Title: A Somatic Cell Genetic System for Dissecting Hemopoietic Cytokine Signal Transduction
Abstract: Somatic cell genetics has proven to be a powerful tool for the dissection of cytokine signal transduction pathways. Here we describe a system in which interleukin-6 (IL-6) signaling may be dissected using myeloid leukemic M1 cells. We utilized two properties of M1 cell differentiation to isolate IL-6-unresponsive mutants. First, M1 differentiation is associated with cessation of cell division. Second, differentiated M1 cells migrate rapidly and form dispersed colonies in agar. Mutant clones that are unresponsive to IL-6 are, therefore, large and compact as compared with clones derived from IL-6-responsive wild type M1 cells. Following spontaneous or chemically induced mutagenesis and selection in a high dose of IL-6, we isolated 27 M1 clones unresponsive to IL-6. Three harbored mutations that acted in a dominant manner, whereas 24 contained recessive mutations. gp130, an IL-6 receptor component, was affected in many mutant clones. We show that these clones display IL-6 and leukemia inhibitory factor receptors with reduced binding affinities and express gp130 at reduced levels. The IL-6-unresponsive phenotype of these mutant clones was fully rescued by the transfection of exogenous gp130 DNA. Therefore, this approach targets components of the IL-6 signaling pathway and may be suitable to study signaling from a variety of cytokines. Somatic cell genetics has proven to be a powerful tool for the dissection of cytokine signal transduction pathways. Here we describe a system in which interleukin-6 (IL-6) signaling may be dissected using myeloid leukemic M1 cells. We utilized two properties of M1 cell differentiation to isolate IL-6-unresponsive mutants. First, M1 differentiation is associated with cessation of cell division. Second, differentiated M1 cells migrate rapidly and form dispersed colonies in agar. Mutant clones that are unresponsive to IL-6 are, therefore, large and compact as compared with clones derived from IL-6-responsive wild type M1 cells. Following spontaneous or chemically induced mutagenesis and selection in a high dose of IL-6, we isolated 27 M1 clones unresponsive to IL-6. Three harbored mutations that acted in a dominant manner, whereas 24 contained recessive mutations. gp130, an IL-6 receptor component, was affected in many mutant clones. We show that these clones display IL-6 and leukemia inhibitory factor receptors with reduced binding affinities and express gp130 at reduced levels. The IL-6-unresponsive phenotype of these mutant clones was fully rescued by the transfection of exogenous gp130 DNA. Therefore, this approach targets components of the IL-6 signaling pathway and may be suitable to study signaling from a variety of cytokines. interferon interleukin-6 leukemia inhibitory factor Janus tyrosine kinase signal transducers and activators of transcription granulocyte macrophage colony-stimulating factor granulocyte colony-stimulating factor oncostatin-M fetal calf serum phosphate-buffered saline Dulbecco's modified Eagle's medium Hank's balanced salt solution phosphoglycerokinase fluorescence-activated cell sorter Genetics has been successfully used to identify and analyze components of signal transduction pathways in a number of systems, including chemotaxis in bacteria (1Boyd A. Simon M. Annu. Rev Physiol. 1982; 44: 501-517Crossref PubMed Scopus (60) Google Scholar), the response to the mating pheromone in yeast (2Marsh L. Neiman A.M. Herskowitz I. Annu. Rev. Cell Biol. 1991; 7: 699-728Crossref PubMed Scopus (154) Google Scholar), the development of the Drosophila melanogaster visual system (3Simon M.A. Carthew R.W. Fortini M.E. Gaul U. Mardon G. Rubin G.M. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 375-380Crossref PubMed Scopus (15) Google Scholar) and nematode vulva (4Eisenmann D.M. Kim S.K. Curr. Opin. Genet. Dev. 1994; 4: 508-516Crossref PubMed Scopus (48) Google Scholar), and the increase in gene transcription in response to interferon (IFN)1 in mammalian somatic cells (5Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar).The study of interferon signaling is of particular interest because it demonstrates that mammalian somatic cells may be used for a genetic analysis of signal transduction. Genetic dissection of interferon signal transduction led to the demonstration that Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs) are key elements in the interferon signal transduction pathway (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar, 23Ichikawa Y. J. Cell. Physiol. 