Isolation of primary bone marrow–derived erythroblasts and EPO dose-dependent proliferation. (A) Erythroid progenitor cells from wild-type marrow were cultured in serum-free SP34-EX medium in the presence of EPO and SCF. At day 3.5, 90% or more of total cells were represented by CD71^high erythroid progenitors (top panels). Lin^pos depletion increased this representation to 98%, including 71% early-stage Kit^posCD71^high cells (bottom panels). (B) For the Lin^pos-depleted erythroblasts represented in panel A, the capacities of low-dose EPO to promote ³HdT incorporation and increases in cell numbers were defined. EPO dose-dependent formation of BrdU-positive cells also was assessed in short-term labeling experiments (ie, 1.5 and 3 hours). (C) Bone marrow progenitor cells were expanded for 3.5 days in SP34-EX medium. Kit^posCD71^high and Kit^negCD71^high cells were then isolated by MACS and analyzed in cytospin preparations. (D) These 2 discrete erythroblast populations were incubated for 5 hours in the absence of hematopoietic cytokines prior to culture in the presence of BrdU and EPO at 0.1 or 1.0 U/mL. At 2 hours, BrdU incorporation levels were assayed. Cells also were costained with 7-AAD to facilitate estimations of cell-cycle phase distributions. Note the increased representation of Kit^posCD71^high cells in S-phase (80%).

CYCLIN G2 protein levels are down-modulated by EPO, and ectopically expressed CYCLIN G2 attenuates UT7epo cell growth and S-phase transition. (A) EPO down-modulation of CYCLIN G2 protein expression. In primary wt-EPOR erythroblasts, EPO's effects on CYCLIN G2 expression levels were assayed by Western blotting, and first were observed to increase 2-fold or more within 6 hours of EPO withdrawal (top subpanels). In addition, EPO expression (in time-course and EPO concentration experiments) was observed to decrease CYCLIN G2 levels (bottom subpanels). Error bars represent standard deviations from the mean for duplicate scans of two ECL exposures (and are normalized for beta-tubulin). (B) Ectopic expression of CYCLIN G2 attenuates proliferation and G1- to S-phase transition in EPO-dependent UT7epo cells. UT7epo cells were transduced with a pBABEpuro-CYCLIN G2 retrovirus (or empty control vector) and were selected in puromycin. Derived lines (UT7epo CYCLIN G2 and UT7epo-vec) then were analyzed for EPO-dependent proliferative capacities and BrdU incorporation profiles. Ectopic expression of CYCLIN G2 first was observed to attenuate EPO-dependent UT7epo cell growth. Here, the cell lines were plated in the presence of EPO at 1 U/mL. Through time, viable cell numbers were monitored. Data shown are representative of 2 independent experiments. Subsequently, time-course analyses of EPO-dependent BrdU incorporation in UT7epo CYCLIN G2 and UT7epo-vec cells were performed. EPO was withdrawn from cultures for a 16-hour period prior to restimulation with EPO (1 U/mL) in the presence of BrdU. At the indicated time points, levels of BrdU incorporation were determined (top right subpanels). (C) Finally, EPO dose–dependent profiles of BrdU incorporation in UT7epo CYCLIN G2 and UT7epo-vec cells were investigated. Here, BrdU experiments were performed but with varying concentrations of EPO exposure. Panels first show raw data for incorporation levels. Bottom panels set gates for low versus high BrdU incorporation populations (gates 2 and 3, respectively), and further illustrate differences in BrdU incorporation profiles for gate 2 and 3 subpopulations for UT7epo CYCLIN G2 and UT7epo-vec cells.

EPO regulation of cyclin G2, cyclin D2, p27, and Bcl6 via minimal EPOR-H and EPOR-HM alleles. (A) EPO up-modulation of cyclin D2 and down-modulation of cyclin G2 depend on EPOR PY343 signals. Kit^posCD71^high cells were prepared from wt-EPOR, EPOR-HM, and EPOR-H erythroblasts. Hematopoietic cytokines were withdrawn for 5.5 hours. Cells then were exposed to EPO (5 U/mL). At 90 minutes, RNA was isolated, and reverse transcribed. Cyclin D2, Cyclin G2, Cdkn1b, and Bcl6 transcript levels then were determined by quantitative PCR. Values are the means (± SD) of n = 3 independent samples. (B) Observed EPO modulation of cell-cycle regulatory genes via wt-EPOR, EPOR-HM, and EPOR-H alleles provided for a mapping of transcriptional circuits to key EPOR subdomains including an EPOR box1-plus-JAK2 pathway to Myc, Nupr1, Egr1, and Nab2; an EPOR/PY343/STAT5 pathway to Cyclin D2 and Nupr1 expression, and Cyclin G2, Cdkn1b, and Bcl6 and Nab2 repression; and EPOR distal domain effects on Gspt1 induction.

At a late Ter119^pos stage, EPOR-HM erythroblasts falter in an apparent EPO dose-dependent G0/G1-phase accumulation. (A) Erythroblasts from marrow preparations were expanded in SP34-EX for 2.5 days, washed, and returned to culture in the absence of SCF and presence of EPO at 0.2 U/mL or 1.0 U/mL. At 24 and 48 hours, erythroblasts were analyzed for Ter119 expression and forward-angle light scatter (data shown are for wt-EPOR erythroblasts). Based on FALS, 3 subpopulations were resolved and are designated A, B, and C. (B,C) For preparations from wt-EPOR, EPOR-HM, and EPOR-H marrow, cell-cycle distributions were determined for Ter119^pos stage-A erythroblasts (panel B) and Ter119^pos stage-B erythroblasts (panel C). Values are means (± SD) for n = 3 independent cultures (from n = 3 mice for EPOR alleles).

Affymetrix array–based discovery of cell-cycle–associated EPO response genes in bone marrow–derived Kit^posCD71^high erythroblasts. (A) Basic steps involved in the discovery of EPO-modulated cell-cycle regulatory genes. Erythroblasts were expanded from the bone marrow of n = 4 replicate mice. For each preparation, purified Kit^posCD71^high cells were subdivided and cultured for 6 hours in the absence of hematopoietic cytokines. Erythroblasts were then exposed to EPO (± 5 U/mL) for 90 minutes and RNA was isolated directly. Biotin-cRNA next was prepared and was used in Affymetrix 430–2.0 array profiling. Transcripts for cell-cycle–associated genes that were modulated approximately 2-fold or more by EPO (with high statistical significance) were then considered. (B) For replicate Affymetrix array data sets, and as analyzed quantitatively (mean modulation due to EPO ± SD), EPO proved to significantly modulate 9 cell-cycle–related genes: Cyclin-D2/Ccnd2, Cyclin-G2/Ccng2, Gspt1, Nupr1, Egr1, Nab2, Myc, p27/Cdkn1b, and Bcl6. (C) In quantitative RT-PCR analyses, EPO regulation of each of the EPO-modulated cell-cycle regulatory genes was confirmed. Values are means and SD for samples (and erythroblasts) from n = 3 mice.

EPO regulation of Egr1, Nab2, Nupr1, and Gspt1 via EPOR-H and EPOR-HM alleles. From wt-EPOR, EPOR-HM, and EPOR-H SP34-EX expanded marrow cell preparations, Kit^posCD71^high erythroblasts were isolated, washed, and plated in IMDM, 0.25% BSA, 25 ng/mL insulin, and 100 μg/mL transferrin. At 5.5 hours, cells were exposed to EPO (± 5 U/mL). At 90 minutes, RNA was isolated and was reverse transcribed. (A) Schematics of knocked-in minimal EPO receptor EPOR-HM and EPOR-H alleles. EPOR-HM is a PY-null allele, while EPOR-H selectively retains a PY343 Stat5 binding site. (B) For each erythroblast preparation, and EPOR allele, levels of EPO-modulated Erg1, Nab2, Nupr1, and Gspt1 transcripts were determined by quantitative PCR.

EPO dose-dependent delays in EPOR-HM erythroblast cell-cycle progression. (A) Defective EPO-dependent cell-cycle progression among EPOR-HM erythroblasts. Erythroid progenitor cells from wt-EPOR, EPOR-HM, and EPOR-H marrow preparations were expanded in SP34-EX. At day 2, erythroblasts were washed and returned to culture in the absence of SCF and presence of EPO at 0.2 U/mL or 1.0 U/mL. At 24 hours, cell-cycle distributions for CD71^highTer119^neg erythroblasts were determined. Values are means (± SD) for n = 3 independent cultures (from n = 3 mice for EPOR alleles). In lower subpanels, cell-cycle distributions are summarized for wt-EPOR versus EPOR-HM versus EPOR-H. (B) Representative primary data for cycle distributions also are shown for wt-EPOR, EPOR-HM, and EPOR-H erythroblasts.