Vol. 11, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 972-978 0270-7306/91/020972-07$02.O00/
Copyright © 1991, American Society for Microbiology
Control of JunB and Extracellular Matrix Protein Expression by Transforming Growth Factor-1l Is Independent of Simian Virus 40 T Antigen-Sensitive Growth-Inhibitory Events MARIKKI LAIHO,' LARS RONNSTRAND,' JYRKI HEINO,1 JAMES A. DECAPRIO.2 TCOHN W. LUDLOW 2 DAVID M. LIVINGSTON,2 AND JOAN MASSAGUEl*
Howard Hughes Medical Institute and Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021,1 and Division of Neoplastic Disease Mechanisms, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 021152 Received 24 September 1990/Accepted 2 November 1990
Treatment of MvILu mink lung epithelial cells with transforming growth factor-,ll (TGF-Il1) prevents phosphorylation of the retinoblastoma susceptibility gene product, RB, in late G1 phase of the cell cycle, which is thought to retain RB in a growth-suppressive state. This effect is paralleled by cell cycle arrest in late G1 (M. Laiho, J. A. DeCaprie, J. W. Ludlow, D. M. Livingston, and J. Massague, Cell 62:175-185, 1990). Arrest can be prevented by expression of simian virus 40 T antigen, which binds to underphosphorylated RB, presumably blocking its growth-suppressive activity. The response of cells to TGF-IV1, however, is complex and includes changes in the levels of expression of genes encoding nuclear transcription factors and extracellular matrix components. To define the relationships among various components of the TGF-IV1 response, we have investigated the ett (-t of TGF-131 on cells whose growth-inhibitory response to this factor is prevented by T antigen. TGF-,1 t4ki,L!tion to exponentially growing MvlLu cells increased the levels of junB mRNA and of three extracellular *iniairix proteins: plasminogen activator inhibitor-1, fibronectin, and thrombospondin. Kinetically, the eftict% onjunB and plasminogen activator inhibitor-i expression occurred faster (half-maximal at 1 to 2 h) than tii; delects on fibronectin and thrombospondin expression (half-maximal at 6 to 10 h). These effects either prest,h:d or overlapped, respectively, the withdrawal of MvlLu cells from the cell cycle. Expression of a tra x fected T-antigen gene in MvlLu cells, however, did not prevent any of these responses to TGF-,B1. The results indicate that TGF- 1 -stimulated expression of junB and extracellular matrix proteins in MvILu cells can occur independently of the T-antigen-sensitive events that lead to growth arrest.
Transforming growth factkr-~(TGF-P) is the designation for a group of paracrine fac-ors that exist in multiple isoforms (TGF-41, -12, and -,B3) and are among the most potent known inhibitors of mamrral'Hn cell proliferation (42, 44, 52, 53). Recent progress in e'u iklating the growth-inhibitory mechanism of TGF-131 hus becn made in studies showing that cell cycle arrest by this fa,tor occurs in late G1 phase (32, 49). In the MvlLu mirnk lung epithelial and MK mouse keratinocyte cell lines, FGF-131 prevents progression through S phase if addec Jutring mid to late G1 but not if added at the G1-S bound-i- (3!, 49). In Mvllu cells. the growth-inhibitory action of TGF-1 has been linked to the ability of this factor to cowtro! the activity of an intracellular growth suppressor, RB, :1 ;;i .duct of the reti,noblastoma susceptibility gene (32). Geiic'Ac evidence has shown that RB has growth suppressor a>i'iLy or functions in a growthsuppressive signal transdusAion pathway(s) (2, 3, 6, i3, 17, 18, 24, 30, 37). RB, a nuclear phosphoprotein (36), is present throughout the cell cycle but its phosphorylation level oscillates in a cycle-dependent manner (5, 8, 12, 19, 41, 43). Under- or unphosphorylated RB prevails in Go cells and during G1, is rapidly phcfphorylated as cells approach S phase, and remains phosphorylated until late M phase. T antigen, the transforming protein of simian virus 40 (SV40), binds underphosphorylated RB (12, 40) and other related proteins (14, 15) via a specific domain (11). It is thought that this interaction, which does not prevent the normal phos*
phorylation of RB, interferes with the growth-suppressive function of RB (12, 40). A hypothesis strongly favored by these observations is that the form of RB that is actively involved in growth suppression is the underphosphorylated form. The growth-suppressive function of RB may therefore be abolished by two mechanisms: cell cycle-dependent phosphorylation and T-antigen binding. In MvlLu cells, TGF-pl inhibits the RB phosphorylation event that is scheduled for late G1 (32), thus retaining RB in its growth-suppressive state (12, 40). That the effect of TGF-1l on RB phosphorylation is linked to the generation of the growth-inhibited state in MvlLu cells is suggested by the fact that SV40 T antigen is able to block the growthinhibitory effect of TGF-,B1 (32). in MvILu cells that express T antigen, TGF-pl still prevents phosphorylation of RB but no longer causes growth inhibition, presumably because of the interference of T antigen with the growth-suppressive activity of RB (32). Thus, inhibition of RB phosphorylation by TGF-,B is not a consequence of but is possibly an event that mediates the cell cycle arrest caused by this factor. These results typify a case in which a prototypic intracellular growth suppressor participates in the mechanism of action of a growth-inhibitory paracrine factor. A similar conclusion has been suggested on the basis of the study of growth inhibition of keratinocytes by 1 GF-1l. In these cells, growth inhibition by TGF-1l occurs with down-regulation of c-myc transcription (49). The silencing of c-myc, which prevents keratinocyte entry into S (49). is prevented by T antigen and other viral products that bind RB and related proteins (50). TGF-pl has many other effects on the cell besides those
Corresponding author. 972
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described above (42, 53). It regulates the expression of nuclear factors, including c-jun (29, 48), junB (23, 48), c-fos (29), and c-myc (10, 16, 50, 57); growth factors, including TGF-pl, and platelet-derived growth factors A and B (38, 46, 56); many components of the cell adhesion apparatus, including extracellular matrix proteins and cell adhesion integrin receptors (27, 33, 42, 53); and various other genes and cellular activities. As progress is made in elucidating the steps in the mechanism of growth inhibition by TGF-pl, it is important to determine how, mechanistically, the diverse effects of TGF-p1 may relate to each other. Genetic and biochemical evidence suggests that many of these effects originate by activation of the same TGF-P receptor complex that mediates suppression of RB phosphorylation and cell
cycle
arrest
(4, 35). Since, directly or indirectly, RB may
influence gene expression (51) and since the expression of certain genes is specifically elevated in the growth-arrested state (55), this has raised the question of whether the effects of TGF-1l on the expression of certain genes lie downstream of the TGF-pl-induced changes in RB function that lead to growth arrest. We have approached this question by examining the ability of TGF-pl to elicit several responses in MvlLu cells and in clones of this cell line that express T antigen, a known inactivator of RB function. MATERIALS AND METHODS
Transfections. MvlLu cells (CCL 64; American Type Culture Collection) were cotransfected by calcium phosphate precipitation with either plasmid pPVU-0, which contains a wild-type SV40 T-antigen gene, or plasmid Kl, which contains a mutant T-antigen gene (28), and with pCD2, which contains a G418 resistance marker (7). G418-resistant colonies were ring cloned, subcloned, and assayed for T expression either by immunostaining with monoclonal antibody PAb419 against SV40 large T antigen (kindly provided by J. Hurwitz, Sloan-Kettering Institute) and rhodamineconjugated goat anti-mouse antibody or by immunoblotting. For immunoblotting, cells were washed with Tris-buffered saline (TBS; 25 mM Tris hydrochloride-150 mM NaCl, pH 8.0) and lysed for 20 min at 4°C with 50 mM Tris hydrochloride buffer, pH 8.0, containing 120 mM NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 1xM Na3VO4, 70 kIU of aprotinin per ml, 0.6 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ,ug of leupeptin per ml. The lysates were cleared by microcentrifugation, and 200-,g aliquots were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions and transferred to Immobilon-P membranes (59). Blots were incubated with 3% bovine serum albumin (BSA) in TBS, probed with monoclonal antibody PAb 419 and rabbit anti-mouse immunoglobulin G, and detected with [1251]-protein A (DuPont, NEN Research Products) and by autoradiography. [1251]iododeoxyuridine incorporation into DNA was determined as previously described (32).
