used in kinase assays. Hsp25 protein was used as a substrate along with [γ-³²P]ATP in the reactions. Subsequently, proteins were analyzed by SDS/PAGE, and the phosphorylated form of Hsp25 was detected by autoradiography.

Fig. 3.

Phosphorylation/activation of p38α, p38δ, and Hsp27 and expression of TNFα during differentiation of erythroid progenitors. (A) During steady-state culture conditions, cells were collected on days 6, 9, and 12 in the presence or absence of Epo and SCF. Total cell lysates were used to perform immunoblot analysis using phospho-specific p38α antibodies to determine the level of phosphorylation on each day of culture. As positive controls, samples were also collected on each day after depriving the cells of Epo and SCF for 4 hours. (B) In vitro kinase assays were performed on samples collected on days as indicated. Proteins were analyzed by SDS/PAGE, and phosphorylated ATF-2 was detected by autoradiography. (C) Cells were collected during differentiation of erythroid progenitors, and 20 μg of total proteins per sample were used for immunoblotting with an anti-phospho Hsp27 antibody. The blot was stripped and immunoblotted with tubulin to demonstrate that cells were undergoing terminal differentiation as Hsp27 was increasingly phosphorylated. (D) RT-PCR analysis using specific primers for TNFα on primary differentiating erythroid progenitors. PCR amplification was performed by using 1/10th of cDNA obtained from the reverse transcriptase reaction. 18S ribosomal RNA gene was also amplified in addition to the positive control sample provided by the manufacturer.

Fig. 4.

p38α MAPK and TNFα promote terminal differentiation of primary erythroid progenitors. (A) Erythroid progenitors were treated with (Lower) or without (Upper) p38α inhibitor SB203580 on day 10 of culture. Cells were collected and analyzed for glycophorin A and CD71 expression by flow cytometry (Right). As controls, cells were also analyzed by using IgG isotype-specific antibodies (Left). (B) Erythroid progenitors (2 × 10⁵ cells) were treated with (Right) or without (Left) neutralizing antibody against TNFα on day 10 and analyzed for glycophorin A and CD71 expression on day 12 by flow cytometry. The percentage of glycophorin A- and CD71-positive cells are indicated in each dot plot.

Fig. 5.

Effects of inhibition of p38α on proliferation and apoptosis of primary erythroid progenitors. (A) Erythroid progenitors cultured in serum-containing media with Epo and SCF were treated with SB203580 inhibitor 24 h before harvesting the cells for proliferation assays. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed on days indicated along with control samples that were not treated with the inhibitor. The data are means ± SE of triplicate measurements. (B) Erythroid progenitors on day 8 were washed to eliminate growth factors (Epo and SCF) and were recultured with the SB203580 inhibitor and growth factors or without the inhibitor treatment but with growth factors. Also, two samples were recultured without growth factors as indicated. After 20 h, cells were harvested and the percentage of apoptotic cells in each sample was determined by flow cytometry using annexin V and propidium iodide as apoptotic markers.