THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 264, No. 4, Issue of February 5,pp. 2272-2278,1989 Printed in U.S. A.
Transforming Growth Factor-@ Inhibition of Epithelial Cell Proliferation Linked to the Expression of a 53-kDa Membrane Receptor* (Received for publication, August 22, 1988)
Frederick T. Boyd and Joan Massague From the Departmentof Biochemistry, University of Massachusetts Medical School, Worcester, Massachusetts01655
Cells whose proliferation is blocked by transforming growth factor-@(TGF-8)express three distinct surface glycoproteins of 5 3 , 7 3 , and 300 kDa that bind TGF-8 with high affinity, but whose function is unknown. We have isolated two classesof chemically-induced MvlLu epithelial cell mutants resistant to growth inhibition by TGF-B. ClassR mutants have selectivelylost expression of the 53-kDa (type I) TGF-&binding protein. They have also lost the ability to respond to TGF-8 with elevated fibronectin expression and cell flattening. Class S mutants bind normally but do not respond to TGF-B. TGF-&resistant mutants retain a contact inhibited, nontransformed phenotype. The properties of S mutants suggest that they are defective in the TGF-B signal transduction mechanism, while the results withR mutants identify the type I TGF-&binding protein as the receptor involved in mediating TGF-j3 actions on cell adhesion and proliferation.
blocks the proliferative response of epithelial cells to epidermal growth factor without altering the binding or signaling capacity of EGF receptors (12, 13). Hence, efforts to identify TGF-@receptors on the basis of their signaling activity have been thus far unsuccessful. However, putative TGF-@receptors have been described based on their ability to bind TGFs@ with high affinity and specificity (14-16). Three distinct types of TGF-@binding components or “receptors” have been identified on the surface of almost all cell types examined. They have been operationally defined as TGF-@ receptor types I, 11, and 111, respectively (4, 17). Two of these receptors are glycoproteins of 53 kDa (type I) and 73-95 kDa (type 11)that display higher affinity for TGF-@l than TGF-@2(18). The third receptor type has the structure of a 300-kDa chondroitin/heparan sulfate membrane proteoglycan with a core protein of 110 kDa that binds TGF-@l and TGF-@2 with similar affinity (18-21). The functional role of these cell surface components is not known. In principle, they could participate in tasks other than signal transduction, including various modes of TGF-@ retenThe concept of negative growth regulation has materialized tion and transport into and across the cell. Preliminary eviin recent years with the identification of diffusible polypepdence to define which one of these binding proteins mediates tides that inhibit mammalian cell proliferation. Transforming the actions of TGF-@ is limited to correlative arguments growth factors @l and @2 (TGF-@’1and TGF-@2) are two linking the differential binding of TGF-Pl andTGF-@Pto the prototypic growth inhibitors whose antimitogenic action in putative receptors and the ligand’s differential potency in vitro affects cells of epithelial, vascular endothelial, lymphocytic, hematopoietic, and neuroectodermal lineages (1,2). Due biological assays. In one case, the higher affinity of TGF-@1 for the type I receptor in mouse hematopoietic progenitor to their multifunctional nature, they control cell differentiacells correlates with the higher potency of TGF-@l toinhibit tion as well as proliferation, the TGFs-@ may function in vivo growth of these cells when compared with TGF-@2 (22). as important effectors in processes of morphogenesis, tissue development, and repair (3-7). At the cellular level, the action Similar lines of correlative evidence have suggested the inof TGFs-@is characterized by changes in the expression of volvement of the type I11 receptor in actions that TGF-@l growth regulatory genes (8, 9)and geneswhose products and TGF-@2elicit with similar potency, such as inhibition of mediate cell adhesion (3, 10, 11).By analogy with the mode epithelial cell proliferation (18), regulation of the expression of action of other polypeptide hormones, TGFs-@may act by of certain phenotypes, and elevated expression of cell adhebinding to cell surface receptors that initiate the transduction sion proteins (11, 23). The three TGF-@ receptor types are of antimitogenic signals and other gene regulatory informa- ubiquitously distributedin cell lines and primary tissues. tion to thenucleus. However, the biochemical components of Their