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The Raf-1 Protein Mediates Insulin-Like Growth Factor-Induced Proliferation of Erythroid Progenitor Cells

^a Departments of Pediatrics and Laboratory Medicine, University of Connecticut School of Medicine;
^b The American Red Cross, Farmington, Connecticut, USA;
^c Department of Medicine, The Bridgeport Hospital, Yale University School of Medicine, Bridgeport, Connecticut, USA

Key Words. Somatomedins • Erythropoietin • Signal transduction • Proto-oncogene

Dr. Marilyn R. Sanders, University of Connecticut Health Center, M.C. 2948, Farmington, CT 06030, USA.

	Abstract

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Previous studies from this and other laboratories have shown that insulin-like growth factor-1 (IGF-I) and insulin-like growth factor-2 (IGF-II) support erythroid colony formation in cultures supplemented with serum substitute and recombinant erythropoietin. Subpopulations of IGF-I- and IGF-II-dependent, erythropoietin-independent colony-forming unit-erythroid (CFU-E)-derived colonies and BFU-E-derived colonies were identified under serum-substituted conditions for adult bone-marrow-derived erythroid progenitors which proliferate in the absence and presence of exogenous anti-erythropoietin receptor monoclonal antibody and in serum-substituted medium that was preadsorbed with anti-erythropoietin IgG. To assess whether Raf-1 is required for the formation of IGF-dependent, erythropoietin-independent human erythroid colonies, 5-15 µM sense or antisense oligomer to raf-1 were added to serum-substituted cultures containing either 2 U/ml recombinant human erythropoietin (rHuEpo) alone or 0-1,000 ng/ml IGF-I or IGF-II with/without 2 U/ml rHuEpo. Both erythropoietin-induced and IGF-induced erythroid colony formation were completely blocked by antisense (but not sense) oligomers to raf-1. Purified human CFU-Es were examined for Raf-1 message and protein. Total RNA was extracted, and raf-1 mRNA was detected on Northern blots. Furthermore, a 74 kD protein, corresponding to Raf-1, was also detected in CFU-Es purified from human adult sources. Together, these studies support the hypothesis that the Raf-1 protein mediates both erythropoietin-induced and IGF-induced signal transduction in human erythroid progenitor cells.

	Introduction

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Recent studies implicate Raf-1, the protein product of the raf-1 proto-oncogene, in post-receptor signal transduction for a variety of growth factors whose receptors are members of both the tyrosine kinase receptor class, e.g., insulin [1, 2], platelet-derived growth factor (PDGF) [3], epidermal growth factor (EGF) [4], Steel factor [5], and colony-stimulating factor-1 (CSF-1) [6], as well as the hematopoietin receptor class, e.g., interleukins 2 and 3 (IL-2, IL-3) [7, 8], interleukin 4 (IL-4) [9], colony-stimulating factors [8, 9] and erythropoietin [10]. This 74 kD cytoplasmic serine/threonine kinase participates in a phosphorylation cascade involving several intermediate proteins and ultimately resulting in induction of nuclear proto-oncogenes (e.g., c-fos and c-jun) whose protein products increase DNA synthesis and enhance cellular proliferation.

Erythropoietin is the primary regulator of terminal differentiation of erythroid progenitor cells exerting maximum activity on the proliferation of colony-forming units-erythroid (CFU-Es) [11]. The Raf-1 protein has been implicated in mediating erythropoietin-induced, post-receptor signal transduction in studies limited to murine erythroid cell lines. Carroll et al. have reported that erythropoietin induces an increase in Raf-1 kinase activity in murine cells and that antisense oligomers to raf-1 inhibit erythropoietin and IL-3-induced DNA synthesis. These results correlated with antisense-mediated diminution of intracellular Raf-1 levels and were associated with phosphorylation of both serine and tyrosine residues [10].

Somatomedins, including insulin-like growth factor-1 (IGF-I) [12-14] and insulin-like growth factor-2 (IGF-II) [14-18], augment erythroid progenitor cell proliferation in the presence of erythropoietin. Concentration-dependent support of human CFU-E-derived colony proliferation under biochemically defined culture conditions has been well documented [14], and receptors for IGF-I have been identified in purified human CFU-Es [19]. Here we have identified an IGF-dependent population of erythroid progenitor cells that proliferates in the complete absence of erythropoietin. We have examined the pattern of raf-1 mRNA and protein production by human erythroid progenitors as well as the ability of antisense oligonucleotides to raf-1 to inhibit the proliferation of erythropoietin-dependent and IGF-dependent, erythropoietin-independent CFU-E-derived colony formation. Together, our results are consistent with the notion that Raf-1 mediates erythropoietin-induced and IGF-induced signal transduction in human erythroid progenitor cells.