Cell labeling and immunoprecipitation. For metabolic labeling, cells were treated as described in the legends to Fig. 2 and 4 and labeled with 100 ,uCi of [35S]cysteine per ml in cysteine-free minimal essential medium (for immunoprecipitations) or with 40 FCi of [35S]methionine per ml in methionine-free minimal essential medium (for extracellular matrix protein analysis). Labeled cell monolayers were rinsed with phosphate-buffered saline, and the cytosolic and nuclear proteins were extracted by subsequent washes with hypotonic buffer and sodium deoxycholate (21). The remaining
973
extracellular matrix proteins were recovered by scraping into electrophoresis sample buffer. [35S]cysteine-labeled cell culture media were mixed with an equal volume of TBS buffer containing 1 mg of BSA per ml, 10 ,ug of leupeptin per ml, 10 ,g of aprotinin per ml, and 1% Triton X-100. The mix was preadsorbed with protein A-Sepharose and then incubated for 18 h at 4°C with either polyclonal anti-human fibronectin antibodies (CalbiochemBehring) or polyclonal anti-human thrombospondin antibodies (kindly provided by R. Nachman, Cornell Medical College). Immunoprecipitates were collected by adsorption to protein A-Sepharose (Pharmacia), washed once with TBS containing 0.1% Triton X-100 and 1 mg of BSA per ml and once with TBS alone, heated in electrophoresis sample buffer containing dithiothreitol, and subjected to SDSPAGE. Gels were fixed and then fluorographed with Enlightening (New England Nuclear). mRNA assays. Total cellular RNA was isolated (9), separated on 1% agarose gels, and transferred to nitrocellulose membranes. junB mRNA was detected by using randomprimed 32P-labeled cDNA (p465.20, obtained from D. Nathans) as a probe. As a control, the filters were probed with 32P-labeled glyceraldehyde phosphate dehydrogenase cDNA (pRGAPDH-13).
RESULTS MvlLu cells are potently growth inhibited by TGF-1l (50% effective dose, 1 pM). Almost complete inhibition of DNA synthesis (98%), accompanied by an enlarged, flattened cell shape, is reached with 10 pM TGF-pl in medium with low serum content (4, 32). In asynchronously growing MvlLu cell populations, inhibition of DNA synthesis can be detected 2 h after the addition of TGF-B1 and is complete in 12 to 16 h as cells accumulate in mid to late G1 (32). In order to study the involvement of RB in the TGF-pl action, MvlLu cells were transfected with plasmid pPVU-0, which encodes SV40 large T antigen, and plasmid Kl, which encodes a mutant large T antigen (28, 32). The Kl cDNA carries a point mutation that renders it unable to bind RB (12, 40). Expression of T and mutant T was detected by immunofluorescence (not shown) and immunoblot (Fig. 1A). Clones with >99% of cells expressing T or mutant T were used for further studies. All cell clones transfected with wild-type T had greatly reduced TGF-pl growth-inhibitory response as measured by [125I]iododeoxyuridine incorporation into DNA (Fig. 1B) (32). The presence of TGF-P receptors was not altered in these transfectants, as all displayed normal levels of type I and II TGF-P receptors and beta-glycan (results not shown). Cells transfected with mutant T still responded to TGF-,1 in the same manner as parental cells (Fig. 1B) (32), indicating an intact growthinhibitory pathway in cells containing undisturbed RB function. The inability of TGF-pl to arrest growth of T-transfected cells is attributed to T binding of RB, rendering it unable to perform functions required for G1 arrest. We used these cells to examine whether other TGF-pl-regulated events were dependent on the presence of active RB. One of the fastest responses to TGF-pl in various cell lines is the stimulation of plasminogen activator inhibitor-i (PAI-1) expression (33). Upon secretion by cells, PAI-1 accumulates at cell-substratum contact points and binds to urokinase, preventing its proteolytic activity (34). In metabolically labeled MvlLu cells, TGF-131 induced a 5- to 20-fold elevation of extracellular matrix PAI-1 (Fig. 2A), a 46-kDa protein whose identity was verified by specific
974
.
MOL. CELL. BIOL.
LAIHO ET AL.
A
T \-
2
1
had occurred 2 to 4 h after TGF-pl addition (data not shown). Immunoprecipitation of [35S]methionine-labeled media also showed increased amounts of PAI-1 after cell treatment with TGF-pl, the increase occurring with similar kinetics in parental, T-transfected, and mutant-T-transfected cells (data not shown). junB is a growth-factor-regulated gene whose expression is elevated by TGF-pl in several cell types, including K562 erythroleukemia cells, A549 adenocarcinoma cells, L6EA rat myoblasts, BC3H1 myocytes, and mouse AKR-2B fibroblasts (23, 39, 48). The addition of TGF-,11 to MvlLu cells caused an approximately fivefold elevation of junB mRNA by 1 h, followed by a rapid decline to basal level over the next 3 to 10 h, as determined by Northern (RNA) hybridization (Fig. 3). Induction ofjunB of a similar magnitude and of similar kinetics was observed in T- and mutant-T-transfected cells (Fig. 3), indicating that elevation ofjunB expression by TGF-pl was not secondary to T antigen-sensitive growthinhibitory events induced by TGF-pl. Previous studies have associated the regulation of expression of extracellular matrix proteins and their receptors to the regulation of cell adhesive properties and of cell phenotype, which may in turn affect cell growth (1, 25, 54). TGF-,B can affect cell adhesion by enhancing synthesis and deposition of extracellular matrix components and by modifying the repertoire of cell surface adhesion receptors (27, 42). The relationship between inhibition of DNA synthesis and regulation of extracellular matrix components by TGF-pl was therefore studied with MvlLu transfectants. In MvlLu cells, TGF-1l elevated fibronectin expression two- to fivefold (Fig. 4A). The kinetics of the fibronectin response were slower than those of the PAI-1 response, with elevated levels of fibronectin detectable 6 to 10 h after addition of TGF-,1 (Fig. 4B). Stimulation of fibronectin production by TGF-pl was independent from growth-inhibitory effects, since simi-
B E A
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53
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FIG. 1. (A) Immunoblotting analyses of T expression in parental MvlLu cells and cells transfected with PVU-0 and Kl plasmids. Cell lysates (200 Rxg) were analyzed by SDS-7.5% PAGE and by Western immunoblotting, and T expression was detected with monoclonal T antibody. Lane 1, Mv1Lu; lane 2, pPVU-0 1.5.3; lane 3, Ki 1.15.2. (B) Effect of TGF-pl on DNA synthesis in MvlLu cells and cells expressing T or mutant T. The effect of increasing concentrations of TGF-41 on DNA synthesis by MvlLu, pPVU-0 1.5.3, and Ki 1.15.2 cells was determined by ['25I]iododeoxyuridine incorporation at the end of the labeling, and the incorporated label was detected by -y-counter. The assays were carried out in triplicate with less than 10% variation.
