JOURNAL OF CELLULAR PHYSIOLOGY 14239-45 (1990)

Distinct Pathways Regulate Transforming Growth Factor p l -Stimulated Proto-Oncogene and Extracellular Matrix Gene Expression PHILIP H. HOWE, MURIEL R. CUNNINGHAM, A N D EDWARD B. LEOF* Department of Cell Biology, Vanderbilt University, Nashville, Tennesee 37232 The effect of pertussis toxin (PT) on transforming growth factor p l (TGF(S1)induced proto-oncogene expression was investigated in AKR-26 fibroblasts. P 1 substantially abolished c-sis and c-myc mKNA expression following TGFPl stiniulation. This inhibitory effect was specific for TCFf3l -stimulated proto-oncogene expression and associated with the ADP-ribosylation of a 41 -kDa substrate. Actinomycin D decay and nuclear run-on experiments demonstrated that the inhibitory effects of PT are a result of decreased transcriptional activation and not to an increased decay of proto-oncogene message. PT did not, however, affect TGFPlstitnulated fibroncctin and collagen mKNA accumulation nor did it have any inhibitory effect on TGFPl -induced morphological transformation. These data indicate that TGFPl -stimulated gene expression is coupled to multiple pathways distinguished by their sensitivity to PT.

Transforming growth factor type P l (TGFp1) is a ubiquitous protein produced by a variety of normal and transformed cells in vitro and in vivo (Moses et al., 1987; Roberts et al., 1986; Silberstein and Daniel, 1987). In general, TGFpl has been shown to be growth stimulatory for mesenchymal derived cells and growth inhibitory for most other cell types (Cheifetz et al., 1987; Shipley et al., 1985; Sporn et al., 1983; Tucker et al., 1984). The mechanism(s) through which TGFpl directly signals a mitogenic response is presently unknown (Chambard and Pouyssegur, 1988; Muldoon et al., 1988). To this end we previously proposed that TGFPl and the ras gene product, p21, might reside within the same (or similar) complementation group (Leof et al., 198713). Because p21 has been shown to exhibit properties similar to other known guanine nucleotide regulatory “G”proteins (Gibbs et al., 1984; Lacal et al., 19861, and TGFpl produces similar phenotypic effects as do transfected ras genes (Leof et al., 1987131, we wished to determine whether agents that uncouple G proteinireceptorieffector system interactions would affect TGFPl action. G proteins are classically defined as heterotrimeric complexes consisting of a,p, and y subunits (Neer and Clapham, 1988). The a subunit is capable of binding GTP and undergoing reversible dissociation from the (Sy complex (Ui, 1986). This results in activation of any associated effector system(s) followed by GTP hydrolysis and reassociation with Py to generate the inactive heterotrimer. Previous work has shown that many receptor/G protein coupled systems can be inhibited by PT (Chambard et al., 1987; Murayama and Ui, 1987). Since recent publications (Howe and Leof, 1989; Murthy et al., 1988) have suggested that a PT-sensitive G protein is coupled to TGFpl receptor binding, we (c~1990 WILEY-LISS, INC

wished to expand upon these results and determine whether TGFpl-stimulated gene expression is similarly affected. Expression of those genes associated with cellular proliferation (i.e., c-sis, c-myc, c-fos, and JE) are inhibited by PT whereas extracellular matrix gene expression (i.e. fibronectin, collagen, and integrin), and morphological transformation are not. The results indicate that multiple pathways, dependent upon their sensitivity to PT, are coupled to TGFpl receptor binding.

MATERIALS AND METHODS Cell Culture AKR-2B cells were grown in McCoys 5A medium supplemented with 5% fetal bovine serum (FBS). Cultures were plated a t 5 x lo3 cells/cm2 and grown for 7 days a t 37°C. The density-arrested quiescent cells were then restimulated with the indicated reagents in serum-free MCDB 402 medium containing 10 p,g/ml BSA or used a s a source for plasma membrane purification. Membrane Preparation Quiescent cultures were scraped with a rubber policeman and cells collected by centrifugation (235g for 5 minutes at 4°C). The cell pellet was lysed by Dounce homogenization in a hypotonic buffer containing 10 mM Tris (pH 7.4), 10 mM NaC1,l.O mM EGTA, and 2.0 mM MgC1,. The homogenate was made 0.125 M sucrose and the nuclei removed by centrifugation (1,400g

Received February 3, 1989; accepted August 29, 1989.