1969; 74: 223-234Crossref PubMed Scopus (417) Google Scholar). Subsequent biochemical studies have also implicated these molecules in the signal transduction pathways of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), LIF, oncostatin-M (OSM), and IL-6 (6Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar).Clearly, a somatic cell genetic system that would enable dissection of the signal transduction events controlling cytokine-induced hemopoietic differentiation would be valuable. The murine myeloid leukemic cell line M1 represents a useful model of normal macrophage differentiation and provides the starting point for establishing such a system. Like normal hemopoietic progenitor cells, M1 cells express the receptor tyrosine kinase flt3/flk2 (7Brasel K. Escobar S. Anderberg R. de Vries P. Gruss H.J. Lyman S.D. Leukemia (Baltimore). 1995; 9: 1212-1218PubMed Google Scholar), the cell surface marker CD34 (8Fackler M.J. Krause D.S. Smith O.M. Civin C.I. May W.S. Blood. 1995; 85: 3040-3047Crossref PubMed Google Scholar), and the transcription factors Scl (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar) and Myc (10Neckers L.M. Tsuda H. Weiss E. Pluznik D.H. J. Cell. Physiol. 1988; 135: 339-344Crossref PubMed Scopus (13) Google Scholar). Upon induction of differentiation by a range of cytokines including LIF (11Hilton D.J. Nicola N.A. Metcalf D. Anal. Biochem. 1988; 173: 359-367Crossref PubMed Scopus (121) Google Scholar), OSM (12Bruce A.G. Hoggatt I.H. Rose T.M. J. Immuno. 1992; 149: 1271-1275PubMed Google Scholar), IL-6 (13Lowe D.G. Nunes W. Bombara M. McCabe S. Ranges G.E. Henzel W. Tomida M. Yamamoto-Yamaguchi Y. Hozumi M. Goeddel D.V. DNA (N. Y.). 1989; 8: 351-359Crossref PubMed Scopus (77) Google Scholar), G-CSF (14Hilton D.J. Nicola N.A. Gough N.M. Metcalf D. J. Biol. Chem. 1988; 263: 9238-9243Abstract Full Text PDF PubMed Google Scholar), GM-CSF (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar, 15Smith A. Metcalf D. Nicola N.A. EMBO J. 1997; 16: 451-464Crossref PubMed Scopus (55) Google Scholar), IL-11 (16Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Harrison-Smith M. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO J. 1994; 13: 4765-4775Crossref PubMed Scopus (253) Google Scholar), and thrombopoietin (17Alexander W.S. Maurer A.B. Novak U. Harrison-Smith M. EMBO J. 1996; 15: 6531-6540Crossref PubMed Scopus (78) Google Scholar), these primitive markers are lost, and there is a concomitant increase in the expression of genes characteristic of mature macrophages. Among the proteins expressed by differentiating M1 cells are the transcription factors Myb (18Selvakumaran M. Liebermann D.A. Hoffman-Liebermann B. Mol. Cell. Biol. 1992; 12: 2493-2500Crossref PubMed Scopus (101) Google Scholar) and Fos (19Chiu C.P. Lee F. J. Immunol. 1989; 142: 1909-1915PubMed Google Scholar), Fcγ receptor types I and II (20Lotem J. Sachs L. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3507-3511Crossref PubMed Scopus (158) Google Scholar), the IL-4 receptor α-chain (21Feldman G.M. Ruhl S. Bickel M. Finbloom D.S. Pluznik D.H. Blood. 1991; 78: 1678-1684Crossref PubMed Google Scholar), the receptor for complement component C3b (20Lotem J. Sachs L. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3507-3511Crossref PubMed Scopus (158) Google Scholar), and lysozyme (22Krystosek A. Sachs L. Cell. 1976; 9: 675-684Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Additionally, like primary macrophages, differentiated M1 cells are vacuolated, phagocytic, capable of extensive movement (23Ichikawa Y. J. Cell. Physiol. 1969; 74: 223-234Crossref PubMed Scopus (417) Google Scholar), and dependent on macrophage colony-stimulating factor for survival (24Metcalf D. Leukemia (Baltimore). 