Cloning and Functional Analysis of Erythropoietin-, Interleukin-3- and Thrombopoietin-Inducible Genes

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1 Cloning and Functional Analysis of Erythropoietin-, Interleukin-3- and Thrombopoietin-Inducible Genes Alan D. D Andrea, Yuan Zhu Division of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA Key Words. Hematopoiesis Cytokines Erythropoietin Thrornbopoietin Abstract. The receptor for thrombopoietin (TPO) is a member of the cytokine receptor superfamily. This superfamily also includes the receptors for erythropoietin (EPO), interleukin 3 (IL-3), GM-CSF and several other cytokines. Stimulation of cytokine receptors with their cognate ligands results in the activation of multiple signal transduction pathways and ultimately in the induction of new genes. The cloning of these specific genes provides one approach for analyzing cytokine-specific responses. In the current study, we have designed a strategy for isolating inducible genes. While the strategy has been used to identify EPO-specific and IL-3-specific inducible genes, the strategy can be extended to clone TPOinducible genes. Stem Cells 1996;14(supp11):82-87 Introduction Hematopoietic stem cells have the capacity to divide and differentiate into several different hematopoietic cell lineages [ 11. Hematopoietic growth factors (cytokines) promote both the growth and differentiation of stem cells and committed progenitor cells. Cytokines bind and activate discrete receptors expressed on the surface of stem cells, thereby influencing cell-fate decisions. Polypeptide receptors, such as the receptors for erythropoietin (EPO), interleukin 3 (IL-3) and thrombopoietin (TPO), are members of the cytokine receptor superfamily [2]. Following ligand stimulation, these receptors activate several downstream signal transduction pathways. For instance, all cytokine receptors activate the Ras/Raf/Map kinase signaling pathway 131 and the JAWSTAT signaling pathway Correspondence: Dr. Alan D. D Andrea, Dana- Farber Cancer Institute, Pediatric Oncology, 44 Binney Street, Boston, MA , USA. Accepted for publication July 22, OAlphaMed Press /96/$5.00/0 [4, 51. How these different mitogenic pathways contribute to cytokine-specific growth and differentiation remains unclear. The specificity of cytokines could, in principle, be determined by two alternative models. In the first model (stochastic model), a stem cell may initially differentiate by some random event into a committed progenitor cell, such as an erythroid progenitor. Stimulation of the EPO receptor (EPO-R) expressed in this progenitor may result in the activation of general mitogenic signals. However, because the cell is committed, these general biochemical signals will be transmitted as a lineage-specific differentiation response. According to the second model (instructive model), unique signaling pathways are found directly downstream of each cytokine receptor. In this case, a specific cytokine such as EPO drives the lineage into a specific differentiative pattern. The identification of differentiation-specific pathways downstream of cytokine receptors has been hampered by the absence of cell lines that respond to one growth factor or another. Recently, we have characterized one cell line, Ba/F3- EPO-R, that responds to two different growth factors [6]. Ba/F3-EPO-R cells proliferate in response to IL-3, but proliferate and differentiate into hemoglobin-bearing cells in response to EPO (Fig. 1). Presumably, the different biological responses to IL-3 and EPO are determined by the induction of different gene products. In the current study, we designed a strategy of differential display to clone cdnas that are specifically induced by either IL-3 or EPO [7,8]. We cloned several specific cdnas from Ba/F3-EPO-R that were induced as either immediate-early genes or delayed-response genes, in response to these cytokines. The identification of these cdnas supports a model of discrete signaling pathways downstream of each STEM CELLS 1996;14(~~ppl1):82-87

