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. 2013 May 2;8(5):e62303.
doi: 10.1371/journal.pone.0062303. Print 2013.

Caspase-3 is involved in the signalling in erythroid differentiation by targeting late progenitors

Daniela Boehm  1 , Christelle Mazurier, Marie-Catherine Giarratana, Dhouha Darghouth, Anne-Marie Faussat, Laurence Harmand, Luc Douay
Affiliations

Caspase-3 is involved in the signalling in erythroid differentiation by targeting late progenitors

Daniela Boehm (V体育平台登录) et al. PLoS One. .

Abstract (VSports手机版)

A role for caspase activation in erythroid differentiation has been established, yet its precise mode of action remains elusive. A drawback of all previous investigations on caspase activation in ex vivo erythroid differentiation is the lack of an in vitro model producing full enucleation of erythroid cells. Using a culture system which renders nearly 100% enucleated red cells from human CD34(+) cells, we investigated the role of active caspase-3 in erythropoiesis. Profound effects of caspase-3 inhibition were found on erythroid cell growth and differentiation when inhibitors were added to CD34(+) cells at the start of the culture and showed dose-response to the concentration of inhibitor employed VSports手机版. Enucleation was only reduced as a function of the reduced maturity of the culture and the increased cell death of mature cells while the majority of cells retained their ability to extrude their nuclei. Cell cycle analysis after caspase-3 inhibition showed caspase-3 to play a critical role in cell proliferation and highlighted a novel function of this protease in erythroid differentiation, i. e. its contribution to cell cycle regulation at the mitotic phase. While the effect of caspase-3 inhibitor treatment on CD34(+) derived cells was not specific to the erythroid lineage, showing a similar reduction of cell expansion in myeloid cultures, the mechanism of action in both lineages appeared to be distinct with a strong induction of apoptosis causing the decreased yield of myeloid cells. Using a series of colony-forming assays we were able to pinpoint the stage at which cells were most sensitive to caspase-3 inhibition and found activated caspase-3 to play a signalling role in erythroid differentiation by targeting mature BFU-E and CFU-E but not early BFU-E. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Caspase-3 inhibition affects erythroid proliferation, differentiation and viability.
a) Left: Dose-response of SIT treatment on erythroid cell expansion (day 5). Reduction of cell expansion as function of the inhibitor concentration (results shown as mean ± SEM, n = 3 experiments). All inhibitor concentrations showed significantly reduced expansion compared to control (* p<0.05) and also differed significantly (p<0.05) from each other (exception 50 µM to 25 µM). Right: Effect of caspase-3 inhibitor treatment on expansion of CD34+-derived erythroid cultures. Expansion curves of erythroid cultures under control (0.05% DMSO, open circles) or SIT-treated (filled circles) conditions showed growth reduction when caspase-3 is inhibited. b and c) Dose-response of erythroid differentiation to caspase-3 inhibitor treatment. Expression of surface antigens CD36 and CD71 in erythroid culture (day 13, b) and distribution between stages of erythroid differentiation (day 11, c) according to SIT treatment (one representative experiment shown). Proerythroblast (ProE), basophilic erythroblast (Baso), polychromatic erythroblast (Polychrom), orthochromatic erythroblast (Orthochrom), reticulocyte (Retic). d) Erythroid phenotype after caspase-3 inhibitor treatment. Almost all cells (day 11) were erythroid (CD36+/CD71+) but delayed differentiation was apparent under SIT treated conditions (bottom) compared to control (top) in respect to expression of surface markers CD71, CD45 and GlyA as assessed by flow cytometry (one representative experiment shown). e) Erythroid enucleation after caspase-3 inhibitor treatment. Enucleation progressed comparably at day 14 (upper panel) in cultures treated with 50 µM SIT (right) or DMSO (left) but a high degree of cell lysis was observed after day 21 (lower panel) in SIT treated cultures (right) compared to DMSO treated control (left) (one representative experiment shown). f) Reduction of viability in late stages of culture. Viabilities up to day 14 did not differ significantly between 50 µM SIT and controls (0.05% DMSO or untreated) but differed significantly (p<0.05) from control on day 18 and from control and DMSO on day 21 as determined by trypan blue exclusion. Flow cytometry analysis using AnnexinV-FITC and PI on day 14 showed higher percentages of early apoptotic (AnnexinV+) and late apoptotic (AnnexinV+/PI+) cells in SIT treated cultures (inset, bottom) vs. control (inset, top) (one representative experiment shown). g) Effect of caspase-3 inhibitor on mature cells. The effect of SIT was tested on native red blood cells (RBC), native reticulocytes and cultured reticulocytes using the calcein AM viability assay. Results are expressed as mean fluorescence intensity (MFI) or percent of cells (%) (one representative experiment shown).
Figure 2
Figure 2. Caspase-3 inhibition modulates cell cycle progression in the erythroid lineage.