presence as well as the presence of growth inhibitory responses to TGF-@ innormal human retina cells but not in this pathway are virtually unknown. retinoblastoma cell lines has suggested that one or more of This problem derives in part from the fact that the TGF-@ receptor-signaling mechanism appears to be different from these TGF-@-binding proteinsmight mediate growth inhibithose of other hormone and growth factor receptors. The tion, the loss of receptors leading to loss of growth control in antiproliferative action of TGFs-@ can take place without retinoblastoma cells (24). These findings have left unsolved the issue of which if any interference with the biochemical events that immediately of the TGF-/3 binding proteins identified to date is directly followcell stimulation by mitogens. For example, TGF-@1 involved in mediating the action of TGF-@. Herewe report * This work was supported by National Institutes of Health Grant that selection of lung epithelial cell mutants defective in CA 34610 The costs of publication of this article were defrayed in growth inhibitory responses to TGF-@results with high frepart by the payment of page charges. This article must therefore be quency in the isolation of cell clones that have selectively lost hereby marked “aduertisement” in accordance with 18 U.S.C. Section expression of functional type I TGF-@receptors, a finding 1734 solely to indicate this fact. * The abbreviations used are: TGF-0, transforminggrowth factor- that strongly implicates this receptor type as the mediator of TGF-P action. @; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
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TGF-/3Receptor Identification EXPERIMENTALPROCEDURES
Culture Conditions-Parental MvlLu mink lung epithelial cells (American Type Culture Collection CCL 64) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Mutant stocks were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum supplemented with 100 pM TGF-(31. Cultures were grown at 37 “C in a humidified atmosphere containing 5% co,. Production of TGF-P-insensitiueMutants-Pilot studies were done to determine the appropriate cell density for maximal inhibition of MvlLu cell proliferation by TGF-P1 and concentrations of ethyl methanesulfonate necessary to achieve 70-90% cell killing. Cells were seeded in 100-mm plastic tissue culture dishes at a density of 1.3 X lo5cells/cm2in 10ml of growth medium. The next day, cultures were treated with ethyl methanesulfonate (Sigma) at either 0.1 pl/ml or 0.5 pl/ml of media for 24h. Culture media was changed and 48 h later, 100 PM human TGF-01 was added to theculture media. Cultures were fedwith growth media supplemented with 100 pM TGF-01 every 3 days. When macroscopically visible colonies had arisen, colonies were ring cloned, transferred into individual culture vessels, and propagated in the presence of 100 PM TGF-P1. A total of 26 TGF-8resistant colonies were cloned from 18 culture dishes. Colonies isolated from plates treated with 0.5 pl/ml ethyl methanesulfonatehave a “5”as thesecond character in their designation and colonies isolated from plates treated with 0.1 pl/ml ethyl methanesulfonate have a “1.” Cell lines which demonstrated stable resistance to TGF-0were subcloned by limiting dilution and all subsequent experiments used clonal lines. Affinity Labeling of Mutant Lines-Subconfluent monolayers of various cell lines were affinity labeled as previously described (16). Briefly, cultures were incubated with ‘251-TGF-01in the presence or absence of competing TGF-(31 or TGF-02 as indicated for 3.5 h at 4 “C. Cultures were washed extensively and cross-linked using the bifunctional reagent disuccinimidyl suberate (Pierce Chemical Co.). Cells were then solubilized in a buffer containing 1%Triton X-100, 1 mM EDTA, 10 mM Tris,pH 7.4, and protease inhibitors. Gel electrophoresis of the Triton-solubilized receptors reduced with 50 mM dithiothreitol was performed according to theprocedure of Laemmli (25) on 6% polyacrylamide gels. Gels were stained for protein with Coomassie Blue, dried, and exposed to Kodak XAR film with enhancing screens (Du Pont Lightning Plus). Receptor Binding Assays-Binding of ‘z51-TGF-01to cell monolayers was performed as described previously (16). Near confluent monolayers of various lines were incubated in binding buffer (128 mM NaCl, 5 mM KCl, 5 mM MgSO,, 1.3 mM CaC12, 50 mM Hepes, pH 7.4, containing 5 mg/ml of bovine serum albumin) for 30 min, a t 37 “C to wash off serum and endogenous proteins. Cultures were subsequently incubated with varying