	Materials and Methods

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Collection of Fetal, Neonatal and Adult Samples
Erythroid progenitor cells were purified from adult bone marrow of normal adult volunteers, neonatal umbilical cord blood obtained at full-term scheduled cesarean section or from fetal livers at 13-21 weeks' gestation obtained at therapeutic termination. Adult bone marrow cells of healthy, paid volunteer donors were obtained from aspirations of the posterior iliac crest and were immediately placed in Iscove's modified Dulbecco's medium (IMDM) containing 20 units preservative-free heparin per ml or from filters obtained from normal adult donor marrow harvests intended for transplantation.

Twenty-five to 75 ml of umbilical cord blood were drawn at delivery from the fetal side of the placenta of term, uncomplicated pregnancies. Kleihauer-Betke staining revealed less than 2.0% contamination with maternal red blood cells. The cord blood was placed immediately in IMDM with 20 units preservative-free sodium heparin per ml (GIBCO Laboratories; Grand Island, NY).

Fetal liver tissue was removed shortly after death and was gently teased apart using forceps into a solution of IMDM with 1 U/ml Dispase, 0.5 mg/ml collagenase (Type IV-S, Sigma; St. Louis, MO), 1% Fungizone, and 1% penicillin-streptomycin. The cell suspension was incubated for 30 min at 37°C with 5% CO₂ to allow for hepatic parenchymal digestion. All protocols were reviewed by the Institutional Review Committee at the University of Connecticut Health Center (adult/fetal tissue) or Hartford Hospital (neonatal blood) with informed consent obtained as required.

Progenitor Cell Processing and Enrichment
Mononuclear cells were separated twice using a Ficoll Paque density gradient (s.g. = 1.077) and washed with IMDM. Cells were suspended at a density of 5 x 10⁶/ml in IMDM with 20% fetal calf serum (FCS) and incubated overnight in plastic 75 cm² flasks at 37°C, 5% CO₂ to permit monocyte adherence. Nonadherent cells were decanted, and an additional monocyte depletion was carried out using the technique of Reisner et al. as follows [20]: The monocyte-depleted population was suspended in IMDM (2 x 10⁸/ml) and incubated with soybean agglutinin, 2 mg/ml (Vector Laboratories; Burlingame, CA.) for 10 min. This suspension was then layered on sterile 5% bovine serum albumin (BSA) at 25°C for 10 min. The interface, containing a cell population further depleted of T, B, myeloid, fibroblast, and stromal cells, was transferred to a new polypropylene tube and washed twice with 0.2 M D-galactose and phosphate-buffered saline (PBS). T lymphocytes were further depleted by sheep red blood cell rosetting using a 0.25% sheep red blood cell solution. The cell suspension was centrifuged at 800 RPM for 10 min, and the pellet was incubated with 75 µl AB serum, 4°C for 1 h. After disruption, the cell pellet was underlayered with 2 ml Ficoll-Paque and centrifuged at 2,000 RPM for 15 min. The interface layer, containing mononuclear cells enriched for progenitor cells through this series of cell depletions, was washed, counted, and suspended to the desired cell density.

Hematopoietic Cell Culture
For dose-response studies, adult bone-marrow-derived mononuclear cells were plated in 125-µl aliquots at densities of 5-6 x 10⁵/ml or as indicated in table legends, using modifications of a previously described serum-substituted fibrin clot technique [21]. Cultures contained IMDM supplemented with BSA Cohn Fraction V (Calbiochem; LaJolla, CA), transferrin, ferric chloride, and 2 U/ml recombinant human erythropoietin (rHuEpo) (Connaught Laboratory; Ontario, Canada; specific activity of [rHuEpo] 100,000 U/mg diluted to 20 U/mg with purified human albumin and freeze-dried). Albumin was adsorbed with activated charcoal prior to addition to culture [22] in order to remove growth factors and other peptides that bind to this protein. Levels of IGF-I and IGF-II are routinely less than 25 ng/ml, respectively, after adsorption of purified human serum albumin with activated charcoal. In some cases, murine anti-human erythropoietin receptor monoclonal antibody (kindly provided by Dr. A. D'Andrea, Dana Farber Cancer Institute; Boston, MA) or control mouse anti-human IgG was added to culture at a final concentration of 60 nmol/L. The antibody used is specific for erythropoietin receptor [23].

In other cases, serum-substituted medium was incubated with anti-erythropoietin monoclonal antibody [24] and freed of antigen-antibody complexes by addition of a fourfold excess of staphylococcal protein A (i.e., preadsorbed medium), as previously described [25]. Briefly, 200 mg IgG was incubated with a 10% Staph A solution (100 ml capable of binding 200 mg IgG) at 37°C for 45 min. Mixtures were centrifuged at 1,200 x g, washed three times and added to serum-substitute. Following incubation at 37°C for 30 min., the solid phase was pelleted by centrifugation.

After seven days of incubation, the cells were removed from fibrin clots, fixed with glutaraldehyde, and stained with benzidine and hematoxylin. CFU-E-derived colonies were recognized as clusters of 8-49 nucleated benzidine-staining cells. BFU-E-derived colonies appearing after 12 days of incubation contained 50 benzidine-positive cells.

For studies involving oligodeoxynucleotides, adult bone-marrow-derived mononuclear cells were incubated in serum-substituted fibrin clots prepared as above containing recombinant IGF-I (0-1,000 ng/ml) or IGF-II (0-1,000 ng/ml) (R & D; Minneapolis, MN) in the absence and presence of 2 U rHuEpo/ml. Fibrin clots containing erythropoietin, IGF-I, or IGF-II were established with cells pretreated for 12-14 h with sense, antisense or nonsense oligomers (5-15 µM). Cell viability by trypan blue dye exclusion was 95% after the overnight incubation with oligomers. In IGF studies, cultures were supplemented daily with oligomers. All studies were terminated after 7 or 12 days of incubation, 5% CO₂ at 37°C. Mean ± SE CFU-E-derived and BFU-E-derived colonies appearing in quadruplicate 125-µl clots were determined, and data sets were compared by analysis of variance, as previously described [26]. Colony counts are recorded based upon a final culture volume of 125 µl.

For cultures of enriched populations of CFU-Es from fetal, neonatal, and adult specimens, the depleted cell populations were suspended in 0.9% methylcellulose with 30% FCS, rHuEpo, 1% Fungizone, and 1% penicillin-streptomycin, and cultured in sterile, 12-well plates at 37°C with 5% CO₂ for seven days. Cells enriched in CFU-Es were then harvested by successive dilutions with IMDM. The cells were washed, counted, and diluted to appropriate density for use in RNA extraction and protein studies as specified below.

Cell Lines
ECO-NIH 3T3 cells (courtesy of Dr. Ulf Rapp, National Cancer Institute) were cultured in Dulbecco's minimal essential medium with 10% FCS, 5% CO₂ and grown to confluence prior to use as positive control cells that contain RNA for the Raf-1 protein.

Oligodeoxynucleotide Preparation
Sense (5'-ATGGAGCACATACAGGGA-3') and antisense (5'-TCCCTGTATGTGCTCCAT-3') oligodeoxyribonucleotides corresponding to codons 1-6 of murine c-raf or a randomly generated nonsense oligomer control (5'-TTTTTGCACCAGCTTGCC-3') with the same overall base composition as the antisense oligomer were prepared using an Applied Biosystems Model 392 DNA Synthesizer (Applied Biosystems, Inc.; Foster City, CA). Oligomers were purified by reverse phase chromatography using standard methods and dissolved to dryness in a Speed Vac Concentrator (Savant; Farmingdale, NY). The antisense oligomer specifically inhibits Raf-1 and has no effect upon the expression of Raf-A [9].

RNA Extraction
RNA was extracted from day 7 CFU-Es and ECO-NIH 3T3 cells with RNAzol (Tel-Test, Inc.; Friendswood, Texas) utilizing manufacturer's instructions. Samples were stored at –20°C until ready for use. For Northern analysis, 5-10 µg total RNA was electrophoresed on a 1% agarose gel with formaldehyde and standard saline citrate (SSC) buffer. RNA was transferred to Gene Screen Plus (NEN; Boston, MA), a nylon membrane using a positive pressure blot apparatus and immobilized by ultraviolet linkage. Greater than 75% transfer of RNA is achieved using this method. The Raf-1 cDNA (kindly provided by Dr. Ulf Rapp) and control actin cDNA were labeled with [³²P]dCTP using a random priming method as per the manufacturer's instructions (Boehringer-Mannheim; Mannheim, West Germany). The RNA blot was then prehybridized for three h at 42°C in SSC with deionized formamide, polyvinyl-pyrrolidine, Ficoll 400, BSA, SDS, glycine, sodium phosphate, and salmon sperm DNA. Hybridizations with labeled probe were carried out for 18-36 h at 45°C in prehybridization solution without glycine/with Dextran. The filter was then washed in SSC/SDS at 25°C and 45°C. Signals were quantified using a Betascope Model 603 blot analyzer and autoradiography at –70°C with Kodak X-OMAT x-ray film for one to two days.