1989; 3: 349-355PubMed Google Scholar).In the experiments described, we demonstrate that M1 cells provide the basis for a somatic cell genetic approach to dissect the signal transduction pathways used by cytokines to stimulate macrophage differentiation. Two spontaneous and 25 ICR-191-induced IL-6-unresponsive clones of M1 cells were selected in agar based on their ability to form tightly packed colonies, rather than small clusters of dispersed cells, in the presence of high concentrations of this cytokine. These lines were at least 2 orders of magnitude less responsive to IL-6 than wild type M1 cells. Somatic cell fusion experiments demonstrated that the majority of the mutations were recessive. Furthermore, among the recessive cell lines, one group appears to lack functional gp130, consistent with the important role that this receptor component plays in IL-6 signal transduction.DISCUSSIONSomatic cell genetics is a powerful tool by which cellular processes may be investigated. Previously, the genes required for interferon signal transduction have been investigated using a specially engineered fibroblast cell line capable of producing hypoxanthine-guanine phosphoribosyltransferase in response to type I and type II interferons (33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Following mutagenesis of this cell line, positive and negative selection allowed the identification of IFN-unresponsive mutants. In the first experiment in which complementation was successful, a cDNA library was transfected, and clones that regained responsiveness to IFN were isolated. Using this strategy, Tyk2, a previously isolated cytoplasmic tyrosine kinase and a member of the Janus kinase family, was shown to be essential for IFN signaling. In subsequent experiments, complementation of the defect was achieved using candidate genes, including the other members of the JAK family, the STATs, and receptor components (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar, 23Ichikawa Y. J. Cell. Physiol. 1969; 74: 223-234Crossref PubMed Scopus (417) Google Scholar, 33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).In this study, rather than producing a cell line in which the promoter of a cytokine-responsive gene drives expression of a selectable marker, we have taken advantage of an existing biological response, terminal macrophage differentiation, in M1 cells. As with the studies of Stark, Kerr and colleagues, we found that the spontaneous rate of cytokine unresponsiveness to be very low and have demonstrated that it was possible to dramatically elevate this rate following multiple rounds of treatment with the frameshift mutagen ICR-191. Consistent with the expected effects of ICR-191 in inducing loss of function mutations, most of the mutations in the IL-6-unresponsive lines arising from ICR-191 treatment behaved in a recessive manner. In contrast, the two spontaneous IL-6-unresponsive cell lines that were isolated in the initial optimization of the system and one of the mutants isolated following ICR-191 treatment contained mutations that acted in a dominant manner. These might be loss of function mutations that act in a dominant-negative manner, or alternatively, mutations that result in overproduction of a negative regulator, such as SOCS1 or SOCS3, which have been shown to be capable of inhibiting IL-6 signal transduction in M1 cells (17Alexander W.S. Maurer A.B. Novak U. Harrison-Smith M. EMBO J. 1996; 15: 6531-6540Crossref PubMed Scopus (78) Google Scholar).To demonstrate the utility of the M1 cell system for identifying genes important in cytokine signal transduction, we determined whether any of the mutant cell lines lacked the crucial IL-6 receptor component gp130. Together with the IL-6 receptor α-chain and the IL-6 ligand, gp130 forms part of the IL-6 receptor complex in which two gp130 molecules are required for activation of signaling (29Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1091) Google Scholar). Flow cytometry and Western blot analysis revealed that gp130 expression was reduced or undetectable in 14 out of 27 mutants (UR4, UR14, UR16, UR17, UR18, UR21, UR24, UR25, UR31, UR39, UR41, UR42, UR43, UR45). The transfection of wild type FLAG-tagged gp130 restored the IL-6-responses of these gp130-deficient cells; however, it should be stated that final proof that the causative mutation lies in gp130 will require sequencing of the gp130 gene in these