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 described (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, hypoxanthine/aminopterin/thymidine; 6TG, 6-thioguanine; PAI-1, plasminogen 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. 1D). TGFP at concentra- manufacturer's protocol. Cell lysates were also assayed for tions up to 10 ng/ml (400 pM) was without effect on the growth ,B-galactosidase activity, which was used as an internal standard of the HT1080 cells (Fig. 1D). By contrast, the mink lung to epithelial CCL64 cell line is potently inhibited by TGF,3 with normalize for transfection efficiencies. All determinations an ID50 of approximately 0.1 ng/ml (4 pM; Fig. ID). To assess were performed in triplicate. whether the HT1080 (HPRT-) cells were transcriptionally Northern and Southern Blot Analysis. Total RNA was responsive to TGFf3, we employed a transient transfection isolated by the guanidinium isothiocyanate method as de- assay with a TGFI3-responsive luciferase reporter construct scribed (25). Total RNA (20 ,ug) was electrophoresed in 1.2% p3TPLux (generously provided by Joan Massague; refs. 11 and agarose/formaldehyde gels and transferred to nitrocellulose. 16). The promoter of this construct is comprised of three Prehybridization, hybridization, and washing conditions were repeats of the TPA response element (TRE) from the human as described (26). Southern blot analysis was carried out as collagenase gene and approximately 100 bp of the PAI-1 described (24). Hybridization was carried out as for the promoter linked to a luciferase reporter (Fig. 1A). In MvLu Northern blots. The HindIII/EcoRV fragment excised from cells this promoter construct has been shown to mediate high p3TPgpt was used to probe gpt expression and was random- inducibility by TGF,B of the luciferase reporter and appears to labeled with [32P]-a-dCTP using a Pharmacia labeling kit. be relatively specific to induction by TGFf3 (11, 16). Similarly, Affinity Crosslinking of [ -251]-labeled TGFI81. Purified we found significant inducibility of this construct when tran- TGFo1 (R & D Systems) was iodinated by the chloramine-T siently transfected into the MvLu cells as well as a comparable method as described (27). Affinity labeling of confluent cell induction of luciferase activity in the HPRT- HT1080 cells of monolayers with [125I]TGFP3 and disuccinimidyl suberate 26- and 17-fold, respectively (Fig. 1C). Therefore, it appears (Pierce) was performed as described (28). Detergent-soluble that although the growth of HT1080 cells is not affected by affinity-crosslinked complexes were then resolved on 6% TGF,B, the pathways bywhich TGFf3 activates transcription are acrylamide gels and were visualized by autoradiography. intact in these cells. Thus, we decided to utilize this promoter Cell Labeling, Extracellular Matrix Preparation, and Im- construct to drive expression of the gpt gene in our genetic munoprecipitation. For the plasminogen activator inhibitor-1 selection strategy. (PAI-1) assay, 2 x 105 cells were plated in 6-well plates. The Generation and Characterization ofa TGFI8-Regulated Cell cells were treated with various doses of TGFJ3 for 2 h at which Line. To generate the TGFI,-regulated gpt construct, the time [35S]methionine was added (40 ,uCi/ml; 1 Ci = 37 GBq) promoter from the p3TPLux vector was amplified by PCR and for an additional 2 h. Extracellular matrices were prepared ligated into the promoterless pSV2gpt vector to obtain p3TPgpt exactly as described (12). The samples were analyzed by (Fig. 1B). The p3TPgpt construct was transfected into HPRT- SDS/PAGE in 8% acrylamide gels, subjected to fluorography, HT1080 cells as described in Materials and Methods. One and visualized by autoradiography. TGF13-regulated clone was obtained, designated BAHgpt, For the fibronectin assay, 4 x 105 cells were plated in 6-cm which exhibits the appropriate growth characteristics in the dishes. Cells were treated overnight with TGFf3 in methionine- four selection media: no growth in HAT alone, growth in HAT free media. The next day [35S]methionine was added to 40 + TGF,B, growth in 6TG alone, and no growth in 6TG + ,Ci/ml for an additional 4 h. The media was removed and TGF13. The BAHgpt clone has maintained its strict regulation subjected to immunoprecipitation with a monoclonal anti- of gpt expression over many passages demonstrating the suit- human fibronectin antibody (clone I, 3E1; GIBCO/BRL). The ability of this cell line for mutagenesis and subsequent comple- samples were analyzed on 6% acrylamide SDS/PAGE gels, mentation analysis. Downloaded by guest on September 25, 2021 Cell Biology: Hocevar and Howe Proc. NatL. Acad. Sci. USA 93 (1996) 7657