immunoblotting and immunoprecipitation (data not shown). A similar induction of PAI-1 by TGF-,B occurred in all pPVU-0 and Kl clones tested (Fig. 2A). Furthermore, parental MvlLu cells and transfected clones showed similar responsiveness to various TGF-pi concentrations (Fig. 2B). Kinetic analyses of the responses indicated that considerable elevation of PAI-1 deposition into the extracellular matrices
iTGF-31
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FIG. 2. Induction of PAI-1 to extracellular matrices by TGF-p in MvlLu cells and cells expressing T and mutant T. (A) Analyses of PAI-1 induction in MvlLu (WT) and in individual clones (denoted by clone number codes) of T-transfected cells (pPVU-0) and mutant-T-transfected cells (Ki). Cells were incubated with or without TGF-pl (200 pM) for 4 h and labeled for the last 2 h with [35S]methionine; extracellular matrix extracts were prepared as described in Materials and Methods. [35S]methionine-labeled extracellular matrix proteins were analyzed by SDS-8% PAGE followed by fluorography. (B) Concentration dependence of PAI-1 induction. MvlLu, pPVU-0 1.5.3, and Kl 1.15.2 cells were incubated with the indicated concentrations of TGF-p1 for 4 h and labeled with [35S]methionine for the last 2 h and matrices were then prepared. The matrix proteins were analyzed by SDS-8% PAGE followed by fluorography.
VOL.
11, 1991
CELL REGULATION BY TGF-p
Mvl Lu
i
|Time (h)
0
1
3
6
10
I
Kl 1.15.2
|
0
1
pPVU-0 1.5.3
6
1
0
6
junB
FIG. 3. Expression of junB mRNA in response to TGF-,B1. MvlLu, pPVU-0 1.5.3, and Kl 1.15.2 cells were treated with 100 pM for the indicated times, and total cellular RNA was prepared as described in Materials and Methods. RNA was separated on agarose gels and transferred to nitrocellulose, and the filter was probed with 32P-labeledjunB cDNA probe.
TGF-,1l
lar induction was detected in both pPVU-0 and Kl transfectants (Fig. 4A). The induction was detectable at TGF-p1 concentrations 5- to 10-fold higher than those required for inhibition of DNA synthesis (results not shown). No change in the concentration dependence or the kinetics of the
4m
..
no
m
oO-a
FIG. 4. (A) Stimulation of fibronectin and thrombospondin by
TGF-pl. MvlLu, pPVU-0 1.5.3, and Kl 1.15.2 cells were incubated with
TGF-pl (100 pM) for 16 h.
Cells
were
labeled with[35S]cysteine
for the last 4 h, and medium proteins were immunoprecipitated with either polyclonal antifibronectin antibodies (a-FN) or polyclonal antithrombospondin antibodies (a-TSP). The immunoprecipitates were analyzed by SDS-6% PAGE and fluorography. (B) Kinetics of fibronectin induction by TGF-pl. Cells were treated with TGF-pl (100 pM) for the indicated time and labeled with [35S]methionine for the last 2 h; extracellular matrix extracts were then prepared. The labeled matrix proteins were detected by SDS-8% PAGE and fluorography. The relevant portion of the fluorogram is shown. Note that lane Kl 1.15.2 at time 0 was slightly overloaded.
975
fibronectin-inductive response was detected in T-transfected cells in comparison with parental MvlLu (Fig. 4B) cells. Thrombospondin is a multifunctional extracellular matrix protein produced by a variety of cell types. It plays a role in tissue remodeling and healing processes but also restricts the growth of lung capillary and endothelial cells (58). Elevation of thrombospondin mRNA by TGF-pl in mouse fibroblasts has been observed (47). TGF-fi1 elevated thrombospondin production in both parental and T-transfected MvlLu cells, as detected by immunoprecipitation (Fig. 4A). Fibronectin coprecipitated with thrombospondin by using antithrombospondin antibodies, which is consistent with the known ability of these proteins to form a complex (31). However, no thrombospondin was detected in precipitates with antifibronectin antibodies, possibly because of dissociation of the complex by these antibodies. Kinetic analysis of the induction of thrombospondin indicated that it occurred parallel to that of fibronectin (data not shown). Thus, the regulation of neither fibronectin nor thrombospondin was secondary to the cell cycle arrest induced by TGF-,11 in MvlLu cells. DISCUSSION In MvlLu cells, cell cycle arrest in late G1 by TGF-pl is associated with the inhibition of phosphorylation of a nuclear growth suppressor, the RB gene product (32). In addition to inhibiting DNA replication, TGF-pl is a major regulator of several other cellular functions. These include the expression of various components of the extracellular matrix whose levels generally increase in response to TGF-,B (26, 33, 42). Another class of cellular components whose expression is increased by TGF-P are transcription factors, including members of the Jun family as well-known examples (23, 29, 39, 48). In the present study, we have characterized the effect of TGF-pl on the expression of these two classes of components in MvlLu cells. The results show that in parallel with the growth-inhibitory effect, TGF-pl increases the level of junB mRNA and the levels of PAI-1, fibronectin, and thrombospondin in MvlLu cells. The effects of TGF-3 on fibronectin and PAI-1 levels appear to be mediated by the same membrane TGF-13 receptor complex that mediates the growth-inhibitory response; evidence for this has been provided by the concomitant loss of all these responses in mutant MvlLu cells that have specific defects in TGF-P receptors (4, 35). The evidence favors a model of TGF-,B action in which a single type of receptor complex mediates multiple effects, some of which culminate in positive or negative proliferative or differentiative responses, depending on the cell type (42). The problems become, therefore, how the various effects of this factor might be mechanistically linked to each other, which of these effects might be sufficient to mediate the ultimate proliferative or phenotypic response, and which of them might be secondary to such responses. The present results address some of these problems with a cell system in which growth inhibition by TGF-pl can be prevented by SV40 T antigen. First, the results show that the effects of TGF-,B on the expression of several nuclear and extracellular matrix components remain unperturbed when the growth-suppressive response is inhibited by the presence ofT antigen. T antigen binds specifically the underphosphorylated form of RB, and it has been proposed that this interaction blocks the growthsuppressive function of RB (5, 12, 40). RB may play a central role in the growth-inhibitory action of TGF-p1, since this factor retains RB in its unphosphorylated, growth-suppressive state (32). RB may also act as a transcription regulator
976
LAIHO ET AL.