cell lines. Indeed, mutations in other genes might indirectly be responsible for reduced expression of gp130, and it is conceivable that overexpression of gp130 might also compensate for such defects. For example, the IFN-unresponsive U1 cell line had severely impaired IFN-α binding, suggesting a reduced receptor number; however, the phenotype was complemented by overexpression of Tyk2, a downstream tyrosine kinase (30Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (315) Google Scholar, 31Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar). In studies of signal transduction in Caenorhabditis elegans, loss of function of the lin-2 gene resulted in abnormal vulval development but was found to encode a protein involved in localization of the LET-23 receptor (32Kim S.K. Curr. Opin. Cell Biol. 1995; 7: 641-649Crossref PubMed Scopus (99) Google Scholar).Since very few cell lines that lack gp130 are available, the gp130-deficient cell lines derived from these studies may also become valuable vehicles with which to study the structure and function of gp130. For example, the UR21 cell line has been used to examine various mutants of gp130 that either lacked the Ig domain or contained either the Ig domain from the GM-CSF receptor or the fibronectin type III repeats from the G-CSF receptor (33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 34Hammacher A. Wijdenes J. Hilton D.J. Nicola N.A. Simpson R.J. Layton J.E. Biochem. J. 2000; 345: 25-32Crossref PubMed Scopus (29) Google Scholar). In this way, one can examine the function of gp130 in a cell line in which it is normally expressed. This has an advantage over using a non-native cell line for studying cytokine signaling as these may not contain all the necessary components or the correct environment for signaling to occur.The IL-6-responsive cell lines expressing normal levels of gp130 may also prove to be of interest. We aim to determine how many complementation groups are represented among these cell lines by performing pairwise fusions and assessing the capacity of somatic cell hybrids to respond to IL-6. In parallel, we also aim to examine the expression of other proteins, including JAK1 and STAT3, known to be important in IL-6 signal transduction. Finally, where mutation in the genes of known components of the signal transduction pathway cannot explain the unresponsive phenotype, it may be possible to identify the defective gene by complementation with cDNAs from a library, as occurred with Tyk2 and IFN signal transduction (31Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar). Genetics has been successfully used to identify and analyze components of signal transduction pathways in a number of systems, including chemotaxis in bacteria (1Boyd A. Simon M. Annu. Rev Physiol. 1982; 44: 501-517Crossref PubMed Scopus (60) Google Scholar), the response to the mating pheromone in yeast (2Marsh L. Neiman A.M. Herskowitz I. Annu. Rev. Cell Biol. 1991; 7: 699-728Crossref PubMed Scopus (154) Google Scholar), the development of the Drosophila melanogaster visual system (3Simon M.A. Carthew R.W. Fortini M.E. Gaul U. Mardon G. Rubin G.M. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 375-380Crossref PubMed Scopus (15) Google Scholar) and nematode vulva (4Eisenmann D.M. Kim S.K. Curr. Opin. Genet. Dev. 1994; 4: 508-516Crossref PubMed Scopus (48) Google Scholar), and the increase in gene transcription in response to interferon (IFN)1 in mammalian somatic cells (5Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar). The study of interferon signaling is of particular interest because it demonstrates that mammalian somatic cells may be used for a genetic analysis of signal transduction. Genetic dissection of interferon signal transduction led to the demonstration that Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs) are key elements in the interferon signal transduction pathway (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar, 23Ichikawa Y. J. Cell. Physiol. 