concentrations of lZ5I-TGF-pl and unlabeled TGF-8 for 3.5 h at 4 “C. Cultures were washed four times with binding buffer and solubilized with 1%Triton X-100, 1 mM EDTA, 10 mM Tris, pH 7.4. Nonspecific binding was measured in the presence of 10 nM unlabeled TGF-P and subtracted from total binding to determine specific binding. Assay of Thymidine Incorporation-Subconfluent cultures of various cell lines were incubated with 100 p~ TGF-01 for 30 h. Cultures were incubated with 1 pCi/ml of [3H]thymidine (Amersham Corp.) for 3 h. Radioactivity incorporated into cellular trichloroacetic acidprecipitable material was assayed as previously described (12). Assay of Fibronectin Production by Mutant Lines-The effect of TGF-8 on production of fibronectin in various cell lines was assayed as previously described (10). Subconfluent monolayers of various cell lines were treated with 100 p~ TGF-01 for 15 h and subsequently biosynthetically labeled in cysteine-free, serum-free medium containing 10 pCi/ml of [35S]cysteinein the presence or absence of 100 PM TGF-P1 for 3 h. The resulting labeled medium was incubated with gelatin-Sepharose beads (Pharmacia LKB Biotechnology Inc.) for 16 h at 4 ‘C to isolate fibronectin. The beads were washed, heated in sample buffer, and analyzed by electrophoresis on 7% polyacrylamidesodium dodecyl sulfate gels and fluorography using Enlightning fluid (Du Pont-New England Nuclear). Anchorage-independent GrowthAssays-Cell lines were assayed for anchorage-independent growth as previously described (26). Approximately 1000 cells from various mutant and parental lines were plated into 35-mm tissue culture wells in a suspension of 1.5 ml of Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and 0.4% agar on a base layer of the same medium containing 0.5% agar. Dishes were incubated at 37 “C in a humidified 5% COz atmosphere
2273
for 3 weeks. Cultures were assayed by microscopic examination. TumorigenesisAssays-Subconfluent cultures of various cell lines were dissociated from culture plates with physiological saline, 5 mM EDTA. Cells were pelleted and resuspended in physiological saline. 1 X IO7 cells in a volume of 0.2 ml were injected subcutaneously into the lower abdomen of nude mice. Mice wereexamined every 3-5 days until tumors developed or for 10 weeks if no tumors developed. RESULTS
Isolation of Mutant Cell Lines Resistantto TGF-@-The strategy to isolate cell mutants resistant to growth inhibition by TGF-@was based on maintaining sparse cell cultures in the continuous presence of TGF-B1 and isolating cell colonies that grow under these conditions (27). For these studies, we chose the diploid MvlLu mink lung epithelial cell line whose proliferation is arrested by TGF-@l orTGF-P2acting at picomolar concentrations (15, 18). The spontaneous colony forming efficiency of the MvlLuparental cell line maintained in the presence of TGF-B1 was -1/106. Ten such colonies were picked and individually propagated for several weeks. They alleventually reverted to thewild type, TGF-@-inhibited phenotype. The colony forming efficiency of MvlLu cells mutagenized with ethyl methanesulfonate and maintained in the presence of TGF-@lwas also -1/106 cells. Most of these colonies and their subclones were significantly growth inhibited by TGF-@upon expansion of the cultures, as determined by [3H]thymidine incorporation assays. With time in culture, these clones also regained full sensitivity to TGF-@. These clones probably represented the small subpopulation of MvlLu cells that becomes spontaneously resistant to TGF-@ for a limited number of generations. However, of 26 colonies originally isolated from 2 x lo7mutagenized MvlLu cells, six independently isolated colonies as well as all their subclones have remained completely resistant toTGF-@ for over 6 months in culture, and have been characterized in detail. TGF-Pl inhibited incorporation of [3H]thymidineinto DNA almost completely in the parental MvlLucell line, but did not decrease this parameter in anyof the six mutant cell lines (Fig. 1).A concentration TGF-Pl 1000-fold higher than that required for a significant inhibitory effect in the parental 150
1
i a 0
Parental R1B R5C
A A
R5D R5L
0
0
0
0
1
10
100
1000
-I
10000
[TGF-OI, pM
FIG. 1. Cell mutants resistant to growth inhibition by TGF0. [3H]Thymidineincorporation into DNA was determined in parental MvlLu cells and in the six mutant cell lines after they had been incubated for 30 h in the presence of the indicated concentrations of TGF-01. The response of two cell lines to 100 p~ TGF-82 (02) is also shown.