a) Immunoprecipitation of activated caspase-3 from cell lysates of cultured erythroid cells at day 7. Whole cell lysate from 10×106 cells was immunoprecipitated with 1 µg of the rabbit anti-active caspase-3 antibody. Lane 1: erythroid cells at day 7 of culture (d7), lane 2: untreated Jurkat cells, lane 3: Jurkat cells incubated with DMSO for 18 h and lane 4: Jurkat cells treated with 10 µM staurosporin (STS) for 18 h. The activated 23 kDa caspase-3 band seen in control Jurkat cells induced into apoptosis by STS is clearly visible in erythroid cells (lane 1). b) Cell cycle analysis on day 7 of cultured erythroid cells. Cells were treated with SIT 50 µM or not and harvested after 2, 6, 8 and 24 hours. Controls consisted of cultured cells supplemented with 0.05% of DMSO. Controls are plotted as empty bullets, treated cells are plotted as black squares. Connecting curves represent mean of results. * indicates a statistically significant result with p<0.05 according to a paired t-test. 3 independent experiments were performed and are plotted on one graph. c) Caspase-3 inhibitor delays progression through G2/M phase in erythroid cultures. Representative histograms of cell cycle profiles of cells treated with 50 µM SIT or controls (0.05% DMSO), 4 h after SIT or DMSO treatment.
Figure 3
Figure 3. The inhibition of caspase-3 induces apoptosis in the myeloid lineage.
a) Myeloid expansion and differentiation. Expansion curve of a myeloid culture (one representative experiment and photographs of the cells on days 7 and 10 of myeloid culture after May-Grünwald-Giemsa staining (X100)). b) Cell cycle analysis on day 7 of cultured myeloid cells. Cells were treated with SIT 50 µM or not and harvested after 6 and 24 hours. Controls consisted of cultured cells supplemented with 0.05% of DMSO. 3 independent experiments were performed. Controls are plotted as empty bars, treated cells are plotted as black bars. Results are expressed as mean ± SEM (3b, top). Representative histograms of cell cycle profiles for control (0.05% DMSO) and treated cells (50 µM SIT) 6 h and 24 h after treatment (3b, bottom). Results indicate that caspase-3 inhibition induced apoptosis in myeloid cultures. Photograph of treated cells (day 10) 24 h post treatment after May-Grünwald-Giemsa staining (X100) (3b, bottom left). c) FACS analyses of CD13 and CD34 marker expression. Expression of surface antigens CD13 and CD34 in myeloid culture (day 10) for both 50 µM SIT and control (0.05% DMSO). Results in percent (%) are expressed as mean ± SEM, n = 3; * indicates a statistically significant result with p<0.05 according to a paired t-test.
Figure 4
Figure 4. Caspase-3 inhibition affects the production of later erythroid progenitors but not primitive progenitors.
a) Effect of caspase-3 inhibition on CD34high cells. CD34high were plated in methylcellulose colony-forming assays supplemented with DMSO or 50 µM SIT. Colony formation was assessed after 14/18 days. Results shown are the mean of 6 independent experiments and are expressed as mean ratio of colony formation in 50 µM SIT conditions to controls. * indicates a significant difference (p<0.05). b) Caspase-3 inhibition in the erythroid lineage did not modulate the production of primitive progenitors but decreased the production of later progenitors. Cells from each of the first 7 days of erythroid culture were plated in methylcellulose colony-forming assays supplemented with DMSO (top) or SIT (bottom). Colony formation was assessed after 14/18 days in respective conditions (results shown are the mean of duplicate plates from a pool of 3 cord blood units). Significant differences (p<0.05) in colony formation compared to control cultures are marked by # for BFU-E, * for CFU-GM.
Figure 5
Figure 5. Caspase-3 inhibition decreases the production of late BFU-E and CFU-E.
a) CD36+/CD34low and CD36+/CD34neg sorted cells derived from 7 days of untreated erythroid liquid culture. The CD34low (pink) and CD34neg (grey) fractions were sorted using a FACS MofloAstrios after labelling with APC-CD34 antibody. Cells were collected in IMDM medium+20% of FCS then washed in PBS before cultivation in semi-solid methylcellulose medium. b) Different types of progenitors obtained in methyl cellulose assays. Top left: a non-haemoglobinized colony scored at day 14 and identified as a myeloid progenitor. Top right: an early BFU-E consisting of a very large colony composed of haemoglobinized sub-units and scored at day 14/18. Bottom left: a mature BFU-E identified by its restricted size. Bottom right: a CFU-E identified as a very small single unit that is haemoglobinized at day 7 of the assay. c, d, e) Caspase-3 inhibition in the erythroid lineage did not modulate the production of primitive progenitors but decreased the production of late BFU-E and CFU-E. Cells from the first 7 days of erythroid culture (unsorted fraction (c), CD36+/CD34low sorted cells (d) or CD36+/CD34neg cells (e) were plated in methylcellulose colony-forming assays supplemented with DMSO (empty bars) or SIT (black bars). Colony formation was assessed after 14/18 days in respective conditions. * indicates significant differences (p<0.05) in colony formation compared to control cultures (paired t-test).
Figure 6
Figure 6. Caspase-3 inhibition does not affect the production of early BFU-E.
a) Dose-response of SIT treatment on CFU-GM formation. Reduction of CFU-GM formation as function of the inhibitor concentration (results shown as mean ± SEM, n = 3 experiments). b) The effect of caspase-3 inhibition on early BFU-E and CFU-GM. Cloning efficiency of untreated cells for the first 10 days of a myeloid culture. Results are expressed as number of colonies (CFU-GM and BFU-E) obtained per 1000 seeded cells, c) Cells from each of the first 10 days of myeloid culture were plated in methylcellulose colony-forming assays supplemented with DMSO (as control) or 50 µM SIT. Results were expressed as the ratio of colony formation in SIT to control. A linear fit was extrapolated for each type of progenitor. A paired Student's t-test established a significant difference for the impact of SIT on CFU-GM formation.
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