Isolation and characterization of mutant cell lines defective in ...

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)

is thus formed, which initiates a downstream signaling cascade

To isolate and characterize effector moleABSTRACT cules of the transforming growth factor 13 (TGF.8) signaling pathway we have used a genetic approach involving the generation of stable recessive mutants, defective in their TGFf3 signaling, which can subsequently be functionally complemented to clone the affected genes. We have generated a cell line derived from a hypoxanthine-guanine phosphoribosyltransferase negative (HPRT-) HT1080 clone that contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to a TGFI8-responsive promoter. This cell line proliferates or dies in the appropriate selection medium in response to TGFI3. We have isolated three distinct TGFg3-unresponsive mutants following chemical mutagenesis. Somatic cell hybrids between pairs of individual TGF38unresponsive clones reveal that each is in a distinct complementation group. Each mutant clone retains all three TGFj3 receptors yet fails to induce a TGF13-inducible luciferase reporter construct or TGF,B-mediated plasminogen activator inhibitor-i (PAI-1) expression. Two of the three have an attenuated TGF.8-induced fibronectin response, whereas in the other mutant the fibronectin response is intact. These TGFf3-unresponsive cells should allow selection and identification of signaling molecules through functional complementation.

(10, 11).

Recent cloning of the receptors for the other members of the TGF superfamily reveals many interesting parallels with the TGF/3 system. For activin signaling, as for TGF,3 signaling, the type I receptor requires the type II receptor to bind ligand followed by the formation of a heteromeric complex that initiates signaling (12). The type I receptors for the bone morphogenetic proteins and the decapentaplegic gene product are capable of binding ligand on their own; however, it appears that signaling in these systems also involves complex formation between the type I and type II receptors (13-15). Despite much research, the mechanism of TGF,B signal transduction between the cell surface and the nucleus remains unclear. Therefore, we wished to establish a genetic system to directly identify these intracellular effectors of the TGFf3 signaling pathway. We have engineered a cell line derived from HPRT- HT1080 cells that contains the selectable marker Escherichia coli gpt linked to a TGF,B-responsive promoter. Mutagenesis followed by selection has allowed the identification of three stable recessive mutant cell lines. We report here on the isolation and characterization of these cells and demonstrate that they are deficient in distinct TGF,B signaling components that constitute separate complementation groups.

Transforming growth factor /3 (TGF,B) is a multifunctional cytokine that mediates a diversity of responses in many types of cells (1-4). TGFf3 is representative of the TGF gene superfamily whose members consist of structurally similar but functionally distinct regulatory proteins. Five different forms of TGF/3 have been cloned, designated TGF/3 1-5, which exhibit between 60 and 80% sequence homology (4). TGF,B1, a homodimer of 25 kDa, obtained its name for its ability to induce anchorage-independent growth in normal fibroblasts (5). TGFf3 stimulates a weak proliferative response in a few cell lines of mesenchymal origin; however, in nonmesenchymal cells it is actually the most potent polypeptide growth inhibitor identified (6). The other members of the TGF13 family consist of the mammalian activins and inhibins, Mullerian inhibiting substance, bone morphogenetic proteins, the Drosophila decapentaplegic gene complex, and the Xenopus Vgl protein (4). The transduction of the TGF,3 signal is initiated by its binding to three cell surface receptors, designated the TGFI types I, II, and III receptors. Recent cloning of these receptors has provided some insight as to how the TGFf3 signal is transmitted (for reviews see refs. 7-9). While the type III receptor contains no obvious signaling motif in its sequence, both the TGFI3 type I and II receptors contain serine/ threonine kinase domains. The most recent signaling model proposes that ligand binding to the type II receptor induces complex formation with the type I receptor, which results in trans-phosphorylation of the type I by the type II (10). An activated heteromeric complex between the two receptor types