Identification TGF-p Receptor
2274
TGF-I31
-
+TGF-Ol -TGF-Ol
+
Parental R1 B R5C
b
R5D
P-"
R5L
a
S1 A
SIB FIG. 2. Mutations block the fibronectin response to TGF-8. Parental MvlLucells and six mutant cell lines were incubated for 18 h with medium alone or containing 100 PM TGF-Dl. The last 3 h of incubation were in the presence of [35S]cysteine.The radiolabeled fibronectin produced by the cells and released into the medium was displayed by gel electrophoresis and fluorography. Shown are autoradiogram sections that contain the 230-kDa-labeled fibronectin band. The autoradiography of gels containing samples from parental cells was relatively underexposed to illustrate the induction of fibronectin by TGF-P1.
FIG.3. Effect of TGF-@on cell morphology. Parental MvlLu cells, R1B mutants, and S1B mutants were incubated with medium alone or containing 100 PM TGF-61. Phase-contrast photomicrographs were taken after 3 days in culture. The mutant cells failed to respond to TGF-01 with an enlarged and flattened morphology. All photomicrographs are shown a t the same magnification.
six MvlLu cell mutants inresponse to TGF-P1. The intensity of the fibronectin response to TGF-P1 canbe affected by the density of the cell culture^.^ However, the MvlLucell mutants failed to respond to TGF-/3 a t sparse as well as confluent cell density (not shown). Cell flattening and enlarged morphology are typically obcell line was without inhibitory effect on any of the mutant served in association with the strong growth inhibitory recell lines. The mutants resistant to TGF-Pl were also resistant sponse of epithelial and vascular endothelial cells to TGF-P to TGF-P2 (Fig. 1, and data not shown). The proliferation rate of the six TGF-P-resistant mutantcell lines was the same (9,12). Fig. 3 illustratesthis phenomenon in parental MvlLu cells and thelack of it in two representative mutant cell lines, in the presence or absence of TGF-P (notshown). The mutant phenotype in these cell lines is remarkably S1B andR1B. The other four mutant cell lines also failed to stable. Using a replica filter assay to screen for cell colonies respond to TGF-/3 with morphological changes. Inhibition of DNA replication, elevation of fibronectin synthat do not incorporate[3H]thymidine in the presence of TGF-P,2 no spontaneous revertants to the wild type phenotype thesis, and changes in morphology represent separate aspects could be detectedin stock culturesof mutants propagated for of a pleiotropic response to TGF-P. The failure of the six 3 months in the absence of TGF-P. Furthermore, norevertant MvlLu mutant cell lines to display any of these responses clones could be isolated after exposing mutant clones (R1B sdggested that the mutation(s) in each of these cell lines and R5L) to cytosine arabinoside in the presence of TGF-P affected a central component in the TGF-/3 signal transducfor four consecutive cycles to selectively kill the TGF-P- tion mechanism. Therefore, we examined the ability of these resistant population. Thus, mutationalevents inthe six cells to bind TGF-8. Selective Loss of Type I Receptor in TGF-P-resistant MuMvlLu clones are theprobable cause of the stable loss of cell functions required for the growth inhibitory response to TGF- tants-Scatchard analysis of '251-TGF-/31equilibrium binding to parental MvlLu cells indicated the presence of-9,000 P l and TGF-P2. Mutations Block Multiple Responses to TGF-@-Two other surface-binding sites/cell. The curvilinear nature of the Scatresponses of MvlLu cells to TGF-P areincreased production chard plot (Fig. 4) indicated the presence of multiple classes of extracellular matrix proteins and morphological changes of binding sites defined by K d values ranging between 25-300 that lead to a flat and grossly enlarged cell morphology. The PM according to the two limit slopes of the curve. These elevation of fibronectin expression is a response to TGF-P results agree well with the presence of three types of TGF-Pseen in many other cell lines of epithelial and mesenchymal bindingproteins in MvlLu cells, andtheir respective K d origin and is commonly accompanied by marked changes in values deduced from receptor affinity labeled experiments the expression of other matrix proteins and cell adhesion (18; and see Figs. 5 and 6 below). Binding of '251-TGF-P1to receptors (3,10,11,28). TGF-P1 increased the production of monolayers of the six TGF-P-resistant mutants was not extracellular fibronectin %fold over basal levels in the paren- grossly different from the parentalcells, as seen in Fig. 4 for tal MvlLucell line (Fig. 2). This increase contrasted with the four of the six mutant cell lines. However, alteration of a lack of a detectable change in fibronectin production by the given subpopulation of binding sitesmight not be detected by -
