Development 114, 245-252 (1992) 245 Printed in Great Britain © The Company of Biologists Limited 1992 Combined action of c-Kit and erythropoietin on erythroid progenitor cells OSAMU OHNEDA, NOBUAKI YANAI and MASUO OBINATA* Department of Cell Biology, The Research Institute for Tuberculosis and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980, Japan *Author for correspondence Summary Mutations at the murine dominant-white spotting locus effective only at the early phase (within 6 hours after (WO (c-lrii) affect various aspects of hematopoiesis. We erythropoietin addition) before the cells start to prolifer- have made antibodies against c-Kit with the synthetic ate induced by erythropoietin. During the early phase, peptides deduced from the murine c-kil gene and erythropoietin down-regulated c-kit gene expression. examined the role of c-Kit in erythropoiesis. The These results suggest a mechanism of combined action of antibody inhibited the stromal cell-dependent large c-Kit with erythropoietin on the lineage-restricted colony formation of the erythroid progenitors. In the erythroid progenitor cells. culture of erythropoietin-responsive erythroid progeni- tors of the anemia-inducing Friend virus-infected mouse spleen, the antibody inhibited only proliferation, but not Key words: c-Kit, erythroid progenitor cells, differentiation of the progenitor cells. The inhibition was erythropoietin, W mutation. Introduction expression was found to be depressed, presumably because of a reduction in c-M-expressing erythroid cell Mutations at the murine dominant-white spotting locus populations in the fetal liver, as a result of the mutation. (W) affect various aspects of hematopoiesis. The defect Whereas levels of the erythroid progenitor cells (CFU- exerted by W mutations is intrinsic to hematopoietic E) were markedly reduced, normal numbers of the cells and the hematopoietic microenvironment in these granulocyte-macrophage progenitor cells (CFU-GM) mice is unaffected and able to support hematopoiesis. were observed in the W/W fetal liver (Nocka et al., Recently, Chabot et al. (1988) have mapped the proto- 1989) consistent with earner observations of only minor oncogene c-kit to the W locus on mouse chromosome 5 effects on granulopoiesis in W mutant mice (Russell, and Geissler et al. (1988) subsequently showed that the 1979). c-kit gene is rearranged in two different W mutants. The Apparently, because of the different microenviron- c-kit gene encodes a transmembrane kinase similar to ments in fetal liver, adult spleen and bone marrow, W CSF-1R and PDGFR (Qui et al., 1988; Yarden et al., 3 mutations affect erythropoiesis to different degrees at 1987). A 145 xlO MT tyrosine-specific transmembrane different stages of differentiation in the various hemato- kinase has been identified and characterized in brain poietic organs. Thus, it is necessary to examine the tissue extracts and in human glioblastoma cell line direct role of c-Kit on the erythropoietic progenitor (Yarden et al., 1987). Recently, SCF or 5/ factor has cells in vitro. Recently, we have demonstrated the in been identified as a candidate for the ligand of the c-Kit vitro reconstruction of a microenvironment adequate protein (receptor) (Zsebo et al., 1990; Martin et al., for a rapid expansion of erythroid cells in fetal liver 1990; Huang et al., 1990; Williams et al., 1990; Flanagan using established fetal liver stromal cells (Ohneda et al., et al., 1990). This is consistent with an earlier conjecture 1990). In this work, we examined the role of c-Kit on that the W locus encodes a cell-surface receptor that the proliferation and differentiation of the late eryth- functions in cell-to-cell interactions during develop- roid progenitors from fetal livers on the supporting ment and hematopoiesis (Russell, 1979). Most W stromal cell layers and from the anemia-inducing Friend mutations cause macrocytic anemia and are known to virus-infected mouse spleens. affect the erythroid progenitors (Gregory and Eaves, 1978; Iscove, 1978; Russell, 1979). Expression of c-kit was found to concur with the sites of active erythropoi- Materials and methods esis in yolk sac (Orr-Urtreger et al., 1990), fetal liver, adult bone marrow, and in Friend virus-transformed Preparation of antibody to c-Kit peptides cell lines (Nocka et al., 1989). In addition, c-kit Two oligopeptides (KIT1; FKTYFNEMVENKKNEW, 246 O. Ohneda, N. Yanai and M. Obinata KIT2; AQVKHNSWHRGDFNYERQE) deduced from the Detection of genotypes of the littermates by RT-PCR murine c-kit gene sequence (Qui et al., 1988) were synthesized method by a peptide synthesizer (Applied Biosystems Inc.). Two The genotypes of the littermates obtained by the mating of rabbits were immunized by subcutaneous injection with 500 WB-W/+ x WB-W/+ were determined by reverse transcrip- /jg of the oligopeptides coupled with keyhole limpet hemocya- tase-polymerase chain reaction (RT-PCR). The primers used nin emulsified in Freund's complete adjuvant, followed by