(51). Regardless of whether the latter function relates to the growth-suppressive function of RB, the present results indicate that TGF-r1 can exert a normal effect on the expression of junB, PAI-1, fibronectin, or thrombospondin in cells whose RB protein and growth-inhibitory response are repressed by the presence of SV40 T antigen. We conclude that the elevation of junB and extracellular-matrix-component expression by TGF-f1 is not secondary to the growthsuppressive activity of RB that is controlled by TGF-pl and is perturbed by T antigen. A second conclusion from the present results is that the elevated expression of junB or extracellular matrix proteins by TGF-g31 is not sufficient to cause growth arrest, at least in MvlLu cells expressing T antigen. This point is of interest because, as a modulator of cell adhesion and extracellular matrix composition, TGF-P may affect the morphology, differentiation, or growth characteristics of cells. These events are susceptible to regulation by cell-matrix interactions (1, 25, 54). TGF-3 increases expression of extracellular matrix proteins, including fibronectin, collagens, proteoglycans, and many others (42), and it increases expression of cell-adhesion receptors (27, 42) and concomitantly decreases extracellular matrix degradation by inducing inhibitors of proteolytic enzymes (33). Indeed, there is evidence that by stimulating extracellular matrix production and by controlling the cell adhesion apparatus, TGF-r can affect the ability of NRK rat fibroblasts to form colonies in semisolid medium (45), the ability of an osteosarcoma cell line to adhere to laminin (22), or the ability of skeletal-muscle myoblasts to differentiate (23). More relevant to the present studies, it has been suggested that the growth-inhibitory action of TGF-P could be mediated by accumulation of extracellular matrix. For example, the presence of a layer of type I collagen, whose production is induced by TGF-3 in NRK fibroblasts, partially inhibits the proliferation of these cells in monolayer culture (45). In addition, thrombospondin has been shown to restrict the proliferation of murine lung capillary cells and bovine heart endothelial cells (58) and to inhibit angiogenesis which may in turn suppress tumor growth (20). However, we could not find evidence that elevated levels of fibronectin, thrombospondin, or PAI-1 were associated with the rapid and profound inhibition of DNA synthesis caused by TGF-,B1 in MvlLu cells. Elevated expression of these proteins in T-antigen-expressing MvlLu cells in response to TGF-,13 was associated with a very limited growth-inhibitory response, even after prolonged incubations with TGF-pl (31a). Extracellular matrix accumulation in response to TGF-,1l might contribute to the long-term maintenance of the growth-arrested state in MvlLu cells or to the characteristically enlarged, flattened cell shape that becomes apparent after prolonged culture of these cells with TGF-pl (4). Extracellular matrix accumulation in MvlLu cells, however, is neither secondary to nor sufficient for the acute growthsuppressive action of TGF-p1 that involves T-antigen-sensitive events. The latter branch of TGF-3 action can be modified (by T antigen) while the other signaling pathways are left operative in their native fashion. ACKNOWLEDGMENTS This study was supported by National Institutes of Health grants CA 34610 and CA 39240 (J.M.) and by National Cancer Institute grant CA 50661 (D.M.L.). L.R. is the recipient of a European Molecular Biology Organization postdoctoral fellowship. M.L. and
MOL. CELL. BIOL.
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25. 26.
27. 28. 29.
authenticity of the human retinoblastoma gene. Science 236: 1657-1661. Furukawa, Y., J. A. DeCaprio, A. Freedman, Y. Kanakura, M. Nakamura, T. J. Ernst, D. M. Livingston, and J. D. Griffin. 1990. Expression and state of phosphorylation of the retinoblastoma susceptibility gene product in cycling and noncycling human hematopoietic cells. Proc. Natl. Acad. Sci. USA 87: 2770-2774. Good, D. J., P. J. Polverini, F. Rastinejad, M. M. Le Beau, R. S. Lemons, W. A. Frazier, and N. P. Bouck. 1990. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. USA 87:6624-6628. Hedman, K., M. Kurkinen, K. Alitalo, A. Vaheri, S. Johansson, and M. Hook. 1979. Isolation of the pericellular matrix of human fibroblast cultures. J. Cell Biol. 81:83-91. Heino, J., and J. Massague. 1989. Transforming growth factor-, switches the pattern of integrins expressed in MG-63 human osteosarcoma cells and causes a selective loss of cell adhesion to laminin. J. Biol. Chem. 264:21806-21811. Heino, J., and J. Massague. 1990. Cell adhesion to collagen and decreased myogenic gene expression implicated in the control of myogenesis by transforming growth factor P. J. Biol. Chem. 265:10181-10184. Huang, H.-J., J.-K. Yee, J.-Y. Shew, P.-L. Chen, R. Bookstein, T. Friedmann, E. Y.-H. P. Lee, and W.-H. Lee. 1988. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science 242:1563-1566. Hynes, R. 0. 1987. Integrins: a family of cell surface receptors. Cell 48:549-554. Ignotz, R. A., and J. Massaguk. 1986. Transforming growth factor-p stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261:4337-4345. Ignotz, R. A., and J. Massague. 1987. Cell adhesion protein receptors as targets for transforming growth factor-P. Cell 51:189-197. Kalderon, D., and A. E. Smith. 1984. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology 139: 109-137. Kim, S.-J., P. Angel, R. Lafyatis, K. Hattori, K. Y. Kim, M. B. Sporn, M. Karin, and A. B. Roberts. 1990. Autoinduction of transforming growth factor p1 is mediated by the AP-1 complex. Mol. Cell. Biol. 10:1492-1497.