F. Boyd and J. MassaguC, unpublished observations.
K. Collart and J. MassaguC, unpublished observations.
Identification TGF-8 Receptor
2275 A.
O
'
*
6. Parental
j
R5C +
S1A
x
S1B
Type 111
-
116
-
Type II-
Type I.
I
!
0.0
5
0
10
15
FIG. 6. TGF-8 binding to receptor types I1andI11 is not affected by loss of type I receptors. A, parental MvlLu cells or Bound, fmolll0 cells R1B mutants were affinity labeled with a low (25 PM) concentration FIG. 4. 12"I-TGF-@1 binding to parental and TGF-@-resist- of lZ5I-TGF-@lalone or with the indicated concentrations of native ant MvlLu cells. Equilibrium binding saturation assays were per- TGF-81 or TGF-82. The autoradiogram from an electrophoresis gel formed with each one of the indicated cell lines by incubation with containing the labeled samples is shown. The R1B samples loaded on increasing concentrations of '251-TGF-81.Specific binding was then the gels were obtained from twice as many cells as the parental cell determined and plotted according to Scatchard (38).The curvilinear samples. B, the same two cell lines were affinity labeled with a high nature of the plot reflects the presence of a complex population of (100 PM) concentration of '251-TGF-pl to saturate type I TGF-8 binding sites; nogross differences are observed between parental and receptors in parental MvlLu cells. No labeling of this receptor type could be observed in R1B cells even under these labeling conditions. mutant cell lines. The wavy line that runs across all the lanes of this figure between the 45- and 68-kDa marks is due to ananomaly of the electrophoresis running front. Parental RIB R5C R5D R5L SIA SIB 6
-
Type
Ill->
I
+
-
+
-
+
-
+
-
+
-
+
-
+
kDa -200
-116
Type
II-:
Type I-:
-94 -68
-45
FIG. 5. Frequent loss of type I TGF-j3 receptors in TGF-@ resistant mutants. Confluent monolayers of parental MvlLu cells and six mutant cell lines were affinity labeled by incubation in the presence of 1251-TGF-@1 alone (-) or with an excess of native TGF81 (+), followed by treatment with disuccinimidyl suberate. Detergent-soluble extracts of the affinity-labeled cells were displayed by gel electrophoresis and autoradiography. The position of the three labeled TGF-8 receptor types and theposition and molecular weight of protein standards areindicated in the autoradiograms shown. Type I receptors are not detectable in four of the mutant cell lines.
measurement of total TGF-@binding but might become apparent upon visualization of individual receptor types by affinity labeling with '251-TGF-@l. Like other cell lines, parental MvlLucells express on their surface TGF-@-binding components of 53 kDa (type I), 73 kDa (type 11), and 300 kDa (type 111) that form affinitylabeled complexes of 65, 85, and 300 kDa, respectively, by cross-linking to '"I-TGF-@l via disuccinimidyl suberate (Fig. 5, and Ref. 17). Analysis of two TGF-@-resistant mutants,
S1A and SlB, and theirsubclones yielded a patternof affinitylabeled TGF-@components very similar to the pattern in parental MvlLu cells. However, mutant cell lines RlB, R5C, R5D, and R5L and all of their subclones lacked detectable type I TGF-@receptors (Fig. 5). This loss contrasted with the normal appearance of the type I1 and I11 TGF-@-binding proteins in all mutantcell lines. To determine whether the absence of type I TGF-@receptors infour of the six TGF-@-resistant mutantswas a random event due to clonal variability, we analyzed various clones isolated from the parental cell line andthe mutagenized MvlLu cell population. Of 30 clones randomly isolated from the parental cell line, and seven TGF-@-sensitiveclones isolated from the same mutagenized MvlLu population that yieled the six mutants described here, none showed loss of TGF-@receptors. The profiles of surface components affinitylabeled with '251-TGF-@1 in these 37 clones were the same as the profile in the parental MvlLu cell population (Table I). Thus, the selective loss of type I TGF-@receptor in four of six TGF-@-resistant mutants was a nonrandom event, and occurred with high frequency only in these mutants. Unimpaired TGF-@Binding to Receptor Types ZZ and ZZZ in Class R Mutants-To detect possible alterations in the ligandbinding properties of TGF-@receptor types I1 and I11 that might derive from the loss of type I receptors, we examined one of the clones, RlB, in more detail. A feature that distinguishes receptor types I and I1 from receptor type I11 is their ability to discriminate between various forms of TGF-@. Receptor types I and I1 have 10-30 times higher affinity for TGF-@1than TGF-@2(18, 22), a property that can be visualized by the higher potency of TGF-@1to inhibit affinitylabeling of these two receptor types in parental MvlLu cells (Fig. 6A). The type I1 receptor present in R1B cells had an unaltered ability to discriminate between TGF-@1 and TGF-