MATERIALS AND METHODS Materials. Recombinant TGFf31 was purchased from Austral Biological (San Ramon, CA). Hygromycin B, geneticin (G418), puromycin, hypoxanthine/aminopterin/thymidine (HAT), hypoxanthine/thymidine (HT), and 6-thioguanine (6TG) supplements were all purchased from Sigma. Recombinant Plasmids. The vector p3TPLux was generously provided by Joan Massague and has been described (11, 16). To generate the vector p3TPgpt, the promoter region of the vector pSV2gpt (17, 18) was excised with PvuII and HindIII and was replaced by the entire promoter region from p3TPLux. which was amplified by PCR with PvuII/HindIII ends. pSV2hyg, pSV2neo, and pSV2puro vectors were as described (19-21). The expression vectors for the TGFf3 type I (ALK-5) and type II receptors (H2-3FF) were generously provided by Carl-Henrik Heldin and Harvey Lodish, respectively (22, 23). Cell Culture, DNA Transfection, Mutagenesis, and Selections. HPRT- HT1080 cells were cultured in DME/F12 media supplemented with 10% calf serum. To establish the cell BAHgpt line,-HPRT- HT1080 cells were cotransfected with p3TPgpt and pSV2hyg in a 10:1 ratio with 10 ,ug/ml polybrene as described (24). Stable transfectants were selected in media containing 100 ,ug/ml of hygromycin B. Individual hygromycin-resistant clones were isolated and tested for their ability to Abbreviations: TGF,B, transforming growth factor ,3; HAT, hypoxanthine/aminopterin/thymidine; 6TG, 6-thioguanine; PAI-1, plasminogen activated inhibitor-1. *To whom reprint requests should be addressed at: Department of Cell Biology, NC1, Cleveland Clinic Research Institute, Cleveland, OH 44195.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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grow in HAT media supplemented with 5 ng/ml TGFf3. Clones that grew in this media were subcultured in HT media, and then regular media, before being placed in media containing 30 ,uM 6TG. This cycle of media selection was repeated twice. Once established, the clones were maintained in hygromycincontaining media to increase the stability of the transfected DNA. To generate TGFI3-unresponsive mutants, BAHgpt cells approximately 80% confluent in 10 separate dishes (10 cm) were subjected to five rounds of chemical mutagenesis with 7 ,ug/ml of ICR-191 (Sigma), which resulted in cell kill of about 50%. After the last round of mutagenesis, cells were placed in media containing 30 ,uM of 6TG + 10 ng/ml of TGF,3. Individual colonies were ring-cloned and expanded. To generate somatic cell hybrids, subclonal cell lines of each mutant were established that carried the pSV2neo or pSV2puro plasmids. Cell fusions were performed as described (19). Selection in media containing 100 ,ug/ml hygromycin, 500 ,ug/ml geneticin, and 500 ng/ml puromycin was continued for 7-10 days before the hybrids were examined for TGF,Binduced responses. Transient Transfection and Luciferase Assays. For transient transfection assays, 2 x 105 cells were plated in 6-well plates. The next day, cells were cotransfected with 1 ,ug p3TPLux and 1 ,ug RSV,Bgal using 5 ,ul Lipofectamine (GIBCO/BRL) per well as per the manufacturer's protocol. Where indicated in the text, 1 ,ug of the expression plasmids harboring the TGF,B type I (ALK-5), type II (H2-3FF) receptors or blank vector (pcDNA3; Invitrogen) were included in the transfection. After 16-18 h of transfection, 2.5 ng/ml TGF,B was added to the cells for an additional 18 h. Cells were harvested and assayed for luciferase activity using a luciferase assay kit (Promega) as per manufacturer's protocol. Cell lysates were also assayed for ,B-galactosidase activity, which was used as an internal standard to normalize for transfection efficiencies. All determinations were performed in triplicate. Northern and Southern Blot Analysis. Total RNA was isolated by the guanidinium isothiocyanate method as described (25). Total RNA (20 ,ug) was electrophoresed in 1.2% agarose/formaldehyde gels and transferred to nitrocellulose. Prehybridization, hybridization, and washing conditions were as described (26). Southern blot analysis was carried out as described (24). Hybridization was carried out as for the Northern blots. The HindIII/EcoRV fragment excised from p3TPgpt was used to probe gpt expression and was randomlabeled with [32P]-a-dCTP using a Pharmacia labeling kit. Affinity Crosslinking of [ -251]-labeled TGFI81. Purified TGFo1 (R & D Systems) was iodinated by the chloramine-T method as described (27). Affinity labeling of confluent cell monolayers with [125I]TGFP3 and disuccinimidyl suberate (Pierce) was performed as described (28). Detergent-soluble affinity-crosslinked complexes were then resolved on 6% acrylamide gels and were visualized by autoradiography. Cell Labeling, Extracellular Matrix Preparation, and Immunoprecipitation. For the plasminogen activator inhibitor-1 (PAI-1) assay, 2 x 105 cells were plated in 6-well plates. The cells were treated with various doses of TGFJ3 for 2 h at which time [35S]methionine was added (40 ,uCi/ml; 1 Ci = 37 GBq) for an additional 2 h. Extracellular matrices were prepared exactly as described (12). The samples were analyzed by SDS/PAGE in 8% acrylamide gels, subjected to fluorography, and visualized by autoradiography. For the fibronectin assay, 4 x 105 cells were plated in 6-cm dishes. Cells were treated overnight with TGFf3 in methioninefree media. The next day [35S]methionine was added to 40 ,Ci/ml for an additional 4 h. The media was removed and subjected to immunoprecipitation with a monoclonal antihuman fibronectin antibody (clone I, 3E1; GIBCO/BRL). The