30. Knudson, A. G., Jr. 1971. Mutation and cancer: statistical study of retinoblastoma. Proc. Natl. Acad. Sci. USA 68:820-823. 31. Lahav, J., J. Lawler, and M. A. Gimbrone. 1984. Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding. Eur. J. Biochem. 145:151-156. 31a.Laiho, M. Unpublished data. 32. Laiho, M., J. A. DeCaprio, J. W. Ludlow, D. M. Livingston, and J. Massague. 1990. Growth inhibition by TGF-13 linked to suppression of retinoblastoma protein phosphorylation. Cell 62:175-185. 33. Laiho, M., and J. Keski-Oja. 1989. Growth factors in the regulation of pericellular proteolysis. A review. Cancer Res. 49:2533-2553. 34. Laiho, M., 0. Saksela, and J. Keski-Oja. 1987. Transforming growth factor-p induction of type-1 plasminogen activator inhibitor. Pericellular deposition and sensitivity to urokinase. J. Biol. Chem. 262:17467-17474. 35. Laiho, M., F. M. B. Weis, and J. Massague. 1990. Concomitant loss of transforming growth factor-p receptor types I and II in TGF-p resistant cell mutants implicates both receptor types in signal transduction. J. Biol. Chem. 265:18518-18524. 36. Lee, W.-H., J.-Y. Shew, F. D. Hong, T. W. Sery, L. A. Donoso, L.-J. Young, R. Bookstein, and E. Y.-H. P. Lee. 1987. The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature (London) 329:642-645. 37. Lee, W.-H., J.-Y. Shew, F. D. Hong, T. W. Sery, L. A. Donoso, L.-J. Young, R. lookstein, E. Y.-H. P. Lee. 1987. Human retinoblastoma susceptibility gene: cloning, identification, and
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sequence. Science 235:1394-1399. 38. Leof, E. B., J. A. Proper, A. S. Goustin, G. D. Shipley, P. E. DiCorleto, and H. L. Moses. 1986. Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor 1: a proposed model for indirect mitogenesis involving autocrine activity. Proc. Natl. Acad. Sci. USA 83: 2453-2457. 39. Li, L., J.-S. Hu, and E. N. Olson. 1990. Different members of the jun proto-oncogene family exhibit distinct patterns of expression in response to type p transforming growth factor. J. Biol. Chem. 265:1556-1562. 40. Ludlow, J. W., J. A. DeCaprio, C.-M. Huang, W.-H. Lee, E. Paucha, and D. M. Livingston. 1989. SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell 56:57-65. 41. Ludlow, J. W., J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio. 1990. The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation binding to and release from SV 40 large T-antigen. Cell 60:387-396. 42. Massague, J. 1990. The transforming growth factor-p family. Annu. Rev. Cell Biol. 6:597-641. 43. Mihara, K., X.-R. Cao, A. Yen, S. Chandler, B. Driscoll, A. L. Murphree, A. T'Ang, and Y.-K. Fung. 1989. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 246:1300-1303. 44. Moses, H. L., R. F. Tucker, E. B. Leof, R. J. J. Coffey, J. Halper, and G. D. Shipley. 1985. Type 13 transforming growth factor is a growth stimulator and growth inhibitor, p. 65-65. In J. Feramisco, B. Ozanne, and C. Stiles (ed.), Cancer Cells, vol. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 45. Nugent, M. A., and M. J. Newman. 1989. Inhibition of normal rat kidney cell growth by transforming growth factor-p is mediated by collagen. J. Biol. Chem. 264:18060-18067. 46. Paulsson, Y., M. P. Beckmann, B. Westermark, and C.-H. Heldin. 1988. Density-dependent inhibition of cell growth by transforming growth factor-pl in normal human fibroblasts. Growth Factors 1:19-27. 47. Penttinen, R. P., S. Kobayashi, and P. Bornstein. 1988. Transforming growth factor P increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc. Natl. Acad. Sci. USA 85:1105-1108. 48. Pertovaara, L, L. Sistonen, T. J. Bos, P. K. Vogt, J. Keski-Oja, and K. Alitalo. 1989. Enhanced jun gene expression is an early genomic response to transforming growth factor p stimulation. Mol. Cell. Biol. 9:1255-1262. 49. Pietenpol, J. A., J. T. Holt, R. W. Stein, and H. L. Moses. 1990. Transforming growth factor 1-1 suppression of c-myc gene transcription: role in inhibition of keratinocyte proliferation. Proc. Natl. Acad. Sci. USA 87:3758-3762. 50. Pietenpol, J. A., R. W. Stein, E. Moran, P. Yaciuk, R. Schlegel, R. M. Lyons, R. M. Pittelkow, K. Munger, P. M. Howley, and H. L. Moses. 1990. TGF-pl inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming protein with pRB binding domains. Cell 61:777-785. 51. Robbins, P. D, J. M. Horowitz, and R. C. Mulligan. 1990. Negative regulation of human c-fos expression by the retinoblastoma gene product. Nature (London) 346:668-671. 52. Roberts, A. B., M. A. Anzano, L. M. Wakefield, N. S. Roche, D. F. Stern, and M. Sporn. 1985. Type-p transforming growth factor: a bifunctional regulator of cellular growth. Proc Natl. Acad. Sci. USA 82:119-123. 53. Roberts, A. B., and M. B. Sporn (ed.). 1990. Peptide growth factors and their receptors, p. 419-472. Springer-Verlag KG, Berlin. 54. Ruoslahti, E. 1988. Fibronectin and its receptor. Annu. Rev. Biochem. 57:375-413. 55. Schneider, C., R. M. King, and L. Philipson. 1988. Genes specifically expressed at growth arrest of mammalian cells. Cell 54:787-793. 56. Silver, B. J., F. E. Jaffer, and H. E. Abboud. 1989. Plateletderived growth factor synthesis in mesangial cells: induction by multiple peptide mitogens. Proc. Natl. Acad. Sci. USA 86:10561060.
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57. Takehara, K., E. C. LeRoy, and G. R. Grotendorst. 1987. TGF-, inhibition of endothelial cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Cell 49:415-422. 58. Taraboletti, G., D. Roberts, L. A. Liotta, and R. Giavazzi. 1990. Platelet thrombospondin modulates endothelial cell adhesion,
MOL. CELL. BIOL.
motility, and growth: a potential angiogenesis regulatory factor. J. Cell. Biol. 111:765-772. 59. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4353.
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 972-978 0270-7306/91/020972-07$02.O00/
Copyright © 1991, American Society for Microbiology
Control of JunB and Extracellular Matrix Protein Expression by Transforming Growth Factor-1l Is Independent of Simian Virus 40 T Antigen-Sensitive Growth-Inhibitory Events MARIKKI LAIHO,' LARS RONNSTRAND,' JYRKI HEINO,1 JAMES A. DECAPRIO.2 TCOHN W. LUDLOW 2 DAVID M. LIVINGSTON,2 AND JOAN MASSAGUEl*
Howard Hughes Medical Institute and Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021,1 and Division of Neoplastic Disease Mechanisms, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 021152 Received 24 September 1990/Accepted 2 November 1990
Treatment of MvILu mink lung epithelial cells with transforming growth factor-,ll (TGF-Il1) prevents phosphorylation of the retinoblastoma susceptibility gene product, RB, in late G1 phase of the cell cycle, which is thought to retain RB in a growth-suppressive state. This effect is paralleled by cell cycle arrest in late G1 (M. Laiho, J. A. DeCaprie, J. W. Ludlow, D. M. Livingston, and J. Massague, Cell 62:175-185, 1990). Arrest can be prevented by expression of simian virus 40 T antigen, which binds to underphosphorylated RB, presumably blocking its growth-suppressive activity. The response of cells to TGF-IV1, however, is complex and includes changes in the levels of expression of genes encoding nuclear transcription factors and extracellular matrix components. To define the relationships among various components of the TGF-IV1 response, we have investigated the ett (-t of TGF-131 on cells whose growth-inhibitory response to this factor is prevented by T antigen. TGF-,1 t4ki,L!tion to exponentially growing MvlLu cells increased the levels of junB mRNA and of three extracellular *iniairix proteins: plasminogen activator inhibitor-1, fibronectin, and thrombospondin. Kinetically, the eftict% onjunB and plasminogen activator inhibitor-i expression occurred faster (half-maximal at 1 to 2 h) than tii; delects on fibronectin and thrombospondin expression (half-maximal at 6 to 10 h). These effects either prest,h:d or overlapped, respectively, the withdrawal of MvlLu cells from the cell cycle. Expression of a tra x fected T-antigen gene in MvlLu cells, however, did not prevent any of these responses to TGF-,B1. The results indicate that TGF- 1 -stimulated expression of junB and extracellular matrix proteins in MvILu cells can occur independently of the T-antigen-sensitive events that lead to growth arrest.