E F - / 3 Receptor Identification
2276
TABLE I TGF-@responses and binding proteins in parental and rnutugenized MvlLu clones TGF-&binding protein
Responses to TGF-@
Source of clones
I
Nonmutagenized parental MvlLu 30130 3013030130" Mutagenized MvlLu (TGF-@ 717 sensitive)717 717 717 Mutagenized MvlLu (TGF-8resistant) 016 216 a Positive clones/total clones analyzed. * ND, not determined in these clones.
I1
717 016616
TABLE I1 Biological churacterization of TGF-8-resistant mutants Gmwth in Cell line Doubling Cell density at Morphology time
confluence
h
ceL5/cma
soft agar
MvlLu 21 293,000 Normal No 24 S1A 315,000 Normal No 32 S1B 351,000 Normal No 20 RIB 355,000 No Normal 37,w R5C Limited 167,000 Normal 27 R5D 277,000 N o d No 148,000 33 R5L Normal No 17 62M 700.oO0b Transformed Limited Two separate determinations; early passages of R5C cells grew significantly slower. Cells continued to grow and shed into the medium as this value was reached.
@2and showed the same affihity for eachof these two ligands
as type 11 receptors in parental cells (Fig. 6A).The type I11 receptor that displays similar affilnity for TGF-B1 and TGFj32 in the parental cell line also retained this property in R1B cells. Some differenceswere observed betweenthe apparent molecular weight of type I11 receptors from parental cells and some of the TGF-8 resistant cell lines (Figs. 5 and 6A). Similar changes occurin the type I11 receptor from the parental cell linewith time in culture and are due to changes in the composition of chondroitin/heparan sulfate glycosaminoglycan side chains present in the type I11 receptor of MvlLu cells (23). The stability of the structural and ligand-bindingproperties of receptor types I1 and I11 contrasted with the loss of functional type I receptors. Type I receptors could not be detected even whenmutant cells were affiiity-labeled with a high (100 PM) concentration of1251-TGF-/31 (Fig.6B). Under these conditions it should have been possible to detect residual type I receptors if they had suffered as much as a 90% reduction in receptor copy number oraffiity for TGF-81 (Fig. 6B). Mutants Retain a Contact-inhibited, NontransformedPhenotype-The basal proliferation rate of the TGF-@-resistant mutants was similar to or slower than that of the parental cell line (Table 11). The proliferation of all six mutants was arrested when cells reached the confluent state, the number of cells/dish remaining essentially constant thereafter. The saturation density number was similar to or lower than that of parental cells. These growth properties contrasted with the higher growth rate andlack of contact inhibition observed in 62M cells, a clone of MvlLu cells transformed with the v-fms oncogene (29) used here as a positive control for oncogenic transformation. The morphology of the parental cell line and six mutant lines was normal (Table II; see also Fig. 3), although mutant lines S1A and R5C exhibited a spindly or elongated morphology. Only R5C and 62M cells formed slowgrowing colonies that contained 10-20 cells after 3 weeks of
I11
Growth inhibition
Fibronectii induction
Flattened morphology
NDb
ND
ND 717
616
016
culture in soft agar. Subcutaneous injection of R5C cells in nude miceled to the formation of tumors at the site of injection in threeof three animals. These tumors reached up to 18 mm in length 6 weeks after injection. 62M cells generated tumors that reached up to 30 mm in length after 6 weeks in all animals injected. Other TGF-&resistant mutants tested, R5D and SlA, did not produce tumors in nude mice after 10 weeks. DISCUSSION
Two Classes of TGF-@-resistantMutants-Isolation of MvlLu cell clonesthat grow in the presence of TGF-8 yields two general classes of stable mutants, those that have lost type I TGF-/3 receptors (class R mutants) and those that retain these receptors but fail to respond to TGF-8 (class S mutants). Two-thirds of the mutants that we have obtained correspond to the R class. The selective loss of type I TGF-/3 receptor in class R mutants is significant because it occurs frequently in TGF-&resistant mutants butis a veryrare event otherwise. Thus, the type I TGF-@receptor is present in each one of 37 parental or mutagenized MvlLu clones that are sensitive to TGF-8. This receptor is also present in the majority of the 96 cell types and tissues screened in this laboratory. Cell lines that lack detectable type I receptors include various human retinoblastoma lines, and PC12 rat pheochromocytoma cells,none of which showany detectable response to TGFs-8 (24): The sensitivity of class R mutants to growth inhibition by TGF-Bl is at least lo00 times lower than parental cells. Given that parental MvlLu cells have a low number of type I TGF-8 receptor copies on their surface estimated at -1000/cell, it is likely that theloss of functional types I receptors in class R mutants is complete. This loss could be due to lack of receptor expression or expression of a nonfunctional receptor protein. The relatively low frequency of isolation of class S mutants was unexpected given the presumed large size of the target for mutations that could generate this phenotype (the entire postreceptor pathway that leads to growth inhibition by TGFj3) compared with the size of the target that is mutated in class R mutants (the type I receptor gene, or the genes that may specifically regulate its expression). Furthermore, class S mutants express type I TGF-8 receptors but areremarkably similar to class R mutants in their failure to display a range of proliferative, biochemicaland morphological responses to TGF-8. The phenotype of class S mutants suggests that they are defective in a central component(s) of the TGF-&signaling transduction mechanism. The similarities between the two classesof mutants suggests that thedefects in S mutants might bein early events in the type I TGF-p receptor signaling pathway. We do not yet know whether any of the class R and class S mutants belong to the same complementation group, a findingthat would suggestthe presence of mutations in the
TGF-@Receptor Identification type I receptor that block signaling without affecting the ability to bind TGF-8. Expression of extracellular matrix fibronectin is a major target for regulation by TGF-@ (10,30) and isprobably involved ingenerating the characteristic morphology of TGF@-treatedcells. The loss of fibronectin and morphological responses in all mutants resistant to growth inhibition by TGF-(3 is of interest. The only other stable TGF-@-resistant mutant cell line previously described (clone 4, Ref. 27) also shows the concomitant loss of growth inhibitory response and morphological response to
[email protected] concomitant loss of multiple responses to TGF-@suggests that they are either causally linked to each other or they aremediated by separate pathways that diverge at a point downstream from the defective component(s) in class S mutants. TGF-8 Resistance, GrowthInhibition, and the Transformed Phenotype-The TGF-@-resistant cell mutants isolated in this study show that thegrowth inhibitory response of MvlLu epithelial cells to TGF-@is neither required for contactinhibition of growth, nor critical for the maintenance of a normal, nontransformed phenotype. TGF-@was originally identified as a secretory product from dense cultures of BSC1 simian kidney epithelial cells that inhibited the growth of sparse cultures of BSC-1 and other epithelial cell lines (15, 31).However, MvlLu cell mutants that have lost growth inhibitory responses to TGF-@become spontaneously growth arrested when they reach a cell density similar to or even lower than thesaturation density of parental MvlLu cells. We have previously proposed that theloss of TGF-@receptors and growth inhibitory responses seen in many human retinoblastoma cell lines might represent an important step in the acquisition of the tumorigenic potential by these cells (24). The hyperproliferative phenotype of cell lines derived from human breast epithelial carcinoma also correlates with their inability to respond to the growth inhibitory action of TGF-@(32). However, the present results show that loss of type I TGF-@receptors and growth inhibitory responses in MvlLu cells isnot sufficient to induce a transformed phenotype. The morphology and growth properties (generationtime, saturation density, and anchorage dependence) of several TGF-@-resistantcell lines are essentially the same as those of the parental line. Of all TGF-&resistant mutants, only M C cells can grow small colonies in soft agar culture and small, noninvasivetumors in nude mice. The clear lack of correlation between TGF-@resistance and phenotypic transformation in MvlLu cells suggest that the loss ofTGF-j3 receptors or responses is not sufficient to induce the transformed