(TEE)3 PAI-1 E4 A EcoRI HindIII BamHI 1 2 3 A 14- 1.'-*1'-* HId iJ 1 2 3 1 2 3 kb (TRE)3 PMI-1 E4 23- B I-4* -10-I--- gpt I IEH -{ 9.4- C 6.7- 4.4- ;. iE,,. 2.3- I...... ;. .AL:...... '-': .. 2.0- # .... w .. ,. b si ... I -TGF +TGFp -TGF3 +TGFp B cT1080 cells D - 18s 0 0.o 1.0 2.5 5.0 TGF3 (ng/ml) C

.. -18S 0 4 8 16 24 Time (hours) 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 0.04 0.2 0.4 2.0 4.0 20 40 200 400 DNA; lanes 2, BAHgpt DNA; lanes 3, parental HT1080 DNA. (B) [TGFI (pM) 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 FIG. 1. HT1080 cells mediate a transcriptional response to TGF,B. agpt probe. (C) BAHgpt cells were treated with 2.5 ng/ml of TGF,B for Diagrammatic representation of the p3TPLux reporter plasmid (A) the indicated times prior to isolation of total RNA for Northern blot and the p3TPgpt plasmid (B). (C) MvLu and HT1080 cells were analysis as in B. cotransfected with 1 jig ofp3TPLux and pRSV,Bgal as described. After an 18-h exposure to 2.5 ng/ml TGF,3, cells were harvested and assayed to TGFI3, BAHgpt cells were subjected to five rounds of for luciferase activity using a luciferase assay kit (Promega). Light mutagenesis with the frameshift mutagen ICR-191 followed by emission is expressed as relative luminometer units. (D) TGF3-growth inhibition assay with MvLu and HT1080 cells as measured by [3H]thy- selection in media containing 6TG + TGF,B. Multiple rounds midine incorporation after exposure ofcells to various doses of TGFf3. of mutagenesis are needed to increase the frequency of Assay was performed as described in Materials and Methods. obtaining an unresponsive mutant due to the fact that for most genes two alleles need to be inactivated. For the interferon To further characterize the arrangement of thegpt transgene system it was found that the frequency of obtaining an in the TGF,3-regulated BAHIKgpt cell line, genomic DNA iso- unresponsive mutant was about one in 106 following five lated from the cells was subjected to Southern blot analysis rounds of mutagenesis (19), and we also observe approxi- (Fig. 2A). When EcoRI and HindIII, which cut the 3TPgpt mately the same frequency. To identify if the mutation oc- vector once, are used to digest the genomic DNA of BAHgpt curred in cis, affected in the transfected gene only, or in trans, cells, a band of 5.4 kb is observed corresponding to the size of affected in the TGFJ3 pathway, we screened the mutants the linearized vector. In addition, when BamHI is used to obtained in a transient transfection assay using the p3TPLux digest the genomic DNA of BAHgpt cells, a band of the reporter plasmid, which contains the same promoter construct expected size of 1.8 kb is observed in both vector control and that drives expression of the gpt gene in the parental BAHgpt BAHgpt lanes. No signal is detected in lanes corresponding to + we obtained genomic DNA isolated from parental HPRT- HT1080 cells, cells. Of the 80 clones that grew. in 6TG TGFI3, which do not contain the gpt gene. From this analysis wve three mutants from three separate pools that were determined conclude that multiple copies of the p3TPgpt vector are to be in trans,-based on their lack of ability to induce luciferase integrated in tandem, and in fact we estimate that 5-10 copies expression using the p3TPLux reporter (Fig. 3A). The parental are present in the genomic DNA of the BAHgpt cell line. cell line exhibits an average 10-fold induction of luciferase We next investigated the expression of gpt in the BAHgpt activity, whereas the mutants exhibit on the average less than cells by Northern blot analysis (Fig. 2 B and C). No detectable 2-fold induction of luciferase activity (Fig. 3A). Most of the gpt expression is observed in the absence of TGFf3; however, clones isolated were able to induce luciferase at levels com- gpt expression is both dose- and