samples were analyzed on 6% acrylamide SDS/PAGE gels,

Proc. Natl. Acad. Sci. USA 93

(1996)

followed by fluorography, and visualization by autoradiography. TGFf3 growth inhibition assays and DNA synthesis assays were measured by [3H]thymidine incorporation into trichloroacetic acid-insoluble material as described (29).

RESULTS HT1080 Cells Mediate a Transcriptional Response to TGFf3. Chemical mutagenesis of mink lung epithelial (MvLu, CCL64) cells was been used previously to generate mutants in the TGFI3 pathway (28, 30). MvLu cells are exquisitely sensitive to growth inhibition by TGFf3, and the selection of mutant cells was based on the ability of these cells to continue to proliferate in the presence of TGF/3. However, complementation of these mutants is difficult because the complemented phenotype, growth inhibition, is a negative selection. We therefore chose an alternative approach to generate stable, recessive mutants in the TGFB signaling pathway involving the introduction of the selectable marker E. coli gpt into an HPRT- cell line (17, 18). The use of a selectable marker gene whose expression can be easily manipulated facilitates the selection of mutant cells no longer capable of gene induction, as well as the cloning of the affected gene in these mutants by functional complementation. Indeed, this approach has proven to be instrumental in elucidating the interferon signaling pathway (31). The HT1080 human fibrosarcoma cell line chosen for this study is nearly diploid and has been used successfully to generate mutants in interferon signaling. In addition, HT1080 cells are are not growth inhibited by TGFI3 as assessed by [3H]thymidine incorporation (Fig. 1D). TGFP at concentrations up to 10 ng/ml (400 pM) was without effect on the growth of the HT1080 cells (Fig. 1D). By contrast, the mink lung epithelial CCL64 cell line is potently inhibited by TGF,3 with an ID50 of approximately 0.1 ng/ml (4 pM; Fig. ID). To assess whether the HT1080 (HPRT-) cells were transcriptionally responsive to TGFf3, we employed a transient transfection assay with a TGFI3-responsive luciferase reporter construct p3TPLux (generously provided by Joan Massague; refs. 11 and 16). The promoter of this construct is comprised of three repeats of the TPA response element (TRE) from the human collagenase gene and approximately 100 bp of the PAI-1 promoter linked to a luciferase reporter (Fig. 1A). In MvLu cells this promoter construct has been shown to mediate high inducibility by TGF,B of the luciferase reporter and appears to be relatively specific to induction by TGFf3 (11, 16). Similarly, we found significant inducibility of this construct when transiently transfected into the MvLu cells as well as a comparable induction of luciferase activity in the HPRT- HT1080 cells of 26- and 17-fold, respectively (Fig. 1C). Therefore, it appears that although the growth of HT1080 cells is not affected by TGF,B, the pathways by which TGFf3 activates transcription are intact in these cells. Thus, we decided to utilize this promoter construct to drive expression of the gpt gene in our genetic selection strategy. Generation and Characterization of a TGFI8-Regulated Cell Line. To generate the TGFI,-regulated gpt construct, the promoter from the p3TPLux vector was amplified by PCR and ligated into the promoterless pSV2gpt vector to obtain p3TPgpt (Fig. 1B). The p3TPgpt construct was transfected into HPRTHT1080 cells as described in Materials and Methods. One TGF13-regulated clone was obtained, designated BAHgpt, which exhibits the appropriate growth characteristics in the four selection media: no growth in HAT alone, growth in HAT + TGF,B, growth in 6TG alone, and no growth in 6TG + TGF13. The BAHgpt clone has maintained its strict regulation of gpt expression over many passages demonstrating the suitability of this cell line for mutagenesis and subsequent complementation analysis.

