The hematopoietic growth factor erythropoietin (Epo) triggers changes in the expression of genes that encode important regulators of erythroid cell growth and differentiation. We now report that Epo markedly upregulates chop (gadd153) expression and that this transcription factor plays a role in erythropoiesis. Using a differential hybridization assay, we isolated a full-length cDNA ofchop as an Epo upregulated gene in Rauscher murine erythroleukemia cells. RNase protection assays demonstrated that Epo or dimethyl sulfoxide induction increased steady-state mRNA levels 10- to 20-fold after 24 to 48 hours. Western blot analysis confirmed a marked increase in CHOP protein. Among the other c/ebp family members, only c/ebp β was also upregulated during erythroid differentiation. Among normal hematopoietic cells examined, steady-state mRNA levels were highest in erythroid cells, with levels peaking during terminal differentiation. Transient overexpression ofchop in Rauscher cells resulted in a significant increase in Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further linking chop upregulation to erythroid differentiation. Artificial downregulation of chop in normal murine bone marrow cells with antisense oligodeoxynucleotides inhibited colony-forming unit-erythroid (CFU-E)–derived colony growth in a concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)–derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.
Erythroid progenitor growth in vitro is stimulated by exogenous platelet-derived growth factor (PDGF). We now report that both normal and transformed erythroid progenitor cells produce authentic PDGF in vitro and in vivo. Importantly, this production is highly regulated during erythropoiesis. Addition of soluble lysates from Rauscher murine erythroleukemia cells-an erythropoietin-responsive model progenitor cell line-to quiescent BALB/c 3T3 fibroblasts resulted in a mitogenic response identical to that observed with the addition of authentic recombinant PDGF. Polyclonal and monoclonal anti-PDGF antibodies immunoabsorbed 50-100% of this activity. Induction of Rauscher cell differentiation in vitro with dimethyl sulfoxide or erythropoietin for 48-72 hr markedly upregulated PDGF production by 17-to 18-fold and 14-to 38-fold, respectively. Importantly, stimulation of normal erythropoiesis in vivo in mice treated either with phenylhydrazine or with erythropoietin increased PDGF levels in the spleen by 11-to 48-fold and 20-to 34-fold, respectively. These results strongly suggest a role for erythroid cell-derived PDGF in normal erythropoiesis and provide documentation of the regulated production of a pleiotropic cytokine by erythroid cells.The growth and differentiation of erythroid progenitors in vitro are enhanced markedly by the addition of platelet-derived growth factor (PDGF) (1,2). The mechanism through which PDGF exerts this effect is unknown. Stimulation of erythroid cells by PDGF may occur by direct activation of the progenitors themselves or may be mediated through another cell type via a so-called paracrine mechanism. Several types of cells, including some transformed cell lines with erythroid characteristics, produce PDGF-like activity or can be induced to do so (3, 4). In the case of the transformed erythroid cells, it had been speculated originally that the production of PDGF was consequential to their transformation rather than characteristic of their erythroid phenotype.Previously, we demonstrated that both erythropoietin (Epo)-responsive Rauscher murine erythroleukemia cells and the splenic erythroid cells of phenylhydrazine (PHZ)-treated mice produce a PDGF-like activity that stimulates erythropoiesis in vitro (5). We now show that these "PDGF-like molecules" are authentic PDGF. Moreover, our results demonstrate that the production of erythroid cell-derived PDGF is highly regulated during erythropoiesis in vivo and erythroid differentiation in vitro. MATERIALS AND METHODSCells. The continuous Rauscher murine erythroleukemia cell line (6, 7) (clone R404) and murine BALB/c 3T3 fibro-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Control splenocytes, the majority of which are B and T lymphocytes, were obtained from B6C3F1 mice. To induce splenic erythropoiesis, B6C3F1 mice were injected subcutaneously either with recombinant human Ep...
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