were sense primer (1541-1565 in nucleotide number); 5'- additional injections of 500 /ig of the oligopeptide coupled GCCITCTTTAACTTTGCATTTAAAG-3', and antisense with keyhole limpet hemocyanin emulsified in Freund's primer (2656-2637): 3'-CTTGGAGTCGACCGGCATCC-5' incomplete adjuvant after 2, 4 and 6 weeks. The antisera were and these primers were kindly supplied by Dr T. Kunisada of obtained and evaluated by ELISA 2 weeks after the final Kumamoto University Medical School, Kumamoto, Japan. booster. igG was precipitated with 1.34 volumes of saturated The amplified DNA product from the W mutant mRNA is ammonium sulphate and dialyzed against 10 mM sodium shorter than that of the wild-type mRNA as described by phosphate buffer (pH 8.0). The dialyzed materials were Hayashi et al. (1991V For cDNA synthesis, 10 \i% of RNAs applied to a DEAE cellulose column and the flow-through from brains (C57BL/6J-+/+ mouse and WB-W/+ mouse) and fractions were collected. Amounts of IgG in the flow-through from whole embryos (littermates of the mating of WB-W/+ x fractions were estimated by ELISA. W/+) were incubated with 100 pmol of random primer and 200 U of MMLV reverse transcriptase (BRL) in 20 /il reaction Immunoprecipitation and autophosphorylation of mixture containing 0.55 M Tris-HCl, pH 8.3, 0.075 M KC1, 3 c-Kit protein mM MgCk, 10 mM dithiothreitol, 200 mM deoxyribonucleo- tide triphosphates, and 20 U of RNAase inhibitor (Pharma- Immune complex kinase assay was performed essentially as cia) at 37°C for 60 minutes. The cDNA was amplified by 2.5 described previously (Majumder et al., 1988). Briefly, mouse units of Taq DNA polymerase and 40 pmol of sense and brain (400 mg) was homogenized in 2 ml of lysis buffer (50 antisense primers in 20 ml reaction buffer (10 mM Tris-HCl, mM Tris, 150 mM NaCl, 20 mM EDTA, 1% Triton X-100,1% pH 8.3, 50 mM KC1,1.5 mM MgCl2, 0.01% gelatin and 0.5 U deoxycholate, 1% SDS, 1 mM phenylmethylsulfonyl fluoride, of Perfect Match (Stratagene) for 28 cycles in a program 20 jig/ml leupeptin, lmM sodium vanadate at pH 8.4) and Temperature Control System PC 700 (Astec Inc.). The centrifuged at 100,000 g for 45 minutes. The supernatant was amplified DNA products were analyzed by 1.4% agarose gel subjected to wheat-germ-agglutinin chromatography and electrophoresis. pretreated with the non-immune serum. The pretreated materials were immunoprecipitated with KTT1 antibodies that were conjugated with protein A sepharose, and incubated Northern blot analysis of c-kit mRNA and SCF with [y- P]ATP. The labeled proteins were separated by mRNA SDS-PAGE and visualized by autoradiography. The erythroid progenitor cells derived from Friend virus- infected mice were cultured in vitro with 0.1 U/ml erythropoi- etin and the cells were collected at appropriate time intervals Preparation of hematopoietic progenitor cells and (0-12 hours). The cells were homogenized in guanidine coculture of hematopoietic cells on stromal cell layers isothiocyanate, and RNAs were isolated by centrifugation Preparation of hematopoietic progenitor cells and coculture through CsCl. 4 ng of poly (A)+ RNA, obtained by oligo(dT)- of hemopoietic cells on stromal cell layers were performed as Latex (Oligotex-dT30, Nihon Synthetic Rubber Co.), were described previously (Yanai et al., 1989). Hematopoietic separated in 0.7% agarose gel containing formaldehyde, and progenitor cells were prepared from the liver of 13-day transferred to nylon membranes. The membranes were embryos after mating of WB-W/+ x WB-W/+ mice. prehybridized and hybridized with the nick-translated 5.1 kb Hemopoietic progenitor cells were cultured on monolayers of c-kit cDNA (provided by Dr A. Singh, Genentech.Inc). The FLS5 and MSS62 cell lines in multiwell plates (Falcon 3047, amount of c-kit mRNA in each sample was assessed by Becton Dickinson), in Iscove's modified Dulbecco's medium comparison to hybridization with a ^3-actin probe. (IMDM; Gibco), supplemented with 0.4% methylcellulose, For detection of SCF mRNA in the stromal cells, total 30% heat-inactivated FBS, 1% bovine serum albumin, 100 RNAs were isolated from two stromal cell lines, FLS cell lines /JM 2-mercaptoethanol and erythropoietin (recombinant (Ohneda et al., 1990) and MSS cell lines (Yanai et al., 1989). human erythropoietin, generously supplied by Kirin-Amgen The SCF cDNA probe was prepared by reverse transcriptase- Co. Ltd.). After 4 days in culture, erythroid colonies were polymerase chain reaction (RT-PCR) from mRNA of FLS5 scored by direct staining with benzidine. cells with sense primer (16-36 nucleotide number by Zsebo et al., 1990) 5'-ATGAAGAAGACACAAACTTGG-3' and Isolation and culture of splenic erythroblasts antisense primer (598-579) 3'-ACGTCGGTCCGAGG- Anemia-inducing Friend virus (FVA, provided by Dr H. GAATCCT-5'. The sequence of the product of RT-PCR was confirmed after subcloning. Amanuma of RIKEN, The Institute of Physical and Chemical Research, Tsukuba, Japan) was injected intravenously into 6- week-old DBA/2 mice.