*To whom reprint requestsicorrespondence should be addressed.

40

HOWE ET AL.

A

28%

28S>

B

2

28S>

,5h 12h 24h,,5h 12h 24h, I

.la

u2

XFP

PT + TGFP

Fig. 2. PT does not delay expression of TGFpl-stimulated c-sis mRNA. Quiescent AKR-2B cells were treated as described in Figure 1 except that the final concentration of TGFpl was 3.0 ngiml. At the indicated times mRNA was isolated and Northern hybridization performed. The top band is c-sis and the bottom is 1B15.

1as>

18s

CON PT TGFP

PT TGFP

Fig. 1. TGFpl stimulation of c-sis and c-myc mRNA is inhibited by

PT. A demonstrates the effect of PT on TGFpl-stimulated c-sis expression, while B shows the response of c-myc. Loading was normalized to the signal obtained with the constitutively expressed IR15 gene (C) for each condition. Quiescent density-arrested cultures of AKR-2B cells were pretreated for 3 hours at 37°C with MCDB 402 medium alone or containing 100 ngiml PT.TGFpl was directly added to the indicated plates to a final concentration of 5.0 ngiml for a n additional 5 hour incubation at 37°C. Total polyh+ containing mRNA was purified by oligo dT cellulose chromatography and processed for Northern hybridization.

for 10 minutes at 4°C). The supernatant was layered over a pad of 20% sucrose and spun for 60 minutes a t 100,000g. Pelleted membranes were suspended in 50 mM Tris (pH 7.4), 1.0 mM EGTA, 100 pgiml leupeptin (membrane suspension buffer), and protein concentration determined by a dye binding assay (Bio Rad) following solubilization with 0.1 N NaOH for 15 minutes.

ADP Ribosylation in Plasma Membranes and Intact Cells For ADP ribosylation in plasma membrane preparations PT (List Biologicals) was activated by incubation at 37°C for 20 minutes in 20 mM dithiothreitol (Katada et al., 1983). Ribosylation was performed by incubating 1.0 pg of the activated toxin with 50 pg plasma membranes at 37°C for 30 minutes in a final reaction vol-

ume of 200 pl containing 2.5 pM NAD, 1.0 mM ATP, 1.0 mM GTP, 10 mM thymidine, and 25 pCiiml to(-”Pl NAD (1,000 Ciimmole, New England Nuclear). Reactions were terminated by the addition of 100% TCA to a final concentration of 10% and the ADP-ribosylated products separated on 7-15% linear gradient SDS-polyacrylamide gels. For toxin treatment in intact cells, cultures were incubated with the indicated concentration of PT for 3 hours a t 37°C in serum-free MCDB 402 medium prior t o the preparation of plasma membranes.

TGFPl Radioreceptor Assay AKR-2B cells were plated at 2 x lo5 celliwell in 6-well culture dishes (9.6 cm’iwell) in McCoys 5A medium containing 5% FBS. After 2 days at 37°C the serum containing medium was removed and replaced with 1ml MCDB 402 medium containing 1mgiml BSA (binding buffer) and the indicated concentrations of PT. After a 3 hour incubation a t 37°C l”I-labelled TGFpl (0.25 ng) was directly added for 2 additional hours a t room temperature as previously described (Tucker et al., 1984). Nonspecific binding was determined in the presence of a 200-fold excess of cold TGFpl and represented approximately 24% of total binding. Only specific binding is shown.

RNA Preparation and Hybridization Density-arrested cultures were treated with MCDB 402 medium alone or containing 100 ngiml PT for 3 hours at 37°C. Growth factors were directly added to the plates and the incubation continued for the indicated times. Total poly A’ containing RNA was purified a s described (Leof et al., 1986a) and equivalent quantities electrophoresed in 1.2% agarose gels containing 2.2 M formaldehyde. The constitutively expressed 1B15 gene (cyclophilin) (Danielson et al., 1988) was used as a n internal marker to normalize RNA loading. Quantitation was performed using a n LKB U1tra Scan Laser Densitometer by designating the control cell treatment (medium alone) as 1.0 (i.e., sis ex-

PERTUSSIS TOXIN AND TGYPl GENE EXPRESSION

41

A

a 43 49

30

C

f0 0

(L

z:a u)

mar

,l

1

1

0

1

PERTUSSIS TOXIN (ng/ml) Fig. 3. F'T ADP ribosylation of AKR-2B cell membranes. A Membranes (50 Fg) were ADP ribosylated in the presence P T ) or absence (Con) of 1.0 pg activated PT. B: Quiescent AKR-2B cells were pretreated at 37°C for 3 hours with the indicated concentration of PT prior to preparation of membranes and ADP ribosylation. Preparation

of membranes and ribosylation were performed as described in Methods. C. PT does not affect TGFBl binding to AKR-PB cells. Binding was performed as described in Materials and Methods. Data represent the mean of triplicate plates.

pression with medium aloneilB15 expression in medium alone as equivalent to 1.0). Nuclear run-on transcription assays were performed essentially a s described by Ruether et al. (1986) and equivalent TCA precipitable counts (approximately 1 x lo6 cpm) were hybridized to nitrocellulose filters containing 5.0 pg of linearized pUC-8 or pSM-1 (human c-sis cDNA) (Josephs e t al., 1984).

binding. Since previous work had indicated that TGFpl receptor binding was coupled to a guanine nucleotide binding regulatory protein (G protein) (Howe and Leof, 1989; Murthy et al., 1988), we wished to expand upon these studies and determine whether F'T, which prevents receptoriG protein coupling (Ui, 19861, would affect TGFp I-stimulated proto-oncogene expression. Consistent with earlier observations there was a n approximate 8.3-fold induction of c-sis mRNA following addition of TGFP1; however, this increase was substantially reduced (approximately 80%) with prior PT treatment (Fig. 1A). Addition of medium or PT alone had no effect on c-sis mRNA accumulation. An additional proto-oncogene which has previously been shown to be regulated by TGFPl is c-myc. The results of Figure I B indicate that the expression of c-myc mRNA can also be modulated by prior treatment with

RESULTS

Inhibition of TGFpl-Stimulated Proto-Oncogene Expression by Pertussis Toxin One of the earliest known responses of TGFp is its stimulation of gene expression (Daniel et al., 1986; Ignotz et al., 1987; Leof et al., 1986a, 1986b; Makela et al.,1987).In AKR-2B cells the induction of c-sis mRNA is the most proximal reported event following TGFP

HOWE ET AL

results are observed for TGE’p1-stimulated c-myc expression (data not shown).

0-0

Actino

A-A

Actino + PT

I

I

v 150

180

TIME (MINUTES)

B pUC8

c-sis

CON

TGFP

PT

TGFP+PT

Fig. 4. PT decreases TGFpl-stimulated c-sis transcription. A The data depict the densitometric tracing of c-sis mRNA relative to 1B15 mRNA for each time point following addition of actinomycin D I PT. Quiescent cultures of AKR-2B cells were stimulated with 3.0 ngiml TGFpl for 5 hours at 37°C. The medium was removed, the cells were washed with serum-free medium: and fresh medium supplemented with 5.0 p,g/ml actinomycin D was added. Replicate plates also received 100 ngiml PT. At the indicated times cells were harvested and Northern hybridization performed. B Quiescent density-arrested ARK-ZB cells were pretreated in the presence or absence of 100 ngiml PT for 3 hours. The cultures were then stimulated for an additional 3 hours with medium alone or containing 3.0 ngiml TGFp1. Nuclear run-on transcription assays were done as described in Materials and Methods.

nanogram concentrations of PT. The 4.0-fold induction of c-myc by TGFPl is reduced to basal levels by prior PT treatment. The results of Figure 1demonstrate that PT inhibits TGFP1-stimulated c-sis and c-myc mRNA accumulation. In order to determine whether this observed inhibition resulted from a delay in TGFpl-stimulated gene expression, kinetic analyses as shown in Figure 2 were performed. PT pretreated AKR-2B cells were stimulated with TGFPl and mRNA isolated at the indicated times. The increased expression of c-sis mRNA is inhibited by PT at all times examined (Fig. 2). Similar

Effect of Pertussis Toxin on ADP Ribosylation and T G F P l Binding A number of distinct proteins are substrates for PT ADP ribosylation (Casey and Gilman, 1988; Miller, 1988; Senogles et al., 1987). Two of the more extensively characterized proteins, G, and transducin, have been shown to regulate adenylate cyclase and light activated cGMP phosphodiesterase, respectively (Casey and Gilman, 1988; Miller, 1988). Treatment of cell membranes with PT in the presence of 32P-NADresults in the ADP ribosylation of proteins that run in SDS gels with apparent molecular weights of 40-45 kDa. Similarly, we found that treatment of AKR-2B cell membranes with PT resulted in the ADP ribosylation of a protein of approximately 41 kDa which migrates identically to that reported for G, (Fig. 3A). The ADP ribosylation seen in purified cell membranes is not a result of in vitro manipulations, since pretreatment of intact cells with PT results in the specific loss of label from the same band (Fig. 3B). While the data of Figure 3A and 3B did not demonstrate any uniquely migrating substrates for PT, they did indicate that the toxin was acting similarly in the AKR-2B culture system as previously described (Casey and Gilman, 1988; Chambard et al., 1987; Howe and Leof, 1989; Iyengar et al., 1987; Murthy et al., 1988). The data of Figures 1 and 2 indicate that PT decreases TGFpl-stimulated c-sis and c-myc expression. Before a more extensive investigation of these results could begin, it was first necessary to determine whether the inhibition seen in TGFpl proto-oncogene stimulation was secondary to a decrease in TGFpl binding to its specific cell surface receptor(s1. The data presented in Figure 3C demonstrate that PT does not interfere with TGFpl receptor binding and suggest that the results of Figures 1 and 2 occur through a n interruption in TGFpl signal transduction. Effect of Pertussis Toxin on G r o w t h Factor-Stimulated Gene Expression and Morphological Transformation In order to characterize further the effect of PT on TGFpl-stimulated proto-oncogene expression, actinomycin D decay curves and nuclear run-on assays were performed. If PT was inhibiting TGFpl signal transduction, the inability to detect c-sis and c-myc gene expression should result from decreased gene transcription rather than a n increase in message decay. As indicated in Figure 4A, there is essentially no difference in c-sis rate of decay in the presence of PT. The estimated half-life is approximately 40-90 minutes, which is similar to previous published reports (Daniel and Fen, 1988). To determine whether PT transcriptionally inhibited TGFpl-stimulated proto-oncogene expression, nuclear run-ons were performed. As demonstrated in Figure 4B, c-sis transcription is inhibited to a similar extent as shown in the Northern analyses of Figures 1and 2 by prior PT treatment. Decay curves and run-ons have been performed for c-myc expression in the presence and absence of PT and similar results

43

PERTUSSIS TOXIN AND TGFpl GENE EXPRESSION

A

B

MEDIA

MEDIA

+

TGFb

MEDIA + PERTUSSIS TOXIN

PERTUSSIS TOXIN

+

TGFb

Fig. 5. Effect of PT on TGFpl-stimulated fibronectin mRNA (A) and morphological transformation (B). A AKR-2B cells were treated as described in the legend to Figure 2. RNA was harvested following 12 hour stimulation and the fibronectin signal (top band) was normalized to the signal obtained with 1B15 (bottom band). B: Quiescent

AKR-2B cells were treated with the indicated reagents for 24 hours at 37°C. Prior to addition ofTGFp (3.0 ngiml) the plates were pretrcated with MCDB 402 medium alone or containing PT (100 ngimll. The cultures were fixed with lUO% methanol and photographed.

(i.e., no effect on decay, but decreased transcription) were observed (data not shown). The previous data (Figs. 1-41 indicate that PT decreases TGFpl-stimulated proto-oncogene transcription by preventing ligand mediated signal transduction. An obvious question raised by these results is whether this effect on c-sis and c-myc gene expression is seen for other TGFpl-modulated genes or for growth factors which are not believed to be coupled through a toxin sensitive G protein. To address these questions the experiments depicted in Figures 5 and 6 and Table 1 were performed. As shown in Figure 5A, when normalized to 1B15 expression there is no effect of PT on TGFPl-stimulated fibronectin mRNA. In addition, the results of Figure 5B demonstrate that TGFPl mediated morphological transformation (Shipley et al., 1985) is also insensitive to prior treatment with PT. A summary of the PT sensitivity of a variety of genes known to be modulated by TGFPl is shown in Table 1. Expression of those genes believed to be associated with a

proliferative response (i.e., c-sis, c-myc, c-fos, and J E ) are inhibited by PT while extracellular matrix gene expression and morphological transformation are not (Table 1 and Fig. 5B, respectively). Additional specificity to the toxin effect is shown in Figure 6, where epidermal growth factor (EGF)-stimulated c-myc expression is examined. No discernible difference in c-myc mRNA accumulation is observed in either the presence or absence of PT. Since EGF-stimulated proto-oncogene expression is not believed to be G protein coupled (Church and Buick, 1988; Wahl e t al., 1988), these results demonstrate that growth factor-stimulated c-myc expression, per se, is not sensitive to PT.

DISCUSSION TGFp has a variety of biological effects depending upon the culture system used. However, the mechanism(s1 by which the TGFp signal is transduced to the cell nucleus has been difficult to determine (Chambard and Pouyssegur, 1988). Data are presented indicating

44

HOWE ET AL.

TABLE 1. Effect of PT on TGFPI-stimulated mRNA accumulation”

28S>

Gene c-sis c-myc c-fos JE Collagen Fibronectin

18%

Fold increase 10.3 4.0 1.6 5.1 1.4 2.5

o/r Inhibition by uertussis toxin 84 100 100 82 0 0

”Northern analysis and densitometry were performed on AKR-ZB cells as described in the legend to Figure 1.Total pirly A - containing inRNA was harvested after 6 hour stimulation for c-sis. c-myc; c-fob, and J E and after 12 or 24 hour incubation for fibronectin and collagen, respectively. The effect of PT pretreatment on expression of the indicated cDNAs is as shown.

d

2 ,lh

Y

c)

5 h 12h 2 4 h , , l h 5h 12h 24h, I

EGF

I

PT + EGF

Fig. 6. Effect of PT on EGF-stimulated c-myc mRNA. Quiescent cultures of AKR-2B cells were treated with PT as described in the legend to Figure 1.Poly A - containing mRNA was isolated at the indicated times following addition of EGF (final concentration 10 ng/ml). The top band is c-myc and the bottom is 1B15.

that the pathways responsible for TGFp1-stimulated proto-oncogene and extracellular matrix gene expression are distinct. This was accomplished by differentially uncoupling the stimulatory effects of TGFpl with PT, a protein responsible for the ADP ribosylation and inactivation of a number of signal transducing G proteins (Ui, 1986). Expression of the proto-oncogenes c-sis, c-myc, and c-fos in response to TGFpl was shown to be drastically reduced by prior treatment with nanogram concentrations of PT (Figs. 1, 2, 4 and Table 1). This is in contrast to the results seen for EGF-stimulated c-myc expression which was shown t o be insensitive to PT (Fig. 6). A 1 hour treatment with EGF induced c-myc mRNA approximately 13-fold above control; similar levels were obtained following a 3 hour PT treatment (Fig. 6). Since recent studies suggest a direct linkage between the EGF receptor tyrosine kinase and second messenger systems (Wahl et al., 1988), agents that uncouple receptoriG protein interactions would not be expected to have a n inhibitory effect. In addition, the gene J E (isolated from a plateletderived growth factor inducible cDNA library, but responsive t o TGFp1) (Cochran et al., 1983; Leof et al., 198613) was also sensitive to PT. However, PT was unable to inhibit TGFPl -stimulated fibronectin, collagen, and integrin (p subunit of the fibronectin receptor) mRNA accumulation (Fig. 5, Table 1, and data not shown). While Figure 5 only examined maximal fibronectin expression after 12 hour TGFpl stimulation, additional kinetic studies from 1 to 24 hour TGFPl addition showed no discernible difference in either fibronectin or collagen mRNA with PT (data not shown). The mechanism(s) by which PT differentially uncouples TGFpl-stimulated gene expression is unknown. However, distinct toxin sensitive pathways for EGFstimulated proto-oncogene expression and soft agar growth have also been recently reported (Church and Buick, 1988). Our results indicate that multiple pathways, differentiated by PT sensitivity, are induced fol-

lowing TGFpl receptor binding. While it is commonly believed that PT exerts its sole effects on receptoriG protein coupling, it is not unreasonable t o propose additional toxin sensitive events distal to ligand binding. Alternatively, a n additional hypothesis might be t h a t the various TGFp binding species (Cheifetz et al., 1987) are coupled to distinct effector systems, one results in proto-oncogene expression and is PT sensitive, and the other stimulates extracellular matrix gene expression and is PT insensitive. Recent evidence by Boyd and Massague (19891, however, indicate that it is the type 1 ( 5 3 kDa) TGFP binding species which is the signal transducing receptor, suggesting that the latter hypothesis may not be appropriate. Regardless of whether any of these proposed models t u r n out to be correct, the stimulation of proto-oncogenes and extracellular matrix genes by TGFPl appears to be distinctly regulated.