Metabolic Labeling and Identification of Raf-1 Protein
Intact day 7 CFU-Es (~10⁷) were incubated in methionine-free medium with 5% dialyzed FCS using 5% CO₂ at 37°C for one h. Proteins were labeled by incubation with ³⁵S-methionine (0.5 mCi/ml; Trans ³⁵S Label, ICN; Costa Mesa, CA) at 37°C for three h. Cellular proteins were extracted on ice with SDS harvest buffer with phenylmethylsulfonyl fluoride and aprotinin. After boiling and attenuation of excess SDS with 1% Nonidet P-40, the cell lysate was centrifuged in a high-speed microfuge for five min at 4°C, and the supernatant, containing Raf-1 protein, was stored at –70°C until immunoprecipitation. Prior to immunoprecipitation, the cell lysate was precleared with a protein A-agarose bead/non-immune rabbit serum solution at 4°C for 20 min. The precleared cell lysate was immunoprecipitated with NH-7, a Raf-1 antipeptide antisera to residues 638-648 (kindly provided by Dr. Andrew Laudano, University of New Hampshire) [27] for two h at 4°C. Immunoprecipitated samples were loaded on a 9% SDS polyacrylamide gel and resolved by electrophoresis. The signal was visualized using autoradiography at –70°C with Kodak X-OMAT x-ray film for two to five days.

	Results

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IGFs Support Erythropoiesis in the Absence and Presence of Erythropoietin
When added to cultures of adult erythroid progenitors containing 2 U rHuEpo/ml, both IGF-I and IGF-II enhanced CFU-E-derived colony proliferation in a concentration-dependent fashion (Fig. 1). To determine whether IGF-specific induction of CFU-E differentiation in vitro can be identified, IGF-I or IGF-II was added to cultures prepared with serum substitute and no exogenous erythropoietin. Under these conditions, IGFs induced the formation of CFU-E-derived colonies whose cell number and intensity of benzidine staining were identical to those of erythropoietin-dependent colonies. As shown in Figure 1, the relative frequency of this class of progenitors is approximately 5%-20% of that represented by erythropoietin-dependent CFU-Es.

View larger version (14K): [in this window] [in a new window]   Figure 1. Support of CFU-E-derived colony proliferation by IGFs. Shown are mean ± SE erythroid colonies in 125 µl clots for quadruplicate cultures prepared in the absence and presence of 2.0 U rHuEpo/ml. Cultures were prepared with the indicated final concentration of IGF-I or IGF-II.

View larger version (51K): [in this window] [in a new window]   Figure 2. raf-1 mRNA expression in purified human CFU-Es. Equal amounts (5-10 µg) of total RNA from: lane 1: fetal-liver; lane 2: cord blood, and lane 3: adult bone-marrow-derived CFU-Es were loaded in each lane. Northern blot analysis was performed using a raf-1 32P labeled cDNA probe and autoradiography (top panel). The same membrane was stripped and probed with a 32P labeled cDNA probe for ß-actin (bottom panel).

View larger version (22K): [in this window] [in a new window]   Figure 3. Raf-1 protein in purified CFU-Es. Autoradiogram showing a 74 kD protein corresponding to Raf-1 which was immunoprecipitated using NH-7, a Raf-1 antipeptide antisera to residues 638-648, from ECO-NIH 3T3 cells (far left lane) and adult bone marrow-derived CFU-Es (middle lane). Lane 3 (far right lane) shows addition of excess Raf peptide with NH-7 to lysates of adult-derived CFU-Es preventing immunoprecipitation of the Raf-1 protein.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effects of antisense oligodeoxynucleotides on adult bone marrow erythroid colony formation. Results are the average of two experiments (CFU-Es) and three experiments (BFU-Es) for light density mononuclear cell cultures in serum-substituted medium containing rHuEpo and antisense oligomers to raf-1 at the indicated final concentrations. Values represent percentage of inhibition of colony formation compared to control cultures with rHuEpo alone.