1969; 74: 223-234Crossref PubMed Scopus (417) Google Scholar). Subsequent biochemical studies have also implicated these molecules in the signal transduction pathways of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), LIF, oncostatin-M (OSM), and IL-6 (6Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar). Clearly, a somatic cell genetic system that would enable dissection of the signal transduction events controlling cytokine-induced hemopoietic differentiation would be valuable. The murine myeloid leukemic cell line M1 represents a useful model of normal macrophage differentiation and provides the starting point for establishing such a system. Like normal hemopoietic progenitor cells, M1 cells express the receptor tyrosine kinase flt3/flk2 (7Brasel K. Escobar S. Anderberg R. de Vries P. Gruss H.J. Lyman S.D. Leukemia (Baltimore). 1995; 9: 1212-1218PubMed Google Scholar), the cell surface marker CD34 (8Fackler M.J. Krause D.S. Smith O.M. Civin C.I. May W.S. Blood. 1995; 85: 3040-3047Crossref PubMed Google Scholar), and the transcription factors Scl (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar) and Myc (10Neckers L.M. Tsuda H. Weiss E. Pluznik D.H. J. Cell. Physiol. 1988; 135: 339-344Crossref PubMed Scopus (13) Google Scholar). Upon induction of differentiation by a range of cytokines including LIF (11Hilton D.J. Nicola N.A. Metcalf D. Anal. Biochem. 1988; 173: 359-367Crossref PubMed Scopus (121) Google Scholar), OSM (12Bruce A.G. Hoggatt I.H. Rose T.M. J. Immuno. 1992; 149: 1271-1275PubMed Google Scholar), IL-6 (13Lowe D.G. Nunes W. Bombara M. McCabe S. Ranges G.E. Henzel W. Tomida M. Yamamoto-Yamaguchi Y. Hozumi M. Goeddel D.V. DNA (N. Y.). 1989; 8: 351-359Crossref PubMed Scopus (77) Google Scholar), G-CSF (14Hilton D.J. Nicola N.A. Gough N.M. Metcalf D. J. Biol. Chem. 1988; 263: 9238-9243Abstract Full Text PDF PubMed Google Scholar), GM-CSF (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar, 15Smith A. Metcalf D. Nicola N.A. EMBO J. 1997; 16: 451-464Crossref PubMed Scopus (55) Google Scholar), IL-11 (16Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Harrison-Smith M. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO J. 1994; 13: 4765-4775Crossref PubMed Scopus (253) Google Scholar), and thrombopoietin (17Alexander W.S. Maurer A.B. Novak U. Harrison-Smith M. EMBO J. 1996; 15: 6531-6540Crossref PubMed Scopus (78) Google Scholar), these primitive markers are lost, and there is a concomitant increase in the expression of genes characteristic of mature macrophages. Among the proteins expressed by differentiating M1 cells are the transcription factors Myb (18Selvakumaran M. Liebermann D.A. Hoffman-Liebermann B. Mol. Cell. Biol. 1992; 12: 2493-2500Crossref PubMed Scopus (101) Google Scholar) and Fos (19Chiu C.P. Lee F. J. Immunol. 1989; 142: 1909-1915PubMed Google Scholar), Fcγ receptor types I and II (20Lotem J. Sachs L. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3507-3511Crossref PubMed Scopus (158) Google Scholar), the IL-4 receptor α-chain (21Feldman G.M. Ruhl S. Bickel M. Finbloom D.S. Pluznik D.H. Blood. 1991; 78: 1678-1684Crossref PubMed Google Scholar), the receptor for complement component C3b (20Lotem J. Sachs L. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3507-3511Crossref PubMed Scopus (158) Google Scholar), and lysozyme (22Krystosek A. Sachs L. Cell. 1976; 9: 675-684Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Additionally, like primary macrophages, differentiated M1 cells are vacuolated, phagocytic, capable of extensive movement (23Ichikawa Y. J. Cell. Physiol. 1969; 74: 223-234Crossref PubMed Scopus (417) Google Scholar), and dependent on macrophage colony-stimulating factor for survival (24Metcalf D. Leukemia (Baltimore). 1989; 3: 349-355PubMed Google Scholar). In the experiments described, we demonstrate that M1 cells provide the basis for a somatic cell genetic approach to dissect the signal transduction pathways used by cytokines to stimulate macrophage differentiation. Two spontaneous and 25 ICR-191-induced IL-6-unresponsive clones of M1 cells were selected in agar based on their ability to form tightly packed colonies, rather than small clusters of dispersed cells, in the presence of high concentrations of this cytokine. These lines were at least 2 orders of magnitude less responsive to IL-6 than wild type M1 cells. Somatic cell fusion experiments demonstrated that the majority of the mutations were recessive. Furthermore, among the recessive cell lines, one group appears to