phenotype. However, loss of TGF-@growth inhibitory responses might increase significantly therate at which additional events lead to an openly tumorigenic phenotype. Inthis regard, it should be noted that defects in a specific genetic locus (33-35) as well as lack of normal TGF-8 receptor types I1 and I11 accompany the lack of type I TGF-8 receptors in all human retinoblastoma cell lines examined (24). The Type I TGF-@ Receptor as the Mediator of Multiple TGF-@Actions-The present study identifies which one among multiple cell surface TGF-@-bindingproteins is directly involved in mediating TGF-@action. Identification of the type I receptor as the mediator of TGF-@action is based on the frequent and selective loss of this receptor in TGF-@resistant mutants, an event that contrasts with the highly conserved expression of this receptor in the vast majority of cell lines examined. The loss of functional type I receptors occurs without any detectable change in the properties of TGF-@receptor types I1 and 111, suggesting that thedifferent receptors are products of separate genes and contain func-
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tionally independent ligand-binding sites. The link between the pleiotropic growth inhibitory response of MvlLu cells to TGF-8 and the expression of the type I TGF-@receptor was unexpected. TGF-@1 and TGF-82 display similar growth inhibitory potency in MvlLu cells, yet TGF81 has higher affinity than TGF-@2 for the type I receptor. Of the three types of cell surface TGF-@-bindingproteins, only the type I11 receptor has an affinity for TGF-m thatis as high as itsaffinity for TGF-@l. The type I receptor has 1030-fold higher affinity for TGF-@l thanfor TGF-82 (18, 21; this report) independent of the temperature or cell density in the assays. How can the ligand-bindingproperties of the type I receptor be reconciled with the evidence providedby class R mutants that this receptor type is the mediator of TGF-@action in MvlLu cells? There areseveral potential explanations for the discrepancy. An obvious possibility is that TGF-82 might be more effectivethan TGF-@lin activating the signaling function of the receptor upon binding to it. This possibility is essentially untestable until early signaling events of the type I receptor have been identified. A second possibility is that TGF-@2might be more resistant than TGF-@l to degradation in thebiological assays. However,MvlLu cells degrade TGF81 and TGF-@2at similar rates.' A third possibility is that the type I11 TGF-@receptor, which structurally is a membrane heparan/chondroitin sulfate proteoglycan (20,21), might modulate the activity of the type I receptor. Extracellularly, type 111receptors might modulate the response of cells by acting as a nonsignaling binding protein capable of concentrating stores of TGFs-@near the cell surface. As precedents, certain proteoglycans act as reservoirs of growth factors (36), and biologically inactive analogues of polypeptide hormones can display biological activity by inducing the release of active hormone from nonsignaling receptors into the pericellular medium (37). Intracellularly, activation of the type I11 receptor might modulate rate-limiting steps in the type I receptor signaling pathway. Evidence for the hypothesis that thetype I11 receptor may facilitate the action of TGF-82 comes from the observation that TGF-@2 is as potent as TGF-Bl on cells that express type I11 receptors together with type I receptors, such as MvlLu cells (18, 21), but is much less potent than TGF-@lin cells that display only type I receptor (22). Direct testing of this hypothesis is underway. The results obtained with class R and class S mutants indicate that multiple biochemical, proliferative, and morphological responses of epithelial cells to TGF-@originate from activation of a common type I receptor. It is possible that the other receptor types may mediate effects of TGF-@ notassayed in thisstudy, or modulate cell responsivenessto different forms of
[email protected] of other cell types with the approach that we haveused here and characterization of isolated TGF-@-bindingproteins andtheir genes will be needed to extend the emerging concepts on the mechanism of TGF-@action. Acknowledgments-Wethank T. Roberta for 62M cells and J. Rauth for excellent technical assistancein the isolation and analysis of all mutants.
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'J. Rauth and J. Massagub, unpublished observations.
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