THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 262, No. 14, Issue of May 15, pp. 6443-6446,1987 0 1987 by The American Society of Biological Chemists, Inc. Printed in U.S.A.
Communication
age-independent growth of untransformed fibroblasts (2) and promote monolayer growth under certain conditions (8). On the other hand, they can inhibitthe growth of certain tumorderived and normal cells (9,101. However, it has been recently appreciated that TGF-Ps can affect profoundly several pro(Received for publication, December 1, 1986) cesses of cell differentiation. TGF-ps inhibitadipogenesis (11) Ronald A. Ignotz, Takeshi Endo$, and myogenesis (12), while they promote chondrogenesis (13) and Joan Massagu8 and thedifferentiation of epithelial cell lines (14). The effects of TGF-Ps on the expression of specific phenotypes occur From the Department of Biochemistry, University of Massachusetts Medical School, Worcester, Massachusetts frequently in the absence of changes in the rate of cell prolif01605 and the $Laboratory of Molecular and Cellular eration. The mechanism(s) by which TGF-Ps induce these Cardiology, Howard Hughes Medical Institute, Department cellular responses has not been established yet. TGF-P1 and of Cardiology, Children’s Hospital and Department of TGF-P2 bind to threedistinct types of high affinity receptors Pediatrics, Harvard Medical School, (15, 16) linked to as yet unknown intracellular signalling Boston, Massachusetts 02115 devices. All three TGF-P receptor species coexist in most cell Human platelet-derived transforming growth fac- types and tissues examined to date. TGF-P is prototypic of a larger family of polypeptide factors tor+ (TGF-81) increases the accumulation of the extracellular matrix proteins, fibronectin and type I col- that controls tissue development in organisms from Drolagen, in mesenchymal and epithelial cells. To deter- sophila to humans. In addition to TGF-01 and TGF-P2, this mine the basis for this effect, we have examined the family includes inhibins and activins that control pituitary levels of mRNAscorrespondingto fibronectin and &(I) cell functions (17), Mullerian inhibiting substance that conprocollagen in NRK-49 rat fibroblasts and LBEBrat trols the development of the Mullerian duct in mammalian myoblasts treated with TGF-81. TGF-81 increased sev- embryos (18), and the decapentaplegic transcript critically eralfold the levels of mRNAs for both proteins. The involved in various stages of Drosophila development (19). kinetics of this effect were similar for bothmRNA Because of their high abundance in platelets, bone, and despecies. The increase in fibronectin and &(I) procol- veloping tissues (20),TGF-(3s are likely to function in abroad lagen mRNAs was detectable 2 h after addition of TGF- range of activities involved in tissue development and repair. 01 to the cells and their maximal levels remained con- We have examined proteins of the extracellular matrix as stant for several days. Actinomycin D, but not cyclo- biochemical targets of TGF-P action because of the known heximide, inhibited the increase in fibronectin and ability of the extracellular matrix to influence the expression &(I) procollagen mRNA levels induced by TGF-81. The results indicate that TGF-81 controls the compo- of individual phenotypes. TGF-p1 and TGF-P2 increase the sition and abundance of extracellular matrices at least synthesis of the fibronectin (12, 21) and collagen (12, 21, 22) in part by inducing a coordinate increase in the levels by many cell types in culture or i n uiuo (22, 23). In addition, TGF-Pl appears to regulate the production of proteins that of fibronectin and type I collagen mRNAs. can modify the extracellular matrix by proteolytic action, such as plasminogen activator and an inhibitor of plasminogen activator(24), procollagenase (25),and several other Type P transforming growth factors (TGF-P)’ are hormon- secreted proteins of as yet unknown function (26). Thus, ally active polypeptides consisting of two 12-kDa chains TGF-Ps might control the expression of specific phenotypes linked by disulfide bonds (reviewed in Refs. 1 and 2). They in vivo by regulating the composition of extracellular matrices. Previous reports concerning the modulation of extracellular are found in a wide range of normal and transformed cells (3, 4) being particularly abundant in platelets (5, 6) and bone matrix components of TGF-fl have dealt with the measurement of the rates of accumulation of fibronectin and type I (7). Two homodimer forms of TGF-P, termed TGF-Pl and TGF-P2, have been identified to date. These forms are struc- collagen (21, 22). This effect of TGF-P could be mediated by turally and biologically related but display only about 70% changes in the rates of synthesis and/or stabilization of the amino acid sequence identity in the 43 amino-terminal resi- various proteins. In this report, we show evidence that TGFdues and show additional differences in thecarboxyl-terminal P l controls the levels of fibronectin and type I collagen, at domains (6). Porcine platelets contain TGF-P1 and TGF-P2 least in part, by increasing the levels of the respective meshomodimers as well as the heterodimer, TGF-P1.2 (6). To senger RNAs. The increase in fibronectin and type I collagen date, only TGF-P1 has been identified in human platelets (5). mRNA levels isan early response of cells to TGF-P and lasts Initial characterization of TGF-P was based on its effects on as long as the factor is present in the medium. cell proliferation. TGF-Ps from various sources allow anchor-
Regulation of Fibronectin and Type I Collagen mRNA Levels by Transforming Growth Factor+*
MATERIALSANDMETHODS
* This work was supported by National Institutes of Health Grants CA 34610 and GM 33577. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recipient of a Career Development Awardfrom The Juvenile Diabetes Foundation. The abbreviations used are: TGF-P, transforming growth factor6; NRK, normal rat kidney.
The sources and culture conditions of NRK-49F rat fibroblasts and L&& rat myoblasts have been described before (3, 21). TGF-Bl was purified to homogeneity from outdated human platelets as described previously (15). Total cellular RNA was extracted essentially as describedbyChirgwin et al. (27) from subconfluent cultures of NRK-49F cells that had been treated with various reagents. To insure that equal amounts of poly(A)+RNA were subjectedto Northern blot analysis, portions of the total RNA samples were subjectedto hybridization to [3H]polyuridylicacid. Briefly, 10-15 p1 of the sample were
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mRNAs Regulated by TGF-/3
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incubated with [3H]polyuridylic acid (Du Pont-New England Nuclear) in 2 X ssc (ssc= 150 mM NaCI, 15 mM sodium citrate, pH 7.0) for 45 min a t 30 “C. Nonhybridized radioactivity was removed by digestion of the incubation mixtures with 40 pg/ml RNase A for 30 min at room temperature. Sampleswere then precipitated by addition of 10% trichloroacetic acid with bovine serum albumin as a carrier. The precipitates were collected on filters and the radioactivity was measured. RNA samples were adjusted such that amounts of RNA giving equivalent hybridization to the [3H]poly(U) were loaded onto the gels. The discrepancy between RNA values quantitated by AZMl absorbance and [3H]poly(U)hybridization was usually less than 10%. Total cytoplasmic RNA from LJ39 cells was isolated as previously described (12). RNA samples were fractionated by the size on 1% agarose gels containing 2.2 M formaldehyde and transferred to nitrocellulose filters as described (12). The filters were hybridized with nick-translated cDNA probes and processed for autoradiography as described previously (12). The ratfibronectin cDNA (rLF-1) was generouslyprovided by Richard Hynes (Massachusetts Institute of Technology) and the chick a2(I) procollagen cDNA (pCG45) was kindly provided by Helga Boedtker (Harvard University). RESULTS
We examined the effect of TGF-P1 on the levels of mRNAs corresponding to fibronectin and type I collagen in NRK-49F fibroblasts,a cell line responsive to TGF-fis. Fig. 1 shows Northern blot analyses of RNA from NRK-49F cells treated with TGF-P1 for 12 or 24 h. A higher level of hybridization of fibronectin as well as &(I) procollagen cDNA probes was observed in the samples from TGF-P-treated cells. The molecular size of the hybridizing species, 8 and 5.2 kilobases, were those expected for mRNAs corresponding to rat fibronectin and a2(I) procollagen (28, 29), respectively. Thus, the increase in fibronectin and type I collagen proteins is correlated with an increase in the respective mRNAs in response to TGF-P. 12h
24h
12h
The kinetics of the increase of fibronectin and a2(I) procollagen mRNA levels are shown in Fig. 2A. An increase in the levels of these mRNAs was detectable by 2 h, reached a maximum at approximately 10 h, and remained constant for at least 24 h following a single addition of TGF-P1 to the cultures. These kinetics are similar to those of increased production of the proteins (21). The effect of TGF-Pl was concentration-dependent, being half-maximal at 25 PM TGFD l (Fig. 2B). Thus, the elevation of fibronectin and type I collagen mRNAs in NRK-49F fibroblasts is an early, persistent, and potenteffect of TGF-fil. The mechanism of the increase in fibronectin and a2(I) procollagen mRNA in response toTGF-P is not known. However, treatment of cultures with the inhibitor of transcription, actinomycin D, blocked the increase in fibronectin (Fig. 3) and a2(I) procollagen (not shown) mRNAs induced by TGF-P1. This suggests, but does not unequivocally demonstrate, the involvement of transcriptional events in the response. To assess the possible role of protein synthesis in this action of TGF-P1, cultures were treated with TGF-Pl and the protein synthesis inhibitor, cycloheximide, concomitantly. In the presence of cycloheximide, TGF-P1 was still able to increase the mRNA levels (Fig. 3). This result suggests that de novo protein synthesisis notrequired for the induction of fibronectin and collagen mRNAs by TGF-fi1.
A
24h
-+-+
0 2 4 6 81012
24
HOURS WTH TGF- p
r> 28s
-
18s
-
0
FIBRONCTIN
a2(1) COLLAGW
FIG. 1. Analysis of mRNA levels for fibronectin and a2(I) collagen following treatment with TGF-8. NRK cells were grown to near confluency and then placed into serum-free medium with or without 500 PM
[email protected] either 12 or 24 h, cultures were harvested and total RNA was extracted and isolated according to Chirgwin et al. (27). Equal amounts of poly(A)+RNA were analyzed, based on quantitation by hydridization to [3H]polyuridylicacid (see “Materials and Methods”). Amounts of RNA (approximately 15 pg of total cellular RNA) that gave equal levels of [3H]poly(U)cpm hybridizedwere electrophoresed on 1% agarose/formaldehyde gels and transferred onto nitrocellulose. The resulting blots were hybridized to either a fibronectin cDNA probe or ana2(1) collagen cDNA probe that had been 3ZP-labeledby nick translation. The arrows indicate the positions of fibronectin mRNA (-8 kilobases) and a2(I)collagen mRNA (-5.2 kilobases). The positions of 28 S and 18 S rRNAs are indicated.
Q
.
0 50 1 0 0
250
500
TGF-P bM)
FIG.2. Parameters of the TGF-8 effect on fibronectin and collagen mRNA levels. A , cultures of NRK cells were placed into serum-free medium supplemented with 500 PM TGF-@. After the indicated lengths of time, cultures were harvested and total cellular RNA was isolated and analyzed by Northern blotting with hybridization to either the fibronectin probe (0)or the a2(I)collagen probe (0).The blots were subjected to autoradiography, and the relative intensity of the autoradiographic signal was determined by densitometry. The results are expressed as the ratio of mRNA levels in TGF@-treateduersus untreated cells. B , cultures of NRK cells were placed into serum-free medium supplemented with varying concentrations of TGF-@. After 12 h, the RNA was extracted and subjected to Northern blot analysis for fibronectin (0)and collagen (0)mRNAs. The resulting autoradiographs were analyzed by densitometry. The results are expressed as indicated above.
mRNAs Regulated by TGF-p
1 A
FN -
0
6445 "TGF-8 3 6 9
I
+TGFp 6 9
3
I
1
Rescued 6days
3
MHC
,$,y-Actin .a-Actin .Troponin T MLC 2
28S-
B 18S-
FIG. 3. Effect ofcycloheximide and actinomycin D on TGFfl induced increases in fibronectin mRNA levels. Cultures were treated simultaneously with TGF-@(500 pM) and either 2 pg/ml of cycloheximide or 250 ng/ml of actinomycin D. After 6 h of treatment, the cells were harvested and the RNAs were extracted and subjected to Northern blot analysis and autoradiography to detect fibronectin mRNA. (FN).
Another cell type examined in these studies was L6E9rat skeletal muscle myoblasts. Like other lines and primary cultures of myoblasts, L6E9 cells are inhibited by TGF-81 in their ability to undergo terminal differentiation to form myotubes (12).TGF-p prevents expression of the muscle-specific mRNAs and proteins, a-actin, myosin heavy and light chains, a- and P-tropomyosin, and troponin subunits in these cells (12) (see also Fig. 4A).L6E9cells and therelated clonesretain their undifferentiated phenotypeof myoblasts as long as TGFp is present in the medium (12) (Fig. 4A). In addition, it has been shown that manipulation of the extracellular matrix of these cells by addition of exogenous fibronectin inhibits the expression of the muscle phenotype (30) and that treatment of these cells with TGF-P increases the production and accumulation of fibronectin and collagen (12, 21). Thus, rat skeletal muscle myoblasts provide an advantageoussystem to study the role of the extracellular matrix incontrolling expression of developmentally regulated muscle-specific genes and commitment to terminal differentiation. We examined the levels of fibronectin and type I collagen mRNAs, comparing them with those of muscle-specific mRNAs and of P- and yactin mRNAs, during inhibition of myogenesis by TGF-p. TGF-p1 elevated fibronectin and a2(I) procollagen mRNAs strongly (Fig. 4B). The effect lasted as long as TGF-Pl was present in the medium and disappeared gradually after the transfer (rescue) of the differentiation-blocked cells to normal differentiation medium lacking TGF-Dl. The progressive decrease in the levels of these mRNAs was observed during differentiation of L6E9cells (Fig. 4). Thisis in agreement with previous observations in theirrelated myoblast cell lines (31). Interestingly, while fibronectin and collagen mRNAs were greatly induced in TGF-pl-treated myoblasts, the levels of pand y-actin mRNAs declined to some extent in the presence of TGF-P1 (Fig. 4). This finding is in contrast with the TGFP-induced increase in P- and y-actin mRNA levels in AKR2B mouse fibroblasts (32). DISCUSSION
The altered expression of fibronectin and typeI collagen in transformed cells offers a precedent for the involvement of
FIG. 4. Effect ofTGF-/3on induction of fibronectin and crB(1) collagen mRNAs in LsEsrat myoblasts. Cultures of L6E9 rat myoblasts were treated with TGF-@ to inhibit their differentiation into skeletal myotubes as previously described (12). RNA was extracted from differentiating (-TGF-@), TGF-@-treated (+TGF-@), and rescued cultures. A , RNA samples (10 pg) were subjected to Northern blot analysis to detect mRNAs specific for myogenic differentiation (myosin heavy chain ( M H C ) , a-actin, troponin T, and myosin light chain 2 (MLC 2)) as well as @- and y-actinmRNAs (also shown in Ref. 12, Fig. 2). B, the radioactivity of the blot ( A ) was erased by washing the filter in 0.01 X SSC for 30 min a t 90 "C, and then the filter was rehybridized to detect fibronectin ( F N ) and a2(I) procollagen (pro a2(Z))mRNAs. Arrowheads, the 18S and 28 S rRNA positions.
transcriptional aswell as post-transcriptional changes in the regulation of fibronectin and collagen expression. For example, decreased transcription of fibronectin and typeI collagen genes has been observed in cells transformed by Rous sarcoma virus or the oncogene v-mos (28, 33-35). The levels of these proteins can also change due to alterations in theirstability (36). Thus, different ways might existby which TGF-ps exert their previously observed effects on fibronectin and type I collagen expression. The results described here imply that TGF-P1 increasesthe synthesis of fibronectin and type I collagen at least inpart by increasing the levels of their corresponding mRNAs. Events sensitive to actinomycin D appear to be required for this response. These observations together with results from previous biosynthetic labeling experiments (21, 22) indicate that increased synthesis of fibronectin and type I collagen is a major component of the action of TGF-ps on extracellular matrices. These results do not exclude, however, the possibility thatadditional control by TGF-ps could be exerted at the level of post-transcription, translation, or the stabilization of the proteins. The parallel kineticsof the effects of TGF-P1 on fibronectin and type I collagen mRNA levels point to thepossibility that TGF-/3 controls the activity of cellular elements that regulate in a concerted fashionthe expression of fibronectin and type I collagen. TGF-/3 could accomplish this effect by a variety of mechanisms, butthe resultsobtained with cycloheximide argue against the possibility that TGF-/? acts by increasing the synthesis of proteins involved in regulating fibronectin and type I collagen mRNA levels. The possible consequences of altering thecomposition and abundance of extracellular matrices infibroblasts, myoblasts, and other cell types of TGF-ps in. vivo remain to be established. However, it has been found that induction of anchorage-independent growth of cultured NRK-49F cells by TGFfl is preceded by accumulation of cell-derived fibronectin in
mRNAs Regulated by TGF-/3
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the soft agar cultures' andcan be inhibited by synthetic peptides that prevent the assembly of fibronectin-based matrices (21). Likewise, a causal link appears to exist between changes in the extracellular matrix of myoblasts and preadipocytes induced by TGF-(31, and the ability of this factor to prevent myogenic and adipogenic differentiation (11, 12). These observations imply that control of the composition and abundance of extracellular matrices might be a central component of the action of TGF-6s in physiological processes of tissue development and remodeling in which these factors intervene. Acknowledgments-We thank Richard Hynes and Helga Boedtker for cDNAs, Betsy Like for assistance with tissue culture, and Judith Kula for preparation of the manuscript.
REFERENCES 1. Massagu6, J. (1985) Trends Biochem. Sci. 10,237-240 2. Sporn, M. B., Roberts, A. B., Wakefield, L. M., and Assoian, R. K. (1986) Science 233,532-534 3. Assoian, R. K., Roberts, A. B., Wakefield, L. M., Anzano, M. A., and Sporn, M.B. (1985) Cancer Cells 3/Growth Factors and Transformation, pp. 59-64, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 4. MassaguB, J. (1984) J. Biol. Chem. 269,9756-9761 5. Assoian, R. K., Komoriya, A., Meyers, C. A., Miller, D. M., and Sporn, M. B. (1983) J. Biol. Chem. 2 5 8 , 7155-7160 6. Cheifetz, S., Weatherbee, J. A., Tsang, M. L.-S., Anderson, J. K., Mole, J. E., Lucas, R., and Massagui., J. (1987) Cell 48,409-415 7. Seyedin, S. M., Thompson, A.Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Bwl. Chem. 2 6 1 , 5693-5695 8. Shipley, G. D., Tucker, R. F., and Moses, H. L. (1985) Proc. Natl. Acad. Sei. U. S. A . 82,4147-4151 9. Tucker, R. F., Shipley, G. D., Moses, H. L., and Holley, R.W. (1984) Science 2 2 6 , 705-707 10. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D.F., and Sporn, M.B. (1985) Proc. Natl. Acad. Sci. U. S. A . 8 2 , 119-123 11. Ignotz, R. A., and Massaguk, J. (1985) Proc.Natl.Acad.Sci. U. 5'. A . 82,8530-8534 12. MassaguB, J., Cheifetz, S., Endo, T., and Nadal-Ginard, B. (1986) Proc. Natl. Acad. Sci. U. S. A . 83,8206-8210 13. Rosen, D. M., Stempien, S. A., Thompson, A. Y., Brennan, J. E., Ellