Graded Levels of GATA-1 Expression Modulate Survival, Proliferation, and Differentiation of Erythroid Progenitors
2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês
10.1074/jbc.m500081200
ISSN1083-351X
AutoresXiaoqing Pan, Osamu Ohneda, Kinuko Ohneda, Fokke Lindeboom, Fumiko Iwata, Ritsuko Shimizu, Masumi Nagano, Naruyoshi Suwabe, Sjaak Philipsen, Kim-Chew Lim, James Douglas Engel, Masayuki Yamamoto,
Tópico(s)Genomics and Chromatin Dynamics
ResumoTranscription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16INK4A than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation. Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16INK4A than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation. Transcription factor GATA-1 recognizes conserved GATA motifs ((T/A)GATA(A/G)) in the regulatory regions of many genes encoding erythroid-restricted proteins, such as globins, heme biosynthetic enzymes, membrane proteins, and transcription factors (1Weiss M.J. Orkin S.H. Exp. Hematol. 1995; 23: 99-107PubMed Google Scholar, 2Ferreira R. Ohneda K. Yamamoto M. Philipsen S. Mol. Cell. Biol. 2005; 25: 1215-1227Crossref PubMed Scopus (303) Google Scholar). To analyze the in vivo function(s) of GATA-1, GATA-1-deficient mice were generated (3Fujiwara Y. Browne C.P. Cunniff K. Goff S.C. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12355-12358Crossref PubMed Scopus (604) Google Scholar). Disruption of primitive erythropoiesis caused GATA-1 homozygous null mutant embryos to die by embryonic day (E) 1The abbreviations used are: E, embryonic day; FBS, fetal bovine serum; ES, embryonic stem; WT, wild type; Epo, erythropoietin; RT-PCR, reverse transcriptase-PCR; CFU, colony forming unit; FACS, fluorescent activating cell sorter; Rb, retinoblastoma. 1The abbreviations used are: E, embryonic day; FBS, fetal bovine serum; ES, embryonic stem; WT, wild type; Epo, erythropoietin; RT-PCR, reverse transcriptase-PCR; CFU, colony forming unit; FACS, fluorescent activating cell sorter; Rb, retinoblastoma. 11.5, demonstrating that GATA-1 is required for primitive erythropoiesis. This early demise precluded the possibility of analyzing the role of GATA-1 in definitive erythropoiesis. To experimentally circumvent this impediment, chimeric mice were derived using GATA-1-/- ES cells, and this confirmed that the null mutant cells did not contribute to the mature definitive erythroid pool (4Pevny L. Simon M.C. Robertson E. Klein W.H. Tsai S.F. D'Agati V. Orkin S.H. Costantini F. Nature. 1991; 349: 257-260Crossref PubMed Scopus (1031) Google Scholar). Thus, GATA-1 is required for the terminal differentiation of both primitive and definitive erythroid progenitors.We previously utilized an erythroid promoter-specific loss-of-function (knockdown) strategy to generate a GATA-1 hypomorphic (GATA-1.05) allele (5Takahashi S. Komeno T. Suwabe N. Yoh K. Nakajima O. Nishimura S. Kuroha T. Nagasawa T. Yamamoto M. Blood. 1998; 92: 434-442Crossref PubMed Google Scholar). The GATA-1 gene is X-linked, and in GATA-1 hemizygous hypomorphic males (GATA-1.05/Y), GATA-1 mRNA was detected at ∼5% of the normal levels. Hence, we termed these mice GATA-1.05 (6Takahashi S. Onodera K. Motohashi H. Suwabe N. Hayashi N. Yanai N. Nabesima Y. Yamamoto M. J. Biol. Chem. 1997; 272: 12611-12615Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). GATA-1.05/Y embryos exhibited a defective maturation of primitive erythroid cells and died by E12.5 (6Takahashi S. Onodera K. Motohashi H. Suwabe N. Hayashi N. Yanai N. Nabesima Y. Yamamoto M. J. Biol. Chem. 1997; 272: 12611-12615Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), which, like GATA-1-/- embryos, precluded the analysis of the function of GATA-1 in definitive erythropoiesis.Impaired primitive and definitive erythropoiesis in both GATA-1-null and hypomorphic mutant embryos resulted in the generation of extremely limited numbers of erythroid progenitors that could be used for further cytological and molecular analyses. Additionally, we suspected that defective erythropoiesis in the mutant embryos could cause secondary growth retardation, which would in turn affect later hematopoietic development. Under such circumstances where cell-autonomous as well as non-cell-autonomous deficiencies could contribute to the phenotype, it becomes difficult to determine conclusively in vivo how different quantitative levels of GATA-1 may affect the developmental decisions available to an erythroid progenitor cell.The generation of homogenous erythroid populations from ES cells (7Leonard M. Brice M. Engel J.D. Papayannopoulou T. Blood. 1993; 82: 1071-1079Crossref PubMed Google Scholar) is a useful experimental tool for analyzing the definitive erythroid population in instances where gene-targeted mutation leads to embryonic death prior to the onset of definitive erythropoiesis. In a two-step ES cell in vitro differentiation method, the ES cells are cultured in methylcellulose medium containing stem cell factor and erythropoietin (Epo) (7Leonard M. Brice M. Engel J.D. Papayannopoulou T. Blood. 1993; 82: 1071-1079Crossref PubMed Google Scholar). Alternatively, if ES cells are co-cultured with OP9 stromal cells, they differentiate into a hematopoietic cell population that consists of erythrocytes, neutrophils, macrophages, mast cells, megakaryocytes, and lymphoid cells (8Nakano T. Kodama H. Honjo T. Science. 1994; 265: 1098-1101Crossref PubMed Scopus (689) Google Scholar). Subsequently, Nakano et al. (9Nakano T. Kodama H. Honjo T. Science. 1996; 272: 722-724Crossref PubMed Scopus (186) Google Scholar) developed the ES/OP9 cell co-culture system, in which two waves of erythroid (primitive and definitive) cell production were detected after either 6 or 14 days of induction, respectively. Consequently, the ES/OP9 cell co-culture system reflects not only primitive, but also definitive, erythropoiesis in vivo and is a useful tool for dissecting in vitro the functional role of any molecule of interest during erythroid development.Here, we report that both differentiation and apoptosis are inhibited in GATA-1.05-definitive erythroid cells. Although GATA-1-/--definitive erythroid cells are similarly arrested in differentiation, they, unlike the GATA-1.05 cells, preferentially undergo apoptosis. Hence, we propose that although normal levels of GATA-1 promote terminal differentiation, the low level of intracellular GATA-1 is insufficient to block continuous progenitor proliferation but is sufficient to prevent apoptosis. In contrast, the complete absence of GATA-1 favors an apoptotic response. In this way, graded levels of transcription factor GATA-1 modulate multiple facets of erythroid cell physiology, including survival, proliferation, and differentiation.EXPERIMENTAL PROCEDURESCell Culture—E14 ES cells were maintained on embryonic fibroblast cells and kept undifferentiated in the presence of recombinant leukemia inhibitory factor (1000 units/ml, ESGRO, Chemicon International). OP9 cells were cultured as described previously (8Nakano T. Kodama H. Honjo T. Science. 1994; 265: 1098-1101Crossref PubMed Scopus (689) Google Scholar). After the harvest of ES cells from OP9 feeder cells by trypsinization, 7 × 103 cells were plated onto subconfluent OP9 cells grown in α-minimum essential medium supplemented with 10% FBS, mouse vascular endothelial growth factor (10 ng/ml, Peprotech), and human bone morphogenic protein-4 (5 ng/ml, R&D systems). After 4 days of co-culture, ES cells were trypsinized and replated onto fresh OP9 cells in α-minimal essential medium supplemented with 10% FBS, Epo (2 units/ml; generous gift from Chugai Pharmaceutical), and stem cell factor (50 ng/ml, generous gift from Kirin Brewer Co.).Non-adherent cells observed on day 6 and day 11 were analyzed as primitive and definitive erythroid cells, respectively. To isolate erythroid cells, floating cells were incubated with biotinylated c-Kit (2B8), CD11b (Mac-1, M1/70), and Gr-1 (RB6-8C5) antibodies for 30 min on ice. After washing twice with washing buffer (2% FBS in phosphate-buffered saline), cells were incubated with streptavidin-conjugated Dynabeads (M-280, Dynal Biotech) for 20 min on ice. Subsequently, cells were washed once with washing buffer, and non-adherent cells attached to the magnet (VarioMACS, Miltenyi Biotec) were collected for further experiments.On day 11 of differentiation, adherent cells were examined in CFU-OP9 colony formation assay. After washing the OP9/ES cell co-culture dish with phosphate-buffered saline, adherent cells were trypsinized and resuspended in α-minimal essential medium supplemented with 10% FBS and incubated for 1 h to eliminate stromal cells. Non-adherent cells were collected and cultured with OP9 cells for 4–6 days in α-minimal essential medium, 10% FBS in the presence of Epo (2 units/ml) and stem cell factor (50 ng/ml). Cobblestone-like colonies, termed CFU-OP9, developed on OP9 cells and were scored.Flow Cytometry—Cultured ES cells and mouse fetal liver cells (E12.5–E14.5) were harvested and incubated in washing buffer containing Fc block (CD16/CD32; 1:200, BD Pharmingen) for 15 min on ice. Subsequently, cells were washed twice with washing buffer and incubated for 30 min on ice with fluorescence-conjugated antibodies. Then, cells were washed twice and analyzed using FACSCalibur and Vantage (BD Biosciences). The following antibodies were purchased from BD Pharmingen and used for analyses: allophycocyanin-conjugated c-Kit antibody (2B8), phycoerythrin-conjugated TER119 antibody, fluorescein isothiocyanate-conjugated CD71 antibody (C2), and allophycocyanin-conjugated CD44 antibody (IM7). DNA content analysis was performed as described previously (10Zhang H.S. Postigo A.A. Dean D.C. Cell. 1999; 97: 53-61Abstract Full Text Full Text PDF PubMed Google Scholar). Floating cells were gently harvested from ES/OP9 cell co-culture on day 11, and cells were fixed in 70% ethanol. Then, cells were treated with 50-μg/ml propidium iodide and 100-units/ml RNase A. Cell cycle distribution was analyzed using ModFit LT software (Verity Software House).Semiquantitative RT-PCR Assay—Primitive and definitive erythroid cells co-cultured on OP9 stromal cells were harvested, and total RNA was isolated by RNAeasy mini-kit (Qiagen). cDNAs were synthesized using a RT-PCR kit (Clontech) in a 20-μl reaction containing 1 μg of total RNA. An aliquot (1 μl) of synthesized cDNA was amplified in a total volume of 20 μl containing 150 μm dNTP, 0.2 units of Taq polymerase, and 0.12 μg of each pair of primers. The PCR profile consisted of 26–36 cycles of 95 °C for 30 s and 68 °C for 1 min. The PCR products were electrophoretically separated on a 2% agarose gel. Primers used in this experiment are listed as follows: Bcl-xL, sense 5′-ACTGAGGCCCCAGAAGAAACTGAAGCA-3′, antisense 5′-CTGCTGCATTGTTCCCGTAGAGATCCA-3′; Bcl2, sense 5′-GGGTATGATAACCGGGAGATCGTGATGA-3′, antisense 5′-TACTCAGTCATCCACAGGGCGATGTTGT-3′; Bax, sense 5′-GGCCTTTTTGCTACAGGGTTTCATCCAG-3′, antisense 5′-CCACAAAGATGGTCACTGTCTGCCATGT-3′; cyclin D2, sense 5′-ACCGACAACTCTGTGAAGCC, antisense 5′-TTCATCATCCTGCTGAAGCC; p16, sense 5′-CGATTCAGGTGATGATGATGG, antisense 5′-GCTTGAGCTGAAGCTATGCC; p21, sense 5′-AGTTCACCGTCAGCATCACC-3′, antisense 5′-ACAGAACTGACATCCATGGC-3′; p53, sense 5′-ATAAGCTATTCTGCCAGCTGG-3′, antisense 5′-AACACGAACCTCAAAGCTGTC-3′; RB, sense 5′-GTTGACATCGGAGTACAGC-3′, antisense 5′-GCTTGTGTCTCTCTGTATTTGC-3′; p27, sense 5′-ACTGTCTGTGTGCAGTCGC-3′, antisense 5′-GCAGTGCTTCTCCAAGTCC-3′; GATA-1; sense 5′-ACTCGTCATACCACTAAGGT-3′, antisense 5′-AGTGTCTGTAGGCCTCAGCT-3′; erythroid-specific aminolevulinate synthase, sense 5′-GTCCTGTGGAGGAATTGTGT-3′, antisense 5′-GTTTTCCATCATCTGAGGGC-3′; HPRT, sense 5′-GCTGGTGAAAAGGACCTCT-3′, antisense 5′-CACAGGACTAGAACACCTGC-3′; glyceraldehyde-3-phosphate dehydrogenase, sense 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′, antisense 5′-CATGTAGGCCATGAGGTCCACCAC-3′.Construction of Retroviral Vectors and Retroviral Infection—Plasmid for murine stem cell virus-internal ribosomal entry site-enhanced green fluorescent protein (MSCV-IRES-EGFP) was kindly provided by Dr. Akihiko Kume. Murine GATA-1 and p16INK4A cDNAs were independently ligated into the BamHI and EcoRI restriction sites of MSCV-IRES-EGFP. Phoenix-Eco packaging cells were maintained in complete Dulbecco's modified Eagle's medium containing 10% FBS. Phoenix-Eco cells at 80% confluency on 6-cm dishes were transfected with 1 μg of DNA using the FuGENE transfection kit (Roche Applied Science). Retroviral supernatant was collected after 72 h and added to NIH3T3 cells to titrate the virus in the presence of 8 μg/ml Polybrene (Sigma). After 4 days, FACS analysis for enhanced green fluorescent protein fluorescence was performed to measure the virus titer. Titers of viruses used in this study were >1 × 107 infectious particles/ml.To establish retroviral packaging cell lines, supernatant from transfected Phoenix-Eco cells was harvested and used to infect PT67 cells (Clontech) in the presence of 8 μg/ml Polybrene. After 2–4 days, the brightest green fluorescent protein-expressing cells were sorted using a FACS Vantage and expanded in culture. The expression of GATA-1 and p16INK4A in NIH3T3 cells infected with viral supernatants was confirmed by RT-PCR. Floating cells were removed from 11-day ES/OP9 co-culture, and adherent cells on OP9 stromal cells were trypsinized. After a 1-h incubation on the dish to remove stromal cells, non-adherent cells were replated onto fresh OP9 cells and co-cultured in the presence of the viral supernatant with 4 μg/ml Polybrene for 2 days. The cultures were kept for an additional 4 days in fresh α-minimal essential medium, 10% FBS containing Epo and stem cell factor. The number of colonies that developed on OP9 stromal cells was scored. Floating cells were harvested and cytospin samples were stained with May-Grünwald-Giemsa to verify the differentiation stage of erythroid cells.RESULTSDefinitive Erythroid Differentiation of GATA-1.05 and GATA-1-null ES Cells—We previously reported that differentiation in GATA-1.05/Y primitive and definitive erythroid cells was blocked at different stages thereby implicating different requirements for GATA-1 levels during erythroid development in distinct hematopoietic organs, such as the yolk sac and fetal liver (6Takahashi S. Onodera K. Motohashi H. Suwabe N. Hayashi N. Yanai N. Nabesima Y. Yamamoto M. J. Biol. Chem. 1997; 272: 12611-12615Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, there is no clear molecular explanation for how varied GATA-1 levels might differentially affect the development of the primitive and definitive erythroid lineages. To address this question, ES cells carrying WT, GATA-1.05, and GATA-1-null alleles were separately co-cultured with OP9 feeder cells, and the resultant differentiated primitive and definitive erythroid cells were harvested for further analyses.Morphological and gene expression analyses confirmed that ES cells cultured on an OP9 feeder layer differentiated into primitive and definitive erythroid cells from days 6–8 and 11–14, respectively (11Suwabe N. Takahashi S. Nakano T. Yamamoto M. Blood. 1998; 92: 4108-4118Crossref PubMed Google Scholar). Therefore, WT, GATA-1.05, and GATA-1-null ES cells were cultured on OP9 cells for 11 days, and the non-adherent fractions were then subjected to flow cytometric analyses using anti-c-Kit and TER-119 antibodies (Fig. 1, A and B). Mature definitive erythroid cells (c-Kit-TER-119+) were largely absent from GATA-1.05 and GATA-1-null, but not from WT, ES cell cultures (Fig. 1A). In contrast, immature erythroid progenitors (c-Kit+TER-119+) were more abundant in both GATA-1 mutant (GATA-1.05 and GATA-1-null) cultures than in WT cells. Interestingly, the anti-c-Kit signal was ∼10-fold higher in TER-119+GATA-1.05 cells than in WT and GATA-1-null cell equivalents. In addition, GATA-1-null ES cell cultures generated the lowest number of non-adherent erythroid cells (Fig. 1C), hinting at the possibility of reduced proliferation and/or exceptionally high cell death in the null mutant cells (below).We previously reported that the adherent cells in the OP9 co-culture displayed numerous immature hematopoietic cell characteristics based on RT-PCR expression analyses and on morphology of the recovered colonies in methylcellulose medium (11Suwabe N. Takahashi S. Nakano T. Yamamoto M. Blood. 1998; 92: 4108-4118Crossref PubMed Google Scholar). Surprisingly, the number of adherent cells and CFU-OP9 colonies derived from GATA-1.05 ES cells was the highest among the three ES cell types (Fig. 1, D and E), perhaps as a consequence of differentiation arrest and/or increased proliferation in the c-Kit+TER-119+ immature erythroid cells in GATA-1.05 co-cultures.Collectively, we concluded that under conditions that promote erythroid differentiation both varieties of GATA-1 mutant ES cells, which express either no or a small amount of GATA-1 protein, produce predominantly immature proerythroblasts and few mature cells. Furthermore, cells with abnormally high proliferative potential are recovered from GATA-1.05 ES/OP9 cell cultures.Elevated Apoptosis in GATA-1-null Erythroid Cells—May-Grünwald-Giemsa staining revealed that WT ES cells after 6 days in co-culture contained primitive erythroid cells with orthochromatic cytoplasm and large nuclei, whereas immature blast cells with large, prominent nuclei and polychromatic cytoplasm were present in cultures that developed from both kinds of GATA-1 mutant ES cells (Fig. 2, A–C). Uptake of trypan blue dye (indicating dead or dying cells) and nuclear fragmentation (apoptotic intermediates) were most frequently observed in the GATA-1-null ES cell culture (Fig. 2, D and F).Fig. 2Cytological comparison of erythroid cells differentiated from WT or mutant GATA-1 ES cells. Primitive (A–F) and definitive (G–L) erythroid cells harvested on day 6 and 11, respectively, were subjected to May-Grünwald-Giemsa (A–C and G–I), propidium iodide (D–F and J–L), and trypan-blue dye staining. WT primitive erythroid cells with orthochromatic cytoplasm and large nuclei (A) were observed, whereas immature primitive erythroid cells were seen in GATA-1 mutant ES cell cultures (B and C). WT definitive erythroid cells at various differentiation stages (G) were observed, whereas more blast-like cells were seen in GATA-1 mutant ES cell cultures (H and I). Morphologically, these cells appeared to be proerythroblasts. Numerous cells with nuclear fragmentation (black arrow) were noticed in GATA-1-null erythroid cells (I). Propidium iodide staining indicated significantly more apoptotic cells with nuclear fragmentation in 6-day and 11-day (F and L, white arrowhead) GATA-1-null ES cell culture than in WT (D) and GATA-1.05 (E) cultures. #, p < 0.05; *, p < 0.01. Scale bar represents 25 μm. Cells were also stained with trypan-blue solution, and the frequency of dead and dying cells was scored. The number represented (mean ± S.D.) was obtained from four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)WT ES cells, after 11 days in co-culture, contained definitive erythroid cells at various differentiation stages, whereas immature blast cells with large, prominent nuclei were the dominant species recovered in both of the GATA-1 mutant ES cell cultures (Fig. 2, G–I). Using benzidine staining to distinguish terminally differentiated erythroid cells, we noted that the benzidine-positive population was significantly reduced in both GATA-1 mutant, but not in GATA-1+/+ ES cell cultures (WT, 48.7 ± 14.9%; GATA-1.05, 8.1 ± 1.8%; GATA-1-null, 0.4 ± 0.1%). Furthermore, at day 11 of differentiation both trypan blue dye uptake and nuclear fragmentation were observed at the highest frequency in the GATA-1-null ES cell culture (Fig. 2, J–L), indicating unusually high apoptotic activity in GATA-1-/- erythroid progenitors.Taken together, these data indicate that although the low GATA-1 level (5% in the GATA-1.05 mutant cells) is sufficient to avert apoptosis, higher GATA-1 expression levels are necessary to induce terminal erythroid maturation. This observation is consistent with the previous report that GATA-1 could act as a survival factor in committed erythroid cells (12Weiss M.J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9623-9627Crossref PubMed Scopus (263) Google Scholar), although the GATA-1 expression level was not determined in that study. Given the intriguing differences observed in ES cells that express graded levels of GATA-1 when exposed to differentiation stimuli, we investigated the activity of candidate GATA-1 target genes during erythroid proliferation versus differentiation.Bcl-xL Is Highly Expressed in GATA-1.05 ES Cell-derived Definitive Erythroid Progenitors—It has been reported that Epo cooperates with GATA-1 to stimulate Bcl-xL gene expression and to maintain erythroid survival, and that Bcl-xL is essential for normal erythroid differentiation (13Gregory T. Yu C. Ma A. Orkin S.H. Blobel G.A. Weiss M.J. Blood. 1999; 94: 87-96Crossref PubMed Google Scholar). Thus, Bcl-xL seems to be a critical downstream effector of GATA-1- and/or Epo-mediated signals. We therefore investigated the expression of several Bcl-2 family members, including Bcl-xL, in primitive and definitive erythroid populations recovered from ES/OP9 cultures (Fig. 3).Fig. 3Expression profiles of genes involved in apoptosis in erythroid cells expressing varying GATA-1 levels. Total RNA isolated from primitive and definitive erythroid cells harvested from day 6 and day 11, respectively, of co-culture was analyzed by RT-PCR (lower (A) and higher PCR cycles (B)). Compared with WT-definitive erythroid cells, Bcl-xL expression was undetected in GATA-1-null (G1-null)-definitive erythroid cells, but was higher in the GATA-1.05 (G1.05) cell equivalents. HPRT, hypoxanthine quanine phosphoribosyl transferase; ddw, deionized distilled water.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although Bcl-2 mRNA was absent in GATA-1-null primitive erythroid cells, it was present at similar levels in definitive erythroid cells differentiated from all three types of ES cells. Bcl-xL expression was not detected in primitive erythroid cells (at day 6) from both GATA-1 mutant ES cell cultures. Bcl-xL expression was also not found in GATA-1-null definitive erythroid cells. Remarkably, however, a slightly higher than normal Bcl-xL mRNA level was observed in GATA-1.05 definitive erythroid cells. These findings strongly suggest that Bcl-xL, but not Bcl-2, is important for cell survival during definitive erythroid differentiation and that low levels of GATA-1 may be adequate for inducing ROM, expression to protect against apoptosis.In contrast, the expression of apoptotic inducers, such as Bax, remained constant during primitive and definitive erythroid differentiation. The stabilization and accumulation of the tumor suppressor protein, p53, has also been shown to contribute to apoptosis. We therefore examined p53 accumulation in GATA-1-mutant erythroid cells. Lower p53 expression was detected in primitive erythroid cells from both GATA-1.05 and GATA-1-null cells compared with WT cells, underscoring the possibility that the apoptosis observed in primitive erythroid GATA-1-null cells is independent of p53, as reported previously (12Weiss M.J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9623-9627Crossref PubMed Scopus (263) Google Scholar).Erythroid Cells Derived from GATA-1 Mutant ES Cells Accumulate in S Phase—It has been reported that forced expression of GATA-1 alters the length of cell cycle segments (14Whyatt D.J. Karis A. Harkes I.C. Verkerk A. Gillemans N. Elefanty A.G. Vairo G. Ploemacher R. Grosveld F. Philipsen S. Genes Funct. 1997; 1: 11-24Crossref PubMed Scopus (54) Google Scholar) and that especially high levels of GATA-1 were found to lengthen S phase in NIH3T3 cells (15Dubart A. Romeo P.H. Vainchenker W. Dumenil D. Blood. 1996; 87: 3711-3721Crossref PubMed Google Scholar). In addition, Cullen et al. (16Cullen M.E. Patient R.K. J. Biol. Chem. 1997; 272: 2464-2469Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) showed that GATA-1 activity in mouse erythroleukemia cells was low in G1 phase, peaked in mid-S phase, and then diminished again in G2/M phase and showed that the accumulation patterns of GATA-1 protein and mRNA mirrored one another throughout the cell cycle. It is therefore tempting to speculate that GATA-1 may directly regulate the cell cycle and/or that its expression may be tightly regulated during erythroid differentiation.To determine how the expression level of GATA-1 affects the cell cycle during erythroid maturation, flow cytometric analysis of ES cell-derived hematopoietic cells was initiated. Definitive erythroid cells were collected on day 11 postinduction (Fig. 4A). Although mature definitive erythroid cells from differentiating WT ES cells accumulated in G0/G1 phase, immature definitive erythroid cells in both GATA-1 mutant ES cell cultures accumulated in S phase. These results are consistent with the previous data (Fig. 1, A and B), in which c-Kit+TER-119+ immature cells were found to be more abundant in GATA-1 mutant ES cell cultures, whereas mature definitive erythroid cells (c-Kit-TER-119+) were more abundant in control cultures.Fig. 4Accelerated cell cycle progression in the definitive erythroid cells correlate with reduced GATA-1 level.A, cell cycle distribution of the definitive erythroid cells expressing different levels of GATA-1. Cells were stained with propidium iodide and then evaluated for DNA content by flow cytometry. The distribution of cells in G0-G1, S, and G2-M cell cycle phases are enumerated and graphically represented. The left red color peak represents diploid cells in G0-G1 phase, and the right red color peak represents diploid cells in G2-M phase. The blue striped area represents diploid cells in S-phase. The left unshaded peak represents aneuploid cells in G0-G1 phase, and the right unshaded peak represents aneuploid cells in G2-M phase. B, RT-PCR analysis of G1-S phase transition regulators in erythroid cells expressing different levels of GATA-1. Of all the G1-S phase transition regulators examined, only expression of p16INK4A was impaired in GATA-1.05 and GATA-1-null erythroid cells. G3PDH, glyceraldehyde-3-phosphate dehydrogenase; DDW, deionized distilled water.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cell cycle arrest is subject to a variety of regulators, including cyclin D-Cdk complex and INK family inhibitors. We analyzed cyclin D2 mRNA levels in definitive erythroid cells differentiated from GATA-1 mutant ES cells by RT-PCR (Fig. 4B). Cyclin D2 mRNA did not vary significantly among definitive erythroid cells recovered from all three ES cell types. Cyclin D-dependent kinases collaborate with cyclin E-Cdk2 to phosphorylate Rb and its family members, p107 and p130, inactivating their growth inhibitory functions and facilitating S phase entry (17Sherr C.J. McCormick F. Cancer Cell. 2002; 2: 103-112Abstract Full Text Full Text PDF PubMed Scopus (1295) Google Scholar). The
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