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To further characterize the arrangement of the gpt transgene in the TGF,3-regulated BAHIKgpt cell line, genomic DNA isolated from the cells was subjected to Southern blot analysis (Fig. 2A). When EcoRI and HindIII, which cut the 3TPgpt vector once, are used to digest the genomic DNA of BAHgpt cells, a band of 5.4 kb is observed corresponding to the size of the linearized vector. In addition, when BamHI is used to digest the genomic DNA of BAHgpt cells, a band of the expected size of 1.8 kb is observed in both vector control and BAHgpt lanes. No signal is detected in lanes corresponding to genomic DNA isolated from parental HPRT- HT1080 cells, which do not contain the gpt gene. From this analysis wve conclude that multiple copies of the p3TPgpt vector are integrated in tandem, and in fact we estimate that 5-10 copies are present in the genomic DNA of the BAHgpt cell line. We next investigated the expression of gpt in the BAHgpt cells by Northern blot analysis (Fig. 2 B and C). No detectable gpt expression is observed in the absence of TGFf3; however, gpt expression is both dose- and time-dependent upon addition of TGF,3 (Fig. 2 B and C). Maximal gene expression is observed after 8 h of treatment with 2.5 ng/ml TGF,3. Isolation and Characterization of TGFf3 Mutants. To obtain mutants that no longer induce gpt expression in response

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FIG. 2. Characterization of the TGFf3-regulated cell line BAHgpt. (A) Genomic DNA (20 ,ug) from the BAIHVt cell line was isolated and subjected to Southern blot analysis after digestion with the indicated restriction enzymes and probed using a gpt probe. Lanes 1, vector DNA; lanes 2, BAHgpt DNA; lanes 3, parental HT1080 DNA. (B) Total RNA (20 ,ug) isolated from BAHgpt cells treated with the various doses of TGFf3 for 16 h was subjected to Northern blot analysis using agpt probe. (C) BAHgpt cells were treated with 2.5 ng/ml of TGF,B for the indicated times prior to isolation of total RNA for Northern blot analysis as in B.

to TGFI3, BAHgpt cells were subjected to five rounds of mutagenesis with the frameshift mutagen ICR-191 followed by selection in media containing 6TG + TGF,B. Multiple rounds of mutagenesis are needed to increase the frequency of obtaining an unresponsive mutant due to the fact that for most genes two alleles need to be inactivated. For the interferon system it was found that the frequency of obtaining an unresponsive mutant was about one in 106 following five rounds of mutagenesis (19), and we also observe approximately the same frequency. To identify if the mutation occurred in cis, affected in the transfected gene only, or in trans, affected in the TGFJ3 pathway, we screened the mutants obtained in a transient transfection assay using the p3TPLux reporter plasmid, which contains the same promoter construct that drives expression of the gpt gene in the parental BAHgpt cells. Of the 80 clones that grew. in 6TG + TGFI3, we obtained three mutants from three separate pools that were determined to be in trans,-based on their lack of ability to induce luciferase expression using the p3TPLux reporter (Fig. 3A). The parental cell line exhibits an average 10-fold induction of luciferase activity, whereas the mutants exhibit on the average less than 2-fold induction of luciferase activity (Fig. 3A). Most of the clones isolated were able to induce luciferase at levels comparable to the parental cell line and were thus concluded to have sustained mutations in cis. An examination of gpt gene expression by Northern blot analysis in the three trans mutants also revealed the inability of these cells to induce gpt expression following TGFI3 treatment (Fig. 3B).