time-dependent upon addition parable to the parental cell line and were thus concluded to of TGF,3 (Fig. 2 B and C). Maximal gene expression is have sustained mutations in cis. An examination of gpt gene observed after 8 h of treatment with 2.5 ng/ml TGF,3. expression by Northern blot analysis in the three trans mutants Isolation and Characterization of TGFf3 Mutants. To ob- also revealed the inability of these cells to induce gpt expression tain mutants that no longer induce gpt expression in response following TGFI3 treatment (Fig. 3B). Downloaded by guest on September 25, 2021 7658 Cell Biology: Hocevar and Howe Proc. Natl. Acad. Sci. USA 93 (1996)

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FIG. 3. Identification of three TGF,B signaling mutants. (A) Pa- 2 rental BAI-Igpt cells and three mutant cell lines were subjected to transient transfection assay using the TGFI3-responsivemill..-. .. reporter plasmid p3TPLux as described.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~,Luciferase activity is shown as fold- BAHgpt M 1-3 M5-3 M 7-5 induction and values plotted represent the average of three assays performed on separate days. (B) Total RNA was isolated from FIG. 4. TGF,B signaling mutants possess normal type I, II, and III BAHgpt and mutant cell lines after treatment with 2.5 ng/ml of TGF,B TGFf3 receptors. (A) MvLu (lane 1), HT1080 (lane 2), BAHgpt(lane for 16 h. Total RNA (20 ,ug) was subjected to Northern blot analysis 3), mutant 1-3 (M 1-3) (lane 4), mutant 5-3 (M 5-3) (lane 5), and and probed for gpt expression. 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 disuc- Binding ofTGFf to its receptors initiates a signaling cascade cinimidyl suberate as described. -(B) BAIHgpt and mutant cell lines that culminates in transcription of target genes in the nucleus. were transiently transfected with either blank vector (pcDNA3), We therefore wished to assess whether the mutant cell lines we T,BI-R (ALK-5), or T,l3I-R (H2-3FF) along with p3TPLux as de- obtained were deficient in TGF,B signaling due to a mutation scribed. Luciferase activity was determined and results are expressed in the TGFf3 type I, II, or III receptors, which could lead either as fold-induction. to a loss of expression or function of the receptors. To address this we performed receptor-affinity labeling of the cell lines sition of PAI-1 to the extracellular matrix with maximal with [125I]TGFO3 followed by crosslinking with disuccinimidyl induction occurring at a dose of 5 ng/ml (Fig. SA). However, suberate, which reveals three prominent species of 180, 95, and each of the three mutant cell lines displays a striking diminu- 72 kDa, corresponding to the type III, II, and I receptors, tion of PAI-1 induction following TGF,B stimulation (Fig. SA). respectively (Fig. 4A). The well-characterized mink lung epi- In HT1080 cells, TGFI3 also causes an increase in the produc- thelial CCL64 cell receptor profile is shown for comparison. tion of the extracellular matrix glycoprotein fibronectin (33). The BAHgpt cell line as well as the three mutant cell lines When we investigated the secretion of fibronectin into the retain the same receptor profile as parental HT1080 cells. To media triggered by TGFI3, we observed the expected increase determine whether the cell lines had sustained inactivating in the BAHgpt cell line (Fig. 5B). Mutant 1-3 exhibited normal mutations to the intracellular domain of the receptors, the induction of fibronectin, whereas in mutant 5-3 the response human TGFf type I and type II receptors were transiently was not as strong. Mutant 7-5 failed to induce fibronectin transfected into each mutant cell line and restoration of production after TGF,B treatment altogether. This is not due TGF,B-mediated inducibility of the luciferase reporter con- to a mutation in the fibronectin gene itself as dexamethasone, struct was assessed. As shown in Fig. 4B, expression of normal another agent known to induce fibronectin production (33), is type I and type II TGFf3 receptors fails to restore normal capable of eliciting induction of fibronectin in the parental and luciferase induction to any of the mutant cell lines. From these all of the mutant cell lines (data not shown). These results experiments we conclude that the mutant cell lines appear to indicate that the three TGF,B signaling mutants we have possess normal TGF,3 receptors suggesting that the mutations obtained are all deficient in TGFf3-stimulated PAI-1 produc- sustained by these cells are likely to lie in the signaling pathway tion, although exhibiting differing degrees of TGF,3-mediated downstream of the receptors. responsiveness in fibronectin production. Although TGF,B affects the expression of many genes, one Establishment of Complementation Groups Among TGFI8 of the most dramatic increases in gene transcription and Signaling Mutants. To determine the recessive or dominant protein expression is seen with PAI-1 (32). Examination of the nature of the mutations in each of the mutant cell lines, as well [35S]-labeled proteins deposited to the extracellular matrix as to establish complementation groups among the mutants, following stimulation with TGF,B reveals a prominent band of we generated somatic cell hybrids between pairs of the indi- 50 kDa, which has been identified as PAI-1 (32). Stimulation vidual cell lines. Because each of the cell lines already carries of BAHgpt cells with TGF,B reveals a dose-dependent depo- the gene for hygromycin resistance, we established a subclonal Downloaded by guest on September 25, 2021 Cell Biology: Hocevar and Howe Proc. Natl. Acad. Sci. USA 93 (1996) 7659 TGFP, (ng/nil) A A o0 0.01 0.10.5 1.0 5.0 8 ------_ 7 BAUI/gpt t-A -<-- PAI-i

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FIG. 6. The three TGF,3 signaling mutants constitute three sepa- B rate complementation groups. (A) BAHgpt cells and heterokaryons derived from the three mutants were subjected to transient transfection assay with the p3TPLux reporter plasmid. Results are expressed < Fn oW~~~o. 00 as fold-induction of luciferase activity from a representative experi- L ment. (B) PAI-1 induction following TGF,B stimulation with varying doses was assayed in the heterokaryons as in Fig. 5. PAI-1 protein is designated by the arrow.

TGF- + + + + the mode of transduction of the TGF,B signal, they have failed to definitively identify the effectors of the TGFI3 transduction pathway. BAHIgpt M1-3 M5-3 M7-5 We decided to address this issue using a genetic approach to FIG. 5. TGFI3-responsiveness is altered in the three signaling generate stable mutants in the TGF,f signaling pathway from which the wild-type version of the affected gene could be mutants. (A) BAHgpt cells and mutants were examined for [35S]-PAI-1 deposited to the extracellular matrix following treatment with varying cloned by functional complementation. This was achieved by doses of TGF,B for 4 h, as described. PAI-1 is indicated by arrows. (B) the generation of a cell line, BAHlpt, where the selectable [35S]fibronectin secretion into the media was assayed by immunopre- marker E. coli gpt is under control of a tightly regulated cipitation with a monoclonal anti-fibronectin antibody after stimula- TGFI3-responsive promoter. This allows forward selection of tion of the indicated cell lines for 24 h with 2 ng/ml of TGF,B. clones expressing gpt in HAT + TGFI3, while allowing back Fibronectin is indicated by the arrow. selection in 6TG + TGF,B. Because the growth of HT1080 human fibrosarcoma cells is cell line for each that carries either the pSV2neo or pSV2puro not affected by TGF,B, the question arises whether HT1080 which confers neomycin and puromycin resistance plasmids, cells still maintain an intact TGF,B signaling pathway. We first Heterokaryons were then generated and selected respectively. examined this by performing a transient transfection assay with as described in Materials and Methods. Luciferase induction, the TGF,B-responsive reporter plasmid, p3TPLux, which pro- assayed by transient transfection assays with the reporter vided evidence that HT1080 cells can indeed respond to TGF,B p3TPLux, is restored to a normal level in each of the hybrids (Fig. 1C). Induction of endogenous TGFf-responsive genes, generated between mutant cell lines, but not in self-fusions of such as PAI-1 and fibronectin, also occurs in HT1080 cells as individual mutants (Fig. 6A). In addition, the hybrids gener- well as our established BAHgpt cells. This is perhaps not a ated between the cell lines exhibit a normal dose-dependent surprising finding since it has been previously shown that the induction of PAI-1 production after stimulation with TGFj3 introduction of the simian virus 40 large T-antigen into CCL64 (Fig. 6B). Together these results demonstrate the recessive cells, while relieving the cells of their growth inhibition by nature of the mutations sustained in each of the unresponsive TGFO3, did not affect their induction of PAI-1, fibronectin, or cell lines and allow us to conclude that each mutant appears to junB triggered by TGFj3 (36). However, whether growth represent a separate complementation group. inhibition and other TGFB3 responses are mediated through the same signaling molecules remains to be determined. We DISCUSSION believe we can begin to address this question with the subsequent identification of the gene defect in each of the TGFf3 The transduction pathway for TGF,3 remains unclear despite mutants we have generated. All three of the mutants exhibit much research over the past several years. Previous studies altered PAI-1 expression after TGF,3 treatment, which is have utilized biochemical approaches to study TGFf3 signaling, restored upon fusion of individual lines with one another. It most of which have employed specific inhibitors of known has previously been demonstrated that multiple TGFI3- signaling pathways such as staurosporine, H7, pertussis toxin, inducible elements regulate expression of PAI-1 (37). Whether etc., to implicate molecules in the signaling cascade (28, 34, these mutants are defective in regulators of these transcrip- 35). Although these studies have provided some insight into tional elements or in other TGFO3-signaling mediators remains Downloaded by guest on September 25, 2021 7660 Cell Biology: Hocevar and Howe Proc. Natl. Acad. Sci. USA 93 (1996) to be established. In addition, two of the three clones exhibit 15. Ruberte, E., Marty, T., Nellen, D., Affolter, M. & Basler, K. altered TGFf3-induced fibronectin expression. Because each of (1995) Cell 80, 889-897. these mutants constitutes its own complementation group, it is 16. Carcamo, J., Zentella, A. & Massague, J. (1995) Mol. Cell. Biol. most likely that the defect in each of these cells lies in a 15, 1573-1581. separate gene, although other explanations are possible. The 17. Mulligan, R. C. & Berg, P. (1980) Science 209, 1422-1427. 18. Mulligan, R. C. & Berg, P. (1981) Proc. Natl. Acad. Sci. USA 78, differential phenotypes of the mutants reveal the complexity 2072-2076. of the TGF3 system, which can best be sorted out by analysis 19. John, J., McKendry, R., Pellegrini, S., Flavell, D., Kerr, I. M. & of the mutants by a variety of techniques. Stark, G. R. (1991) Mol. Cell. Biol. 11, 4189-4195. TGFf3 has been implicated in a variety of pathological 20. McKendry, R., John, J., Flavell, D., Muller, M., Kerr, I. M. & conditions, characterized by either a lack of or overstimulation Stark, G. R. (1991) Proc. Natl. Acad. Sci. USA 88, 11455-11459. of pathways mediated by TGFJ3 (38-41). Therefore, it be- 21. Pellegrini, S., John, J., Shearer, M., Kerr, I. M. & Stark, G. R. comes imperative to identify the intracellular effectors of the (1989) Mol. Cell. Biol. 9, 4605-4612. TGF,B signaling pathway to begin to understand how these 22. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., disease processes occur. We are hopeful that the genetic Heldin, C.-H. & Miyazono, K (1993) Cell 75, 681-692. system that we have established will identify some of these 23. Lin, H. Y., Wang, X.-F., Ng-Eaton, E., Weinberg, R. A. & molecules thereby providing a better understanding of TGFI3 Lodish, H. F. (1992) Cell 68, 775-785. 24. Maniatis, T., Fritsch, E. F. .& Sambrook, J. (1989) Molecular signaling, which is also likely to contribute to the understand- Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, ing of the signal transduction of the whole TGFI3 superfamily. Plainview, NY), 2nid Ed., pp. 9.45, 16.47. 25. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156- We wish to thank J. Massague and J. Carcamo for the p3TPLux 159. vector, C.-H. Heldin for the ALK-5 plasmid, and H. Lodish for the 26. Howe, P. H., Bascom, C. & Leof, E. B. (1989) Cancer Res. 49, H2-3FF plasmid. We are very grateful to George R. 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