lack functional gp130, consistent with the important role that this receptor component plays in IL-6 signal transduction. DISCUSSIONSomatic cell genetics is a powerful tool by which cellular processes may be investigated. Previously, the genes required for interferon signal transduction have been investigated using a specially engineered fibroblast cell line capable of producing hypoxanthine-guanine phosphoribosyltransferase in response to type I and type II interferons (33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Following mutagenesis of this cell line, positive and negative selection allowed the identification of IFN-unresponsive mutants. In the first experiment in which complementation was successful, a cDNA library was transfected, and clones that regained responsiveness to IFN were isolated. Using this strategy, Tyk2, a previously isolated cytoplasmic tyrosine kinase and a member of the Janus kinase family, was shown to be essential for IFN signaling. In subsequent experiments, complementation of the defect was achieved using candidate genes, including the other members of the JAK family, the STATs, and receptor components (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar, 23Ichikawa Y. J. Cell. Physiol. 1969; 74: 223-234Crossref PubMed Scopus (417) Google Scholar, 33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).In this study, rather than producing a cell line in which the promoter of a cytokine-responsive gene drives expression of a selectable marker, we have taken advantage of an existing biological response, terminal macrophage differentiation, in M1 cells. As with the studies of Stark, Kerr and colleagues, we found that the spontaneous rate of cytokine unresponsiveness to be very low and have demonstrated that it was possible to dramatically elevate this rate following multiple rounds of treatment with the frameshift mutagen ICR-191. Consistent with the expected effects of ICR-191 in inducing loss of function mutations, most of the mutations in the IL-6-unresponsive lines arising from ICR-191 treatment behaved in a recessive manner. In contrast, the two spontaneous IL-6-unresponsive cell lines that were isolated in the initial optimization of the system and one of the mutants isolated following ICR-191 treatment contained mutations that acted in a dominant manner. These might be loss of function mutations that act in a dominant-negative manner, or alternatively, mutations that result in overproduction of a negative regulator, such as SOCS1 or SOCS3, which have been shown to be capable of inhibiting IL-6 signal transduction in M1 cells (17Alexander W.S. Maurer A.B. Novak U. Harrison-Smith M. EMBO J. 1996; 15: 6531-6540Crossref PubMed Scopus (78) Google Scholar).To demonstrate the utility of the M1 cell system for identifying genes important in cytokine signal transduction, we determined whether any of the mutant cell lines lacked the crucial IL-6 receptor component gp130. Together with the IL-6 receptor α-chain and the IL-6 ligand, gp130 forms part of the IL-6 receptor complex in which two gp130 molecules are required for activation of signaling (29Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1091) Google Scholar). Flow cytometry and Western blot analysis revealed that gp130 expression was reduced or undetectable in 14 out of 27 mutants (UR4, UR14, UR16, UR17, UR18, UR21, UR24, UR25, UR31, UR39, UR41, UR42, UR43, UR45). The transfection of wild type FLAG-tagged gp130 restored the IL-6-responses of these gp130-deficient cells; however, it should be stated that final proof that the causative mutation lies in gp130 will require sequencing of the gp130 gene in these cell lines. Indeed, mutations in other genes might indirectly be responsible for reduced expression of gp130, and it is conceivable that overexpression of gp130 might also compensate for such defects. For example, the IFN-unresponsive U1 cell line had severely impaired IFN-α binding, suggesting a reduced receptor number; however, the phenotype was complemented by overexpression of Tyk2, a downstream tyrosine kinase (30Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (315) Google Scholar, 31Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar). In studies of signal transduction in