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Binding of TGFf to its receptors initiates a signaling cascade that culminates in transcription of target genes in the nucleus. We therefore wished to assess whether the mutant cell lines we obtained were deficient in TGF,B signaling due to a mutation in the TGFf3 type I, II, or III receptors, which could lead either to a loss of expression or function of the receptors. To address this we performed receptor-affinity labeling of the cell lines with [125I]TGFO3 followed by crosslinking with disuccinimidyl suberate, which reveals three prominent species of 180, 95, and 72 kDa, corresponding to the type III, II, and I receptors, respectively (Fig. 4A). The well-characterized mink lung epithelial CCL64 cell receptor profile is shown for comparison. The BAHgpt cell line as well as the three mutant cell lines retain the same receptor profile as parental HT1080 cells. To determine whether the cell lines had sustained inactivating mutations to the intracellular domain of the receptors, the human TGFf type I and type II receptors were transiently transfected into each mutant cell line and restoration of TGF,B-mediated inducibility of the luciferase reporter construct was assessed. As shown in Fig. 4B, expression of normal type I and type II TGFf3 receptors fails to restore normal luciferase induction to any of the mutant cell lines. From these experiments we conclude that the mutant cell lines appear to possess normal TGF,3 receptors suggesting that the mutations sustained by these cells are likely to lie in the signaling pathway downstream of the receptors. Although TGF,B affects the expression of many genes, one of the most dramatic increases in gene transcription and protein expression is seen with PAI-1 (32). Examination of the [35S]-labeled proteins deposited to the extracellular matrix following stimulation with TGF,B reveals a prominent band of 50 kDa, which has been identified as PAI-1 (32). Stimulation of BAHgpt cells with TGF,B reveals a dose-dependent depo-

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3), mutant 1-3 (M 1-3) (lane 4), mutant 5-3 (M 5-3) (lane 5), and mutant 7-5 (M 7-5) (lane 6) cells were analyzed for expression of TGF,B receptors after exposure to [1251]TGFf3 and crosslinking with disuccinimidyl suberate as described. -(B) BAIHgpt and mutant cell lines were transiently transfected with either blank vector (pcDNA3), T,BI-R (ALK-5), or T,l3I-R (H2-3FF) along with p3TPLux as described. Luciferase activity was determined and results are expressed as fold-induction.

sition of PAI-1 to the extracellular matrix with maximal induction occurring at a dose of 5 ng/ml (Fig. SA). However, each of the three mutant cell lines displays a striking diminution of PAI-1 induction following TGF,B stimulation (Fig. SA). In HT1080 cells, TGFI3 also causes an increase in the production of the extracellular matrix glycoprotein fibronectin (33). When we investigated the secretion of fibronectin into the media triggered by TGFI3, we observed the expected increase in the BAHgpt cell line (Fig. 5B). Mutant 1-3 exhibited normal induction of fibronectin, whereas in mutant 5-3 the response was not as strong. Mutant 7-5 failed to induce fibronectin production after TGF,B treatment altogether. This is not due to a mutation in the fibronectin gene itself as dexamethasone, another agent known to induce fibronectin production (33), is capable of eliciting induction of fibronectin in the parental and all of the mutant cell lines (data not shown). These results indicate that the three TGF,B signaling mutants we have obtained are all deficient in TGFf3-stimulated PAI-1 production, although exhibiting differing degrees of TGF,3-mediated responsiveness in fibronectin production. Establishment of Complementation Groups Among TGFI8 Signaling Mutants. To determine the recessive or dominant nature of the mutations in each of the mutant cell lines, as well as to establish complementation groups among the mutants, we generated somatic cell hybrids between pairs of the individual cell lines. Because each of the cell lines already carries the gene for hygromycin resistance, we established a subclonal

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