View larger version (12K): [in this window] [in a new window]   Figure 5. Suppression of IGF-dependent, erythropoietin-independent CFU-E-derived colony proliferation by antisense oligomers to raf-1. Shown are mean ± SE CFU-E-derived colony formation in quadruplicate cultures prepared with IGF-I or IGF-II. Similar results were obtained in two additional experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Summary References

Mechanisms of signal transduction subsequent to growth factor binding at the cell surface receptor are under active investigation. An integral role has been shown for the Raf-1 protein in this signaling cascade, which results in the induction of nuclear transcription factors [1-10]. Stimulation of neonatal cardiac myocytes with IGF-I is followed by transient activation of Raf-1 [28]. Since Raf-1 does not associate directly with the cell surface receptor or immediate substrates of the plasma membrane, it is clear that there is a role for other signaling proteins such as those containing an Src homology-2 (SH2) domain [29]. Following insulin stimulation, receptor tyrosine kinase activity leads to tyrosine phosphorylation of the SH2 2 collagen-related protein (SHC) [29]. Tyrosine-phosphorylated SHC associates with the mammalian son-of-sevenless/growth factor receptor-binding protein 2 (SOS-GRB2) complex which causes activation of ras by exchange of GTP for GDP and ultimately results in coupling with Raf-1 [30]. IGF-I induced mitogenesis of fetal rat brown fat adipocytes results in tyrosine phosphorylation of SHC proteins as well as an increase in Raf kinase activity [31]. Using a Balb/c-derived cell line expressing a dominant negative mutant of Raf, Oliver et al. showed that IGF-I stimulation of MAP kinase was eliminated [32]. Of interest is that erythropoietin appears to activate Raf-1 via an SHC-independent pathway in a cell line that expresses the erythropoietin receptor, suggesting that SHC activation is not required for erythropoietin-dependent cell growth [33].

Because of the suggestion that Raf-1 is involved in post-receptor signal transduction in murine hematopoietic cell lines, we sought to better understand its role in growth-factor mediated signaling in primary human erythroid cells. Here, we show that both raf-1 message and Raf-1 protein are present in human CFU-Es (Figs. 2 and 3) and that addition of antisense oligomers to cultures of adult erythroid progenitor cells eliminates the appearance of erythroid colonies in the presence of erythropoietin or IGFs alone. Because neither sense nor nonsense oligomers have an effect on erythroid colony formation, nonspecific toxicity of raf-1 oligomers is unlikely. It is well known that Raf-1 is important for erythropoietin-induced erythroid differentiation [10, 33]. Our observation that Raf-1 function is also required for the proliferative effect of IGF in cultures containing anti-erythropoietin antibody and no exogenous erythropoietin raises the possibility that upregulation of Raf-1 may be important for erythroid differentiation per se. How changes in Raf-1 upregulation in the presence of both erythropoietin and IGFs result in enhanced erythroid colony formation remains unknown. One possibility is that distinct classes of erythroid progenitor cells are "recruited" to differentiate. Alternatively, erythropoietin and IGFs may affect substrates for Raf-1 or downstream regulatory proteins, such as SHC proteins. Accordingly, each class of growth factors may affect a distinct site in the signaling cascade of erythroid progenitor cells. Further investigations are required in order to dissect precise mechanisms by which erythropoietin and IGFs may interact to stimulate signal transduction.

Together, our results provide strong evidence that Raf-1 protein plays a role in both erythropoietin-induced and IGF-induced signal transduction in human erythroid progenitor cells. Additional studies are required to understand how IGF-induced and erythropoietin-induced signal transduction pathways are interrelated.

	Summary

Top Abstract Introduction Materials and Methods Results Discussion Summary References

 
Evidence exists to support the role of Raf-1 protein in somatomedin-induced and erythropoietin-mediated signal transduction in human erythroid progenitor cells. Both the message for Raf-1 and the protein itself have been demonstrated in purified human CFU-Es, and the suppression of erythropoietin-dependent and IGF-dependent, erythropoietin-independent CFU-E-derived colonies and erythroid bursts in the presence of antisense oligomers to raf-1 suggest a specific role of the protein in post-receptor signal transduction.

	Acknowledgments

 
The authors wish to thank the Genetics Division and staff of the postpartum nursing unit at the University of Connecticut Health Center whose cooperation made this study possible.

This work was supported in part by NIH grants #T32-HLO7324 to M.R.S. and DK31060 to N.D.

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Top Abstract Introduction Materials and Methods Results Discussion Summary References

 

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