2 DAndrea, Zhu 83 Figure 1. Schematic representation of Ba/F3-EPO-R cells. BdF3-EPO-R cells have functional receptors for IL-3 and EPO. IL-3 promotes cellular proliferation. EPO promotes proliferation and partial erythroid differentiation 161. The cells were used for the cloning of IL-3- and EPO-inducible genes. cytokine receptor. Moreover, the 5 cis regulatory elements of these inducible genes should help identify signature DNA sequences upstream of cytokine-inducible genes, and identify novel transcriptional activators that bind and activate cytokine-specific gene expression. While the strategy described has been useful in identifying EPO and IL-3-specific genes, it will have general applicability to the study of other cytokines. Results Cloning of IL-3- and EPO-Inducible Genes We have utilized the strategy of differential display [7, 81 to isolate IL-3- and EPO-inducible genes from the Ba/F3-EPO-R cells. Initially, we isolated a partial cdna that was specifically induced by IL-3 but not by EPO (Fig. 2). For these studies, total RNA was isolated from Ba/F3 cells that were starved and restimulated with either murine IL-3 (Fig. 2A, lane 1) or recombinant human EPO (lanes 2, 3 and 4). A specific, partial cdna was identified in IL-3-stimulated cells but not in EPO-stimulated cells (lane 1). In order to confirm differential expression of the mrna, a Northern blot was performed using total RNA samples prepared from Ba/F3-EPO-R grown in the same conditions (Fig. 2B). A 3 kb mrna was identified that was specifically expressed in IL-3-stimulated cells (lane 1) but not in EPO-stimulated cells. The IL-3-inducible cdna was isolated by screening a cdna library prepared from IL-3-stimulated Ba/F3 cells (lanes 2, 3, and 4). We have subsequently identified this mrna as DUB-I, an IL-3-inducible immediate-early gene that encodes a growth regulatory deubiquitinating enzyme [9]. The isolated cdna was used to clone a full-length gene. In this way, we identified a specific cis-acting enhancer element that regulated the IL-3- inducible DUB-I expression [ 101. Interestingly, cells stimulated with EPO for longer periods of time expressed a larger mrna (5 kb) that cross-hybridized with the probe generated by differential display (Fig. 2C). The cdna containing the open reading frame of the DUB-I gene did not hybridize with this 5 kb mrna (data not shown), suggesting that the sequence homology between the IL-3-induced DUB-I mrna and the EPO-induced 5 kb mrna was restricted to the 3 untranslated regions. By screening a cdna library prepared from EPO-induced Ba/F3-EPO-R cells, a fulllength cdna encoding coproporphyrinogen oxidase (CO) was identified (data not shown). Interesting, CO has previously been identified as an erythroid-specific cdna that is upregulated in response to EPO-induced erythroid differentiation [ 111. The region of homology between the DUB-I cdna and the CO cdna is found to be an S1 repeat located in the 3 untranslated regions, as predicted by the hybridization data.

3 84 Cloning Cytokine-Inducible Genes Figure 2. Zdentification of DUB-1, an ZL3 inducible immediate-early gene with deubiquitinating activity. A) Total RNA from BdF3-EPO-R cells treated with IL-3 or EPO wus subjected to differential display analysis as described in the text [7, 81. A single band (indicated by arrow) was isolated and subcloned into pbluescript, The partial cdna was sequenced and shown to contain the polymerase chain reaction primer pair. B) The cloned partial cdna was labeled by the random primed labeling procedure and used to probe a Northern blot with the indicated BdF3-EPO-R total RNA. C) The same labeled cdna used in (B) was used to probe a Northern blot with RNA from (three-day) IL-3 or EPO-stimulated BdF3-EPO-R. IL-3 induced the DUB-I mrna as an immediate-early gene (lane I). EPO induced the mrna for CO [l I] us a delayed response gene. These two mrnas have homologous 3 untrunslated regions and therefore crossreact with the cdna probe. The DUB Gene Family: A Novel Family of Cytokine-Inducible Genes As discussed above, our differential display strategy initially led to the cloning of DUB-I, an IL-3-inducible cdna [9]. The mrna for DUB-I was induced by IL-3, followed by its rapid decline. More recently we have examined the effects of overexpressing DUB-1 in BalF3- EPO-R cells. Interestingly, DUB-1 overexpression resulted in BdF3 cell cycle arrest. Taken together, we conclude that DUB-I is a deubiquitinating enzyme that integrates IL-3R with cell cycle progression. Consistent with our results, recent evidence demonstrates that the ubiquitin-pathway is an important determinant of cell cycle progression [12]. The full-length DUB-I gene has also been cloned and analyzed for its 5 cis regulatory elements [lo]. A 5 enhancer region was identified that responds not only to IL-3R stimulation, but also to GM-CSF-R and IL-5R stimulation. The DUB-I gene is therefore specifically induced by receptors containing the pc-receptor subunit [13].