Transforming growth factkr-~(TGF-P) is the designation for a group of paracrine fac-ors that exist in multiple isoforms (TGF-41, -12, and -,B3) and are among the most potent known inhibitors of mamrral'Hn cell proliferation (42, 44, 52, 53). Recent progress in e'u iklating the growth-inhibitory mechanism of TGF-131 hus becn made in studies showing that cell cycle arrest by this fa,tor occurs in late G1 phase (32, 49). In the MvlLu mirnk lung epithelial and MK mouse keratinocyte cell lines, FGF-131 prevents progression through S phase if addec Jutring mid to late G1 but not if added at the G1-S bound-i- (3!, 49). In Mvllu cells. the growth-inhibitory action of TGF-1 has been linked to the ability of this factor to cowtro! the activity of an intracellular growth suppressor, RB, :1 ;;i .duct of the reti,noblastoma susceptibility gene (32). Geiic'Ac evidence has shown that RB has growth suppressor a>i'iLy or functions in a growthsuppressive signal transdusAion pathway(s) (2, 3, 6, i3, 17, 18, 24, 30, 37). RB, a nuclear phosphoprotein (36), is present throughout the cell cycle but its phosphorylation level oscillates in a cycle-dependent manner (5, 8, 12, 19, 41, 43). Under- or unphosphorylated RB prevails in Go cells and during G1, is rapidly phcfphorylated as cells approach S phase, and remains phosphorylated until late M phase. T antigen, the transforming protein of simian virus 40 (SV40), binds underphosphorylated RB (12, 40) and other related proteins (14, 15) via a specific domain (11). It is thought that this interaction, which does not prevent the normal phos*
phorylation of RB, interferes with the growth-suppressive function of RB (12, 40). A hypothesis strongly favored by these observations is that the form of RB that is actively involved in growth suppression is the underphosphorylated form. The growth-suppressive function of RB may therefore be abolished by two mechanisms: cell cycle-dependent phosphorylation and T-antigen binding. In MvlLu cells, TGF-pl inhibits the RB phosphorylation event that is scheduled for late G1 (32), thus retaining RB in its growth-suppressive state (12, 40). That the effect of TGF-1l on RB phosphorylation is linked to the generation of the growth-inhibited state in MvlLu cells is suggested by the fact that SV40 T antigen is able to block the growthinhibitory effect of TGF-,B1 (32). in MvILu cells that express T antigen, TGF-pl still prevents phosphorylation of RB but no longer causes growth inhibition, presumably because of the interference of T antigen with the growth-suppressive activity of RB (32). Thus, inhibition of RB phosphorylation by TGF-,B is not a consequence of but is possibly an event that mediates the cell cycle arrest caused by this factor. These results typify a case in which a prototypic intracellular growth suppressor participates in the mechanism of action of a growth-inhibitory paracrine factor. A similar conclusion has been suggested on the basis of the study of growth inhibition of keratinocytes by 1 GF-1l. In these cells, growth inhibition by TGF-1l occurs with down-regulation of c-myc transcription (49). The silencing of c-myc, which prevents keratinocyte entry into S (49). is prevented by T antigen and other viral products that bind RB and related proteins (50). TGF-pl has many other effects on the cell besides those
Corresponding author. 972
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11, 1991
CELL REGULATION BY TGF-4
described above (42, 53). It regulates the expression of nuclear factors, including c-jun (29, 48), junB (23, 48), c-fos (29), and c-myc (10, 16, 50, 57); growth factors, including TGF-pl, and platelet-derived growth factors A and B (38, 46, 56); many components of the cell adhesion apparatus, including extracellular matrix proteins and cell adhesion integrin receptors (27, 33, 42, 53); and various other genes and cellular activities. As progress is made in elucidating the steps in the mechanism of growth inhibition by TGF-pl, it is important to determine how, mechanistically, the diverse effects of TGF-p1 may relate to each other. Genetic and biochemical evidence suggests that many of these effects originate by activation of the same TGF-P receptor complex that mediates suppression of RB phosphorylation and cell
cycle
arrest
(4, 35). Since, directly or indirectly, RB may
influence gene expression (51) and since the expression of certain genes is specifically elevated in the growth-arrested state (55), this has raised the question of whether the effects of TGF-1l on the expression of certain genes lie downstream of the TGF-pl-induced changes in RB function that lead to growth arrest. We have approached this question by examining the ability of TGF-pl to elicit several responses in MvlLu cells and in clones of this cell line that express T antigen, a known inactivator of RB function. MATERIALS AND METHODS
Transfections. MvlLu cells (CCL 64; American Type Culture Collection) were cotransfected by calcium phosphate precipitation with either plasmid pPVU-0, which contains a wild-type SV40 T-antigen gene, or plasmid Kl, which contains a mutant T-antigen gene (28), and with pCD2, which contains a G418 resistance marker (7). G418-resistant colonies were ring cloned, subcloned, and assayed for T expression either by immunostaining with monoclonal antibody PAb419 against SV40 large T antigen (kindly provided by J. Hurwitz, Sloan-Kettering Institute) and rhodamineconjugated goat anti-mouse antibody or by immunoblotting. For immunoblotting, cells were washed with Tris-buffered saline (TBS; 25 mM Tris hydrochloride-150 mM NaCl, pH 8.0) and lysed for 20 min at 4°C with 50 mM Tris hydrochloride buffer, pH 8.0, containing 120 mM NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 1xM Na3VO4, 70 kIU of aprotinin per ml, 0.6 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ,ug of leupeptin per ml. The lysates were cleared by microcentrifugation, and 200-,g aliquots were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions and transferred to Immobilon-P membranes (59). Blots were incubated with 3% bovine serum albumin (BSA) in TBS, probed with monoclonal antibody PAb 419 and rabbit anti-mouse immunoglobulin G, and detected with [1251]-protein A (DuPont, NEN Research Products) and by autoradiography. [1251]iododeoxyuridine incorporation into DNA was determined as previously described (32).
Cell labeling and immunoprecipitation. For metabolic labeling, cells were treated as described in the legends to Fig. 2 and 4 and labeled with 100 ,uCi of [35S]cysteine per ml in cysteine-free minimal essential medium (for immunoprecipitations) or with 40 FCi of [35S]methionine per ml in methionine-free minimal essential medium (for extracellular matrix protein analysis). Labeled cell monolayers were rinsed with phosphate-buffered saline, and the cytosolic and nuclear proteins were extracted by subsequent washes with hypotonic buffer and sodium deoxycholate (21). The remaining
973
extracellular matrix proteins were recovered by scraping into electrophoresis sample buffer. [35S]cysteine-labeled cell culture media were mixed with an equal volume of TBS buffer containing 1 mg of BSA per ml, 10 ,ug of leupeptin per ml, 10 ,g of aprotinin per ml, and 1% Triton X-100. The mix was preadsorbed with protein A-Sepharose and then incubated for 18 h at 4°C with either polyclonal anti-human fibronectin antibodies (CalbiochemBehring) or polyclonal anti-human thrombospondin antibodies (kindly provided by R. Nachman, Cornell Medical College). Immunoprecipitates were collected by adsorption to protein A-Sepharose (Pharmacia), washed once with TBS containing 0.1% Triton X-100 and 1 mg of BSA per ml and once with TBS alone, heated in electrophoresis sample buffer containing dithiothreitol, and subjected to SDSPAGE. Gels were fixed and then fluorographed with Enlightening (New England Nuclear). mRNA assays. Total cellular RNA was isolated (9), separated on 1% agarose gels, and transferred to nitrocellulose membranes. junB mRNA was detected by using randomprimed 32P-labeled cDNA (p465.20, obtained from D. Nathans) as a probe. As a control, the filters were probed with 32P-labeled glyceraldehyde phosphate dehydrogenase cDNA (pRGAPDH-13).
RESULTS MvlLu cells are potently growth inhibited by TGF-1l (50% effective dose, 1 pM). Almost complete inhibition of DNA synthesis (98%), accompanied by an enlarged, flattened cell shape, is reached with 10 pM TGF-pl in medium with low serum content (4, 32). In asynchronously growing MvlLu cell populations, inhibition of DNA synthesis can be detected 2 h after the addition of TGF-B1 and is complete in 12 to 16 h as cells accumulate in mid to late G1 (32). In order to study the involvement of RB in the TGF-pl action, MvlLu cells were transfected with plasmid pPVU-0, which encodes SV40 large T antigen, and plasmid Kl, which encodes a mutant large T antigen (28, 32). The Kl cDNA carries a point mutation that renders it unable to bind RB (12, 40). Expression of T and mutant T was detected by immunofluorescence (not shown) and immunoblot (Fig. 1A). Clones with >99% of cells expressing T or mutant T were used for further studies. All cell clones transfected with wild-type T had greatly reduced TGF-pl growth-inhibitory response as measured by [125I]iododeoxyuridine incorporation into DNA (Fig. 1B) (32). The presence of TGF-P receptors was not altered in these transfectants, as all displayed normal levels of type I and II TGF-P receptors and beta-glycan (results not shown). Cells transfected with mutant T still responded to TGF-,1 in the same manner as parental cells (Fig. 1B) (32), indicating an intact growthinhibitory pathway in cells containing undisturbed RB function. The inability of TGF-pl to arrest growth of T-transfected cells is attributed to T binding of RB, rendering it unable to perform functions required for G1 arrest. We used these cells to examine whether other TGF-pl-regulated events were dependent on the presence of active RB. One of the fastest responses to TGF-pl in various cell lines is the stimulation of plasminogen activator inhibitor-i (PAI-1) expression (33). Upon secretion by cells, PAI-1 accumulates at cell-substratum contact points and binds to urokinase, preventing its proteolytic activity (34). In metabolically labeled MvlLu cells, TGF-131 induced a 5- to 20-fold elevation of extracellular matrix PAI-1 (Fig. 2A), a 46-kDa protein whose identity was verified by specific
974
.
MOL. CELL. BIOL.
LAIHO ET AL.
A
T \-
2
1
had occurred 2 to 4 h after TGF-pl addition (data not shown). Immunoprecipitation of [35S]methionine-labeled media also showed increased amounts of PAI-1 after cell treatment with TGF-pl, the increase occurring with similar kinetics in parental, T-transfected, and mutant-T-transfected cells (data not shown). junB is a growth-factor-regulated gene whose expression is elevated by TGF-pl in several cell types, including K562 erythroleukemia cells, A549 adenocarcinoma cells, L6EA rat myoblasts, BC3H1 myocytes, and mouse AKR-2B fibroblasts (23, 39, 48). The addition of TGF-,11 to MvlLu cells caused an approximately fivefold elevation of junB mRNA by 1 h, followed by a rapid decline to basal level over the next 3 to 10 h, as determined by Northern (RNA) hybridization (Fig. 3). Induction ofjunB of a similar magnitude and of similar kinetics was observed in T- and mutant-T-transfected cells (Fig. 3), indicating that elevation ofjunB expression by TGF-pl was not secondary to T antigen-sensitive growthinhibitory events induced by TGF-pl. Previous studies have associated the regulation of expression of extracellular matrix proteins and their receptors to the regulation of cell adhesive properties and of cell phenotype, which may in turn affect cell growth (1, 25, 54). TGF-,B can affect cell adhesion by enhancing synthesis and deposition of extracellular matrix components and by modifying the repertoire of cell surface adhesion receptors (27, 42). The relationship between inhibition of DNA synthesis and regulation of extracellular matrix components by TGF-pl was therefore studied with MvlLu transfectants. In MvlLu cells, TGF-1l elevated fibronectin expression two- to fivefold (Fig. 4A). The kinetics of the fibronectin response were slower than those of the PAI-1 response, with elevated levels of fibronectin detectable 6 to 10 h after addition of TGF-,1 (Fig. 4B). Stimulation of fibronectin production by TGF-pl was independent from growth-inhibitory effects, since simi-
B E A
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53
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FIG. 1. (A) Immunoblotting analyses of T expression in parental MvlLu cells and cells transfected with PVU-0 and Kl plasmids. Cell lysates (200 Rxg) were analyzed by SDS-7.5% PAGE and by Western immunoblotting, and T expression was detected with monoclonal T antibody. Lane 1, Mv1Lu; lane 2, pPVU-0 1.5.3; lane 3, Ki 1.15.2. (B) Effect of TGF-pl on DNA synthesis in MvlLu cells and cells expressing T or mutant T. The effect of increasing concentrations of TGF-41 on DNA synthesis by MvlLu, pPVU-0 1.5.3, and Ki 1.15.2 cells was determined by ['25I]iododeoxyuridine incorporation at the end of the labeling, and the incorporated label was detected by -y-counter. The assays were carried out in triplicate with less than 10% variation.
immunoblotting and immunoprecipitation (data not shown). A similar induction of PAI-1 by TGF-,B occurred in all pPVU-0 and Kl clones tested (Fig. 2A). Furthermore, parental MvlLu cells and transfected clones showed similar responsiveness to various TGF-pi concentrations (Fig. 2B). Kinetic analyses of the responses indicated that considerable elevation of PAI-1 deposition into the extracellular matrices
iTGF-31
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FIG. 2. Induction of PAI-1 to extracellular matrices by TGF-p in MvlLu cells and cells expressing T and mutant T. (A) Analyses of PAI-1 induction in MvlLu (WT) and in individual clones (denoted by clone number codes) of T-transfected cells (pPVU-0) and mutant-T-transfected cells (Ki). Cells were incubated with or without TGF-pl (200 pM) for 4 h and labeled for the last 2 h with [35S]methionine; extracellular matrix extracts were prepared as described in Materials and Methods. [35S]methionine-labeled extracellular matrix proteins were analyzed by SDS-8% PAGE followed by fluorography. (B) Concentration dependence of PAI-1 induction. MvlLu, pPVU-0 1.5.3, and Kl 1.15.2 cells were incubated with the indicated concentrations of TGF-p1 for 4 h and labeled with [35S]methionine for the last 2 h and matrices were then prepared. The matrix proteins were analyzed by SDS-8% PAGE followed by fluorography.
VOL.
11, 1991
CELL REGULATION BY TGF-p
Mvl Lu
i
|Time (h)
0
1
3
6
10
I
Kl 1.15.2
|
0
1
pPVU-0 1.5.3
6
1
0
6
junB
FIG. 3. Expression of junB mRNA in response to TGF-,B1. MvlLu, pPVU-0 1.5.3, and Kl 1.15.2 cells were treated with 100 pM for the indicated times, and total cellular RNA was prepared as described in Materials and Methods. RNA was separated on agarose gels and transferred to nitrocellulose, and the filter was probed with 32P-labeledjunB cDNA probe.
TGF-,1l
lar induction was detected in both pPVU-0 and Kl transfectants (Fig. 4A). The induction was detectable at TGF-p1 concentrations 5- to 10-fold higher than those required for inhibition of DNA synthesis (results not shown). No change in the concentration dependence or the kinetics of the
4m
..
no
m
oO-a
FIG. 4. (A) Stimulation of fibronectin and thrombospondin by
TGF-pl. MvlLu, pPVU-0 1.5.3, and Kl 1.15.2 cells were incubated with
TGF-pl (100 pM) for 16 h.
Cells
were
labeled with[35S]cysteine
for the last 4 h, and medium proteins were immunoprecipitated with either polyclonal antifibronectin antibodies (a-FN) or polyclonal antithrombospondin antibodies (a-TSP). The immunoprecipitates were analyzed by SDS-6% PAGE and fluorography. (B) Kinetics of fibronectin induction by TGF-pl. Cells were treated with TGF-pl (100 pM) for the indicated time and labeled with [35S]methionine for the last 2 h; extracellular matrix extracts were then prepared. The labeled matrix proteins were detected by SDS-8% PAGE and fluorography. The relevant portion of the fluorogram is shown. Note that lane Kl 1.15.2 at time 0 was slightly overloaded.
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fibronectin-inductive response was detected in T-transfected cells in comparison with parental MvlLu (Fig. 4B) cells. Thrombospondin is a multifunctional extracellular matrix protein produced by a variety of cell types. It plays a role in tissue remodeling and healing processes but also restricts the growth of lung capillary and endothelial cells (58). Elevation of thrombospondin mRNA by TGF-pl in mouse fibroblasts has been observed (47). TGF-fi1 elevated thrombospondin production in both parental and T-transfected MvlLu cells, as detected by immunoprecipitation (Fig. 4A). Fibronectin coprecipitated with thrombospondin by using antithrombospondin antibodies, which is consistent with the known ability of these proteins to form a complex (31). However, no thrombospondin was detected in precipitates with antifibronectin antibodies, possibly because of dissociation of the complex by these antibodies. Kinetic analysis of the induction of thrombospondin indicated that it occurred parallel to that of fibronectin (data not shown). Thus, the regulation of neither fibronectin nor thrombospondin was secondary to the cell cycle arrest induced by TGF-,11 in MvlLu cells. DISCUSSION In MvlLu cells, cell cycle arrest in late G1 by TGF-pl is associated with the inhibition of phosphorylation of a nuclear growth suppressor, the RB gene product (32). In addition to inhibiting DNA replication, TGF-pl is a major regulator of several other cellular functions. These include the expression of various components of the extracellular matrix whose levels generally increase in response to TGF-,B (26, 33, 42). Another class of cellular components whose expression is increased by TGF-P are transcription factors, including members of the Jun family as well-known examples (23, 29, 39, 48). In the present study, we have characterized the effect of TGF-pl on the expression of these two classes of components in MvlLu cells. The results show that in parallel with the growth-inhibitory effect, TGF-pl increases the level of junB mRNA and the levels of PAI-1, fibronectin, and thrombospondin in MvlLu cells. The effects of TGF-3 on fibronectin and PAI-1 levels appear to be mediated by the same membrane TGF-13 receptor complex that mediates the growth-inhibitory response; evidence for this has been provided by the concomitant loss of all these responses in mutant MvlLu cells that have specific defects in TGF-P receptors (4, 35). The evidence favors a model of TGF-,B action in which a single type of receptor complex mediates multiple effects, some of which culminate in positive or negative proliferative or differentiative responses, depending on the cell type (42). The problems become, therefore, how the various effects of this factor might be mechanistically linked to each other, which of these effects might be sufficient to mediate the ultimate proliferative or phenotypic response, and which of them might be secondary to such responses. The present results address some of these problems with a cell system in which growth inhibition by TGF-pl can be prevented by SV40 T antigen. First, the results show that the effects of TGF-,B on the expression of several nuclear and extracellular matrix components remain unperturbed when the growth-suppressive response is inhibited by the presence ofT antigen. T antigen binds specifically the underphosphorylated form of RB, and it has been proposed that this interaction blocks the growthsuppressive function of RB (5, 12, 40). RB may play a central role in the growth-inhibitory action of TGF-p1, since this factor retains RB in its unphosphorylated, growth-suppressive state (32). RB may also act as a transcription regulator
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(51). Regardless of whether the latter function relates to the growth-suppressive function of RB, the present results indicate that TGF-r1 can exert a normal effect on the expression of junB, PAI-1, fibronectin, or thrombospondin in cells whose RB protein and growth-inhibitory response are repressed by the presence of SV40 T antigen. We conclude that the elevation of junB and extracellular-matrix-component expression by TGF-f1 is not secondary to the growthsuppressive activity of RB that is controlled by TGF-pl and is perturbed by T antigen. A second conclusion from the present results is that the elevated expression of junB or extracellular matrix proteins by TGF-g31 is not sufficient to cause growth arrest, at least in MvlLu cells expressing T antigen. This point is of interest because, as a modulator of cell adhesion and extracellular matrix composition, TGF-P may affect the morphology, differentiation, or growth characteristics of cells. These events are susceptible to regulation by cell-matrix interactions (1, 25, 54). TGF-3 increases expression of extracellular matrix proteins, including fibronectin, collagens, proteoglycans, and many others (42), and it increases expression of cell-adhesion receptors (27, 42) and concomitantly decreases extracellular matrix degradation by inducing inhibitors of proteolytic enzymes (33). Indeed, there is evidence that by stimulating extracellular matrix production and by controlling the cell adhesion apparatus, TGF-r can affect the ability of NRK rat fibroblasts to form colonies in semisolid medium (45), the ability of an osteosarcoma cell line to adhere to laminin (22), or the ability of skeletal-muscle myoblasts to differentiate (23). More relevant to the present studies, it has been suggested that the growth-inhibitory action of TGF-P could be mediated by accumulation of extracellular matrix. For example, the presence of a layer of type I collagen, whose production is induced by TGF-3 in NRK fibroblasts, partially inhibits the proliferation of these cells in monolayer culture (45). In addition, thrombospondin has been shown to restrict the proliferation of murine lung capillary cells and bovine heart endothelial cells (58) and to inhibit angiogenesis which may in turn suppress tumor growth (20). However, we could not find evidence that elevated levels of fibronectin, thrombospondin, or PAI-1 were associated with the rapid and profound inhibition of DNA synthesis caused by TGF-,B1 in MvlLu cells. Elevated expression of these proteins in T-antigen-expressing MvlLu cells in response to TGF-,13 was associated with a very limited growth-inhibitory response, even after prolonged incubations with TGF-pl (31a). Extracellular matrix accumulation in response to TGF-,1l might contribute to the long-term maintenance of the growth-arrested state in MvlLu cells or to the characteristically enlarged, flattened cell shape that becomes apparent after prolonged culture of these cells with TGF-pl (4). Extracellular matrix accumulation in MvlLu cells, however, is neither secondary to nor sufficient for the acute growthsuppressive action of TGF-p1 that involves T-antigen-sensitive events. The latter branch of TGF-3 action can be modified (by T antigen) while the other signaling pathways are left operative in their native fashion. ACKNOWLEDGMENTS This study was supported by National Institutes of Health grants CA 34610 and CA 39240 (J.M.) and by National Cancer Institute grant CA 50661 (D.M.L.). L.R. is the recipient of a European Molecular Biology Organization postdoctoral fellowship. M.L. and
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