ACKNOWLEDGMENTS We would like to thank Drs. R.T. Abraham, N.E. Olashaw, and W.R. Wharton for many helpful discussions. These studies were supported by NCI grant CA 42836, BRSG RR-05424, and ACS IN-25-29. P.H.H. is supported by NCI postdoctoral fellowship CA 09592. REFERENCES Boyd, F.T., and Massague, J. (1989) Transforming growth factor-p inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor. J. Biol. Chem., 264t2272-2278. Casey. P.J., and Gilman, A.G. (1988) G protein involvement in receptor-effector coupling. J. Biol. Chem., 263r2577-2580. Chambard, J.C., and Pouyssegur, J. (1988) TGFp inhibits growth factor-induced DNA synthesis in hamster fibroblasts without affecting the early mitogenic event. J . Cell. Physiol., 136:lOl-107. Chambard, J.C., Paris, S., L’Allem, G . , andpouyssegur, J. (1987)Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin. Nature, 326:800-803. Cheifetz, S., Weatherbee, J.A., Tsang, M.L.S., Anderson, J.K., Mole, J.E., Lucas, R., and Massague, J. (19x7) The transforming growth factor-beta system, a complex pattern of cross-reactive ligands and receptors. Cell, 48:409-415. Church, J.G., and Buick, R.N. (1988) G-protein mediated epidermal growth factor signal transduction in a human breast cancer cell line: Evidence for two intracellular pathways distinguishable by pertussis toxin. J. Riol. Chem., 263r4242-4246. Cochran, B.H., Reffel, A.C., and Stiles, C.D. (1983) Molecular cloning of gene sequences regulated by plat,elet-derived growth factor. Cell, 33:939-947. Daniel, T.O., and Fen, Z. (19881Distinct pathways mediate transcriptional regulation of PDGF Bic-sis expression. J . Biol. Chem., 26‘3: 19815-19820. Daniel, T.O., Gibbs, V.C., Milfay, D.F., Garovoy, M.R., and Williams, L.T. (1986)Thrombin stimulates c-sis gene expression in microvascular endothelial cells. J. Biol. Chem., 261:9579-9582. Danielson, P.E., Forss-Petter, S.,Brow, M.A., Calavetta, L., Douglass,

PERTUSSIS TOXIN AND TGFpl GENE EXPRESSION

J., Milner, R.J., and Sutcliffe, J.G. (1988) plB15: A cDNA clone of the rat mRNA encoding cyclophilin. DNA, 7t261-267. Gibbs, J.B., Diegal, IS., Poe, M., and Scolnick, E.M. (1984) Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc. Natl. Acad. Sci. USA, 81t5704-5708. Howe, P.H., and Leof, E.B. 11989) Transforming growth factor p l treatment of AKR-2B cells is coupled through a pertussis toxin sensitive G proteinis). Biochem. J . , 26It87Y-886. Ignotz, R.A., Endo, T., and Massague, J. (1987) Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor+. J. Biol. Chem., 262:6443-6446. Iyengar, R., Rich, K.A., Herberg, J.T., Grenet, D., Mumby, S., and Codina, J. (1987) Identification of a new G'rP-binding protein. J. Biol. Chem., 262:9239-9245. Josephs, S.F., Wong-Staal, F., and Gallo, K.C. (1984) Oncogenes, growth factors, and transformation: our lessons from the sis gene. Cancer Surv., 3,266-285. Katada, T., Tamura, M., and Ui, M. (1983) The A protomer of isletactivating protein, pertussis toxin, is an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch. Riochem. Biophys., 224t290 -298. Iacal, J.C., Srivastava, S.K., Anderson, P.S., and Aaronson, S.A. (1986) Ras p21 proteins with high or low GTPase activity can efficiently transform NIH/3T3 cells. Cell, 44t609-617. Leof, E.B., Proper, J.A., Getz, M.J., and Moses, H.L. (1986a) Transforming growth factor type p regulation of actin mHNA. J . Cell. Physiol., 127:83-88. I.eof, E.B., Proper, J.A., Goustin, AS., Shipley, G.D., DiCorleto, P.E., and Moses, H.L. (1986b) Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor-beta: A proposed model for indirect mitogenesis involving automine activity. Proc. Natl. Acad. Sci. USA, 85r24.53-2457. Leof, E.B., Proper, J.A., and Moses, H.L. (1987b) Modulation of transforming growth factor type beta action by activated ras and c-myc. Mol. Cell. Biol., 72649-2652. Makela, T.P., Alitalo, R., Paulsson, Y., Westerrnark, B., Heldin, C.-H., and Alitalo, K. 11987) Regulation of platelet-derived growth Factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines. Mol. Cell. Biol., 7:3656-3662. Miller, R.J. 11988) G proteins flex their muscles. Trends Neurosci., 1114-6. Moses, H.L., Shipley, G.D., Leof, E.B., Halper, J., Coffey, R.J., Jr., and Tucker, R.F. (1987) Transforming growth factors. In: Control of Animal Cell Proliferation, Vol. 11. A.L. Boynton and H.L. Leffert. eds. Academic Press, New York, pp. 75-92. Muldoon, L.L., Rodland, K.D., and Magun, B.E. (1988)Transforming growth factor p modulates epidermal growth factor-induced phos-