4 D Andrea, Zhu 85 Finally, the DUB-1 gene is a member of a gene family (the DUB gene family) that maps to murine chromosome 7. The DUB gene family contains four to six gene (or pseudogenes) members. Interestingly, another member of the DUB family, DUB-2, is specifically induced by IL-2 (unpublished data). Other members of this family may be induced by other hematopoietic growth factors. In summary, our strategy of differential display has led to: A) the cloning of a novel growth regulatory immediate-early gene (DUB-1); B) the identification of a novel cytokine-specific signaling pathway downstream of the pc subunit and upstream of the DUB-I enhancer; and C) the identification of a family of genes that is differently induced by specific cytokines. Other Cytokine-Specific Inducible Genes The strategy of differential display was next used to identify additional mrnas that are induced by either IL-3 or EPO (data not shown). Two partial cdnas were identified that are specifically induced in response to IL-3, and two partial cdnas were identified that are specifically induced in response to EPO. Characteristics of the IL-3- or EPO-induced cdna clones are summarized in Table 1. Some general conclusions can be drawn from these observations. First, IL-3 but not EPO induces the expression of G15. This mrna is highly related to a known interferon-inducible cdna [ 14, 151. These results suggest that members of this gene family are cytokine-inducible and may therefore play a regulatory role in cellular proliferation. Another IL-3 inducible clone (A9) encodes mast cell protease-5 (MP-5). MP-5 has previously been identified as a protein expressed in IL-3-dependent bone marrow mast cells [16]. The cloning of a known IL-3-inducible cdna in our strategy further confirms the fidelity of our system. Two EPO-inducible genes have also been isolated (A5 and G6). Clone A5 has sequence similarity to acetyl coenzyme A synthetase [ 171. Northern blot analysis of multiple hematopoietic cell lines suggests that the A5 mrna has preferential expression in cells with erythroid phenotype (data not shown). Clone G6 is an orphan cdna clone with no known homologies in the gene bank. The possible function of these cdnas in erythroid differentiation is unknown. This general approach of differential display has more recently been used to identify IL-3- and TPO-specific inducible genes. For this purpose, we have utilized the FDCP cell line. FDCP cells are dependent on murine IL-3 for growth. The cells have been transfected with the cdna encoding the TPO-R [ 181. The resulting FDCP- TPO-R cell line can be grown continuously in either murine IL-3 or murine TPO (Fig. 3). Removal of growth factor results in cell death. By using the approach of differential display, we have identified genes that are specifically induced by either IL-3 or TPO in the same cellular background. The analysis of these cytokine-inducible genes is now in progress. Conclusions We have used differential display to identify cytokine-inducible genes in hematopoietic cells. By using a cell line that responds to two discrete cytokines, IL-3 and EPO, we were able to identify cytokine-specific genes. Interestingly, the enhancers of these genes have provided us with tools for identifying cytokinespecific signaling pathways. Finally, the cloned cdnas can now be functionally assessed by their reintroduction into BdF3 cells using constitutive and inducible eukaryotic expression systems. In this way, we can demonstrate the role of the cytokine-inducible genes in cell Table 1. Example of cdnas that are selectively induced by either IL-3 or EPO in the same cell type - Clone mrna cdna Cloned Gene Identity Sequence Homology G15(IL-3) 1 kb 763 bp IFN-regulated gene family A9(IL-3) 0.9 kb 385 bp mast cell protease-5 T14(IL-3) 3 kb 2676 bp deubiquitinating enzyme family AS(EP0) 4kb 2439 bp acetyl-coenzyme A synthetase G6(EPO) 3 kb 800 bp unknown T14(EPO) 3.5 kb 1000 bp coproporphyrinogen oxidase

5 86 Cloning Cytokine-Inducible Genes n O W C 0.I CI 0 J 3 a: lz x 1 - \ FDCP-mpl - 1.o- 1 m w / FDCP I I I I I I TPO (unitdml) Figure 3. Growth characteristics of FDCP-mpl cells. FDCP cells growing in IL-3 were cotransfected with the cdna encoding the TPO-R (rnurine mpl) and the cdna for neomycin resistance. A neomycin resistant subclone was isolated by limiting dilution. The indicated cells were assayed for cytokine-dependent growth and viability by the XTTassay, as previously described [19]. While parental FDCP cells do not grow in TPO, FDCP-mpl cells grow in either TPO (as shown) or IL-3 (not shown). growth and differentiation. The induction of cytokine-specific genes presumably accounts, at least in part, for the specific biological function of different hematopoietic growth factors. Acknowledgments Supported by a grant from NIH (Award R01 DK43889) (A.D.D.). Y.Z. is a fellow of the Leukemia Society of America. A.D.D. is a scholar of the Leukemia Society of America. References 1 D Andrea AD. Cytokine receptors in congenital hematopoietic disease. N Engl J Med 1994;330: , 2 Cosman D, Lyman SD, Idzerda RL et al. A new cytokine receptor superfamily. Trends Biochem Sci 1990; Sato N, Sakamaki K, Terada N et al. Signal transduction by the high affinity GM-CSF receptor: two distinct cytoplasmic regions of the common p subunit responsible for differentiation. EMBO J 1993; 12: Darnel1 JE, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264: Ihle J, Witthuhn BA, Quelle FW et al. Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci 1994; 19: Carroll M, Zhu Y, D Andrea AD. Erythropoietininduced cellular differentiation requires prolongation of the G, phase of the cell cycle. Proc Natl Acad Sci USA 1995:92:

6 D Andrea, Zhu 87 7 Liang P, Pardee AB. Differential display of eukaryotic mrna by means of the polymerase chain reaction. Science 1992;257: Liang P, Averboukh L, Pardee AB. Distribution and cloning of eukaryotic mrnas by means of differential display: refinements and optimization. Nucleic Acids Res 1993;21: Zhu Y, Carroll M, Papa FR et al. DUB-1, a novel deubiquitinating enzyme with growthsuppressing activity. Proc Natl Acad Sci USA 1996;93: Zhu Y, Pless M, Inhorn R et al. The murine DUB-1 gene is specifically induced by the pc subunit of the interleukin-3 (IL-3) receptor. Mol Cell Biol 1996;16: Kohno H, Furukawa T, Yoshinaga T et al. Coproporphyrinogen oxidase. Purification, molecular cloning, and induction of mrna during erythroid differentiation. J Biol Chem 1993;268: Glotzer M, Murray AW, Kirschner MW. Cyclin is degraded by the ubiquitin pathway. Nature 1991 ;349: Nicola NA, Metcalf D. Subunit promiscuity among hemopoietic growth factor receptors. Cell 1991 ;67: Hayzer DJ, Brinson E, Runge MS. A rat p-interferon-induced mrna: sequence characterization. Gene 1992;117: Lewin AR, Reid LE, McMahon M et al. Molecular analysis of a human interferon-inducible gene family. Eur J Biochem 1991;199: McNeil HP, Austen KF, Somerville LL et al. Molecular cloning of the mouse mast cell protease-5 gene. J Biol Chem 1991;266: Priefert H, Steinbuchel A. Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoe) of Alcaligenes eutrophus. J Bacteriol 1992; 174: Lok S, Kaushansky K, Holly RD. Cloning and expression of murine thrombopoietin cdna and stimulation of platelet production in vivo. Nature 1994;369: Barber DL, D Andrea AD. Erythropoietin and interleukin-2 activate distinct JAK kinase family members. Mol Cell Biol 1994;14: