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Isolation and Characterization of Mutant Cell Lines Defective in Transforming Growth Factor (3 Signaling BARBARA A

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Isolation and Characterization of Mutant Cell Lines Defective in Transforming Growth Factor (3 Signaling BARBARA A Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7655-7660, July 1996 Cell Biology Isolation and characterization of mutant cell lines defective in transforming growth factor (3 signaling BARBARA A. HOCEVAR AND PHILIP H. HowE* Department of Cell Biology, Cleveland Clinic Research Institute, Cleveland, OH 44195 Communicated by George R Stark The Cleveland Clinic Foundation, Cleveland, OH, April 12, 1996 (received for review November 1, 1995) ABSTRACT To isolate and characterize effector mole- is thus formed, which initiates a downstream signaling cascade cules of the transforming growth factor 13 (TGF.8) signaling (10, 11). pathway we have used a genetic approach involving the Recent cloning of the receptors for the other members of the generation of stable recessive mutants, defective in their TGF superfamily reveals many interesting parallels with the TGFf3 signaling, which can subsequently be functionally com- TGF/3 system. For activin signaling, as for TGF,3 signaling, the plemented to clone the affected genes. We have generated a cell type I receptor requires the type II receptor to bind ligand line derived from a hypoxanthine-guanine phosphoribosyl- followed by the formation of a heteromeric complex that transferase negative (HPRT-) HT1080 clone that contains the initiates signaling (12). The type I receptors for the bone selectable marker Escherichia coli guanine phosphoribosyl- morphogenetic proteins and the decapentaplegic gene product transferase (gpt) linked to a TGFI8-responsive promoter. This are capable of binding ligand on their own; however, it appears cell line proliferates or dies in the appropriate selection that signaling in these systems also involves complex formation medium in response to TGFI3. We have isolated three distinct between the type I and type II receptors (13-15). TGFg3-unresponsive mutants following chemical mutagenesis. Despite much research, the mechanism of TGF,B signal Somatic cell hybrids between pairs of individual TGF38- transduction between the cell surface and the nucleus remains unresponsive clones reveal that each is in a distinct comple- unclear. Therefore, we wished to establish a genetic system to mentation group. Each mutant clone retains all three TGFj3 directly identify these intracellular effectors of the TGFf3 receptors yet fails to induce a TGF13-inducible luciferase signaling pathway. We have engineered a cell line derived from reporter construct or TGF,B-mediated plasminogen activator HPRT- HT1080 cells that contains the selectable marker inhibitor-i (PAI-1) expression. Two of the three have an Escherichia coli gpt linked to a TGF,B-responsive promoter. attenuated TGF.8-induced fibronectin response, whereas in Mutagenesis followed by selection has allowed the identifica- the other mutant the fibronectin response is intact. These tion of three stable recessive mutant cell lines. We report here TGFf3-unresponsive cells should allow selection and identifi- on the isolation and characterization of these cells and dem- cation of signaling molecules through functional complemen- onstrate that they are deficient in distinct TGF,B signaling tation. components that constitute separate complementation groups. Transforming growth factor /3 (TGF,B) is a multifunctional MATERIALS AND METHODS cytokine that mediates a diversity of responses in many types of cells (1-4). TGFf3 is representative of the TGF gene Materials. Recombinant TGFf31 was purchased from Aus- superfamily whose members consist of structurally similar but tral Biological (San Ramon, CA). Hygromycin B, geneticin functionally distinct regulatory proteins. Five different forms (G418), puromycin, hypoxanthine/aminopterin/thymidine of TGF/3 have been cloned, designated TGF/3 1-5, which (HAT), hypoxanthine/thymidine (HT), and 6-thioguanine exhibit between 60 and 80% sequence homology (4). TGF,B1, (6TG) supplements were all purchased from Sigma. a homodimer of 25 kDa, obtained its name for its ability to Recombinant Plasmids. The vector p3TPLux was gener- induce anchorage-independent growth in normal fibroblasts ously provided by Joan Massague and has been described (11, (5). TGFf3 stimulates a weak proliferative response in a few cell 16). To generate the vector p3TPgpt, the promoter region of lines of mesenchymal origin; however, in nonmesenchymal the vector pSV2gpt (17, 18) was excised with PvuII and HindIII cells it is actually the most potent polypeptide growth inhibitor and was replaced by the entire promoter region from identified (6). The other members of the TGF13 family consist p3TPLux. which was amplified by PCR with PvuII/HindIII of the mammalian activins and inhibins, Mullerian inhibiting ends. pSV2hyg, pSV2neo, and pSV2puro vectors were as de- substance, bone morphogenetic proteins, the Drosophila de- scribed (19-21). The expression vectors for the TGFf3 type I capentaplegic gene complex, and the Xenopus Vgl protein (4). (ALK-5) and type II receptors (H2-3FF) were