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phoinositide metabolism and intracellular calcium levels. J. Biol. Chem., 263:5030-50:73. Murayama, T., and Ui, M. (1987) Possible involvement of a GTPbinding protein, the substrate of islet activating protein, in receptor-mediated signalling responsible for cell proliferation. J. Biol. Chem., 262:12463-12467. Murthy, US., Anzano, M.A., Stadel, J.M., and Greig, R. (1988) Coupling of TGF-p-induced mitogenesis to G-protein activation in AKR-2B cells. Biochem. Biophys. Res. Commun., 1.52:1228-1235. Neer, E.J., and Clapham, D.E. 11988) Roles of G protein subunits in transmembrane signalling. Nature, 333:129-134. Roberts, A.B., Sporn, M.B.: Assoian, R.K., Smith, J.M., Roche, N.S., Wakefield, L.M., Heine, U.I., Liotta, L.A., Falanga, V., Kehrl, J.H., and Fauci, A S . 11986) Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA, 83t41674171. Ruether, J.E., Maderious, A,, Lavery, D., Logan, J., Fu, S.M., and Chen-Kiang, S. (1986) Cell-type specific synthesis of murine immunoglobulin niRNA from an Adenovirus vector. Mol. Cell. Biol., 6. ,123-1 33. Senogles, S.E., Benovic, J.L., Amlaiky, N., Unson, C., Milligan, G., Vinitsky, R., Spiegel, A.M., and Caron, M.G. (1987) The D2- dopamine receptor o f anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein. J. Biol. Chem., 262t4860-4867. Shipley, G.D., Tucker, R.F., and Moses, H.L. (1985) Type p-transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval. Proc. Natl. Acad. Sci. USA, 82:4147-4151. Silberstein, G.B., and Daniel, C.W. (1987) Reversible inhibition of mammary gland growth by transforming growth factor-beta. Science, 237,291-293, Sporn, M.B., Roberts, A.B., Shull, J.H., Smith, J.M., Ward, J.M., and Sodek, J. (1983) Polypeptide transforming growth factors isolated from bovine sources and used for wound healing in vivo. Science, 219t1329-1331. Tucker, R.F.; Shipley, G.D., Moses, H.L., and Holley, R.W. (1984) Growth inhibitor from BSC-1 cells closely related to the platelet type beta transforming growth factor. Science, 226t70.5-707. Ui, M. (1986)Pertussis toxin as a probe of receptor coupling to inositol lipid metabolism. In: Phosphoinositides and Receptor Mechanisms. J.W. Putney Jr., ed. Alan R. Liss, Inc., pp. 163-195. Wahl, M.I., Daniel, T.O., and Carpenter, G. (1988) Anti-phosphotyrosine recovery of phospholipase C activity after epidermal growth factor treatment of A-431 cells. Science, 241t968-970.

Distinct pathways regulate transforming growth factor beta 1-stimulated proto-oncogene and extracellular matrix gene expression.

The effect of pertussis toxin (PT) on transforming growth factor beta 1 (TGF beta 1)-induced proto-oncogene expression was investigated in AKR-2B fibr...
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