Caenorhabditis elegans, loss of function of the lin-2 gene resulted in abnormal vulval development but was found to encode a protein involved in localization of the LET-23 receptor (32Kim S.K. Curr. Opin. Cell Biol. 1995; 7: 641-649Crossref PubMed Scopus (99) Google Scholar).Since very few cell lines that lack gp130 are available, the gp130-deficient cell lines derived from these studies may also become valuable vehicles with which to study the structure and function of gp130. For example, the UR21 cell line has been used to examine various mutants of gp130 that either lacked the Ig domain or contained either the Ig domain from the GM-CSF receptor or the fibronectin type III repeats from the G-CSF receptor (33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 34Hammacher A. Wijdenes J. Hilton D.J. Nicola N.A. Simpson R.J. Layton J.E. Biochem. J. 2000; 345: 25-32Crossref PubMed Scopus (29) Google Scholar). In this way, one can examine the function of gp130 in a cell line in which it is normally expressed. This has an advantage over using a non-native cell line for studying cytokine signaling as these may not contain all the necessary components or the correct environment for signaling to occur.The IL-6-responsive cell lines expressing normal levels of gp130 may also prove to be of interest. We aim to determine how many complementation groups are represented among these cell lines by performing pairwise fusions and assessing the capacity of somatic cell hybrids to respond to IL-6. In parallel, we also aim to examine the expression of other proteins, including JAK1 and STAT3, known to be important in IL-6 signal transduction. Finally, where mutation in the genes of known components of the signal transduction pathway cannot explain the unresponsive phenotype, it may be possible to identify the defective gene by complementation with cDNAs from a library, as occurred with Tyk2 and IFN signal transduction (31Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar). Somatic cell genetics is a powerful tool by which cellular processes may be investigated. Previously, the genes required for interferon signal transduction have been investigated using a specially engineered fibroblast cell line capable of producing hypoxanthine-guanine phosphoribosyltransferase in response to type I and type II interferons (33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Following mutagenesis of this cell line, positive and negative selection allowed the identification of IFN-unresponsive mutants. In the first experiment in which complementation was successful, a cDNA library was transfected, and clones that regained responsiveness to IFN were isolated. Using this strategy, Tyk2, a previously isolated cytoplasmic tyrosine kinase and a member of the Janus kinase family, was shown to be essential for IFN signaling. In subsequent experiments, complementation of the defect was achieved using candidate genes, including the other members of the JAK family, the STATs, and receptor components (9Tanigawa T. Elwood N. Metcalf D. Cary D. DeLuca E. Nicola N.A. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7864-7868Crossref PubMed Scopus (54) Google Scholar, 23Ichikawa Y. J. Cell. Physiol. 1969; 74: 223-234Crossref PubMed Scopus (417) Google Scholar, 33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In this study, rather than producing a cell line in which the promoter of a cytokine-responsive gene drives expression of a selectable marker, we have taken advantage of an existing biological response, terminal macrophage differentiation, in M1 cells. As with the studies of Stark, Kerr and colleagues, we found that the spontaneous rate of cytokine unresponsiveness to be very low and have demonstrated that it was possible to dramatically elevate this rate following multiple rounds of treatment with the frameshift mutagen ICR-191. Consistent with the expected effects of ICR-191 in inducing loss of function mutations, most of the mutations in the IL-6-unresponsive lines arising from ICR-191 treatment behaved in a recessive manner. In contrast, the two spontaneous IL-6-unresponsive cell lines that were isolated in the initial optimization of the system and one of the mutants isolated following ICR-191 treatment contained mutations that acted in a dominant manner. These might be loss of function mutations that act in a dominant-negative manner, or alternatively, mutations that result in overproduction of a negative regulator, such as SOCS1 or SOCS3, which have been shown to be capable of inhibiting IL-6 signal transduction in M1 cells (17Alexander W.S. Maurer A.B. Novak U. Harrison-Smith M. EMBO J. 1996; 15: 6531-6540Crossref PubMed Scopus (78) Google Scholar). To demonstrate the utility of the M1 cell system for identifying genes important in cytokine signal transduction, we determined whether any of the mutant cell lines lacked the crucial IL-6 receptor component gp130. Together with the IL-6 receptor α-chain and the IL-6 ligand, gp130 forms part of the IL-6 receptor complex in which two gp130 molecules are required for activation of signaling (29Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1091) Google Scholar). Flow cytometry and Western blot analysis revealed that gp130 expression was reduced or undetectable in 14 out of 27 mutants (UR4, UR14, UR16, UR17, UR18, UR21, UR24, UR25, UR31, UR39, UR41, UR42, UR43, UR45). The transfection of wild type FLAG-tagged gp130 restored the IL-6-responses of these gp130-deficient cells; however, it should be stated that final proof that the causative mutation lies in gp130 will require sequencing of the gp130 gene in these cell lines. Indeed, mutations in other genes might indirectly be responsible for reduced expression of gp130, and it is conceivable that overexpression of gp130 might also compensate for such defects. For example, the IFN-unresponsive U1 cell line had severely impaired IFN-α binding, suggesting a reduced receptor number; however, the phenotype was complemented by overexpression of Tyk2, a downstream tyrosine kinase (30Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (315) Google Scholar, 31Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar). In studies of signal transduction in Caenorhabditis elegans, loss of function of the lin-2 gene resulted in abnormal vulval development but was found to encode a protein involved in localization of the LET-23 receptor (32Kim S.K. Curr. Opin. Cell Biol. 1995; 7: 641-649Crossref PubMed Scopus (99) Google Scholar). Since very few cell lines that lack gp130 are available, the gp130-deficient cell lines derived from these studies may also become valuable vehicles with which to study the structure and function of gp130. For example, the UR21 cell line has been used to examine various mutants of gp130 that either lacked the Ig domain or contained either the Ig domain from the GM-CSF receptor or the fibronectin type III repeats from the G-CSF receptor (33Hammacher A. Richardson R.T. Layton J.E. Smith D.K. Angus L.J. Hilton D.J. Nicola N.A. Wijdenes J. Simpson R.J. J. Biol. Chem. 1998; 273: 22701-22707Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 34Hammacher A. Wijdenes J. Hilton D.J. Nicola N.A. Simpson R.J. Layton J.E. Biochem. J. 2000; 345: 25-32Crossref PubMed Scopus (29) Google Scholar). In this way, one can examine the function of gp130 in a cell line in which it is normally expressed. This has an advantage over using a non-native cell line for studying cytokine signaling as these may not contain all the necessary components or the correct environment for signaling to occur. The IL-6-responsive cell lines expressing normal levels of gp130 may also prove to be of interest. We aim to determine how many complementation groups are represented among these cell lines by performing pairwise fusions and assessing the capacity of somatic cell hybrids to respond to IL-6. In parallel, we also aim to examine the expression of other proteins, including JAK1 and STAT3, known to be important in IL-6 signal transduction. Finally, where mutation in the genes of known components of the signal transduction pathway cannot explain the unresponsive phenotype, it may be possible to identify the defective gene by complementation with cDNAs from a library, as occurred with Tyk2 and IFN signal transduction (31Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar). We thank Ladina Di Rago, Sandra Mifsud, and Dale Cary for excellent technical assistance, and we thank Dr. Helene Martin for critically reading this manuscript.