generously The transduction of the TGF,3 signal is initiated by its provided by Carl-Henrik Heldin and Harvey Lodish, respec- binding to three cell surface receptors, designated the TGFI tively (22, 23). types I, II, and III receptors. Recent cloning of these receptors Cell Culture, DNA Transfection, Mutagenesis, and Selec- has provided some insight as to how the TGFf3 signal is tions. HPRT- HT1080 cells were cultured in DME/F12 media transmitted (for reviews see refs. 7-9). While the type III supplemented with 10% calf serum. To establish the cell receptor contains no obvious signaling motif in its sequence, BAHgpt line,-HPRT- HT1080 cells were cotransfected with both the TGFI3 type I and II receptors contain serine/ p3TPgpt and pSV2hyg in a 10:1 ratio with 10 ,ug/ml polybrene threonine kinase domains. The most recent signaling model as described (24). Stable transfectants were selected in media proposes that ligand binding to the type II receptor induces containing 100 ,ug/ml of hygromycin B. Individual hygromy- complex formation with the type I receptor, which results in cin-resistant clones were isolated and tested for their ability to trans-phosphorylation of the type I by the type II (10). An activated heteromeric complex between the two receptor types Abbreviations: TGF,B, transforming growth factor ,3; HAT, hypoxan- thine/aminopterin/thymidine; 6TG, 6-thioguanine; PAI-1, plasmino- gen activated inhibitor-1. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department ofCell payment. This article must therefore be hereby marked "advertisement" in Biology, NC1, Cleveland Clinic Research Institute, Cleveland, OH accordance with 18 U.S.C. §1734 solely to indicate this fact. 44195. 7655 Downloaded by guest on September 25, 2021 7656 Cell Biology: Hocevar and Howe Proc. Natl. Acad. Sci. USA 93 (1996) grow in HAT media supplemented with 5 ng/ml TGFf3. Clones followed by fluorography, and visualization by autoradiogra- that grew in this media were subcultured in HT media, and phy. then regular media, before being placed in media containing TGFf3 growth inhibition assays and DNA synthesis assays 30 ,uM 6TG. This cycle of media selection was repeated twice. were measured by [3H]thymidine incorporation into trichlo- Once established, the clones were maintained in hygromycin- roacetic acid-insoluble material as described (29). containing media to increase the stability of the transfected DNA. RESULTS To generate TGFI3-unresponsive mutants, BAHgpt cells approximately 80% confluent in 10 separate dishes (10 cm) HT1080 Cells Mediate a Transcriptional Response to were subjected to five rounds of chemical mutagenesis with 7 TGFf3. Chemical mutagenesis of mink lung epithelial (MvLu, ,ug/ml of ICR-191 (Sigma), which resulted in cell kill of about CCL64) cells was been used previously to generate mutants in 50%. After the last round of mutagenesis, cells were placed in the TGFI3 pathway (28, 30). MvLu cells are exquisitely sensi- media containing 30 ,uM of 6TG + 10 ng/ml of TGF,3. Indi- tive to growth inhibition by TGFf3, and the selection of mutant vidual colonies were ring-cloned and expanded. cells was based on the ability of these cells to continue to To generate somatic cell hybrids, subclonal cell lines of proliferate in the presence of TGF/3. However, complemen- each mutant were established that carried the pSV2neo or tation of these mutants is difficult because the complemented pSV2puro plasmids. Cell fusions were performed as described phenotype, growth inhibition, is a negative selection. We (19). Selection in media containing 100 ,ug/ml hygromycin, 500 therefore chose an alternative approach to generate stable, ,ug/ml geneticin, and 500 ng/ml puromycin was continued for recessive mutants in the TGFB signaling pathway involving the 7-10 days before the hybrids were examined for TGF,B- introduction of the selectable marker E. coli gpt into an induced responses. HPRT- cell line (17, 18). The use of a selectable marker gene Transient Transfection and Luciferase Assays. For transient whose expression can be easily manipulated facilitates the transfection assays, 2 x 105 cells were plated in 6-well plates. selection of mutant cells no longer capable of gene induction, The next day, cells were cotransfected with 1 ,ug p3TPLux and as well as the cloning of the affected gene in these mutants by 1 ,ug RSV,Bgal using 5 ,ul Lipofectamine (GIBCO/BRL) per functional complementation. Indeed, this approach has proven well as per the manufacturer's protocol. Where indicated in the to be instrumental in elucidating the interferon signaling text, 1 ,ug of the expression plasmids harboring the TGF,B type pathway (31). I (ALK-5), type II (H2-3FF) receptors or blank vector The HT1080 human fibrosarcoma cell line chosen for this (pcDNA3; Invitrogen) were included in the transfection. After study is nearly diploid and has been used successfully to 16-18 h of transfection, 2.5 ng/ml TGF,B was added to the cells generate mutants in interferon signaling. In addition, HT1080 for an additional 18 h. Cells were harvested and assayed for cells are are not growth inhibited by TGFI3 as assessed by luciferase activity using a luciferase assay kit (Promega) as per [3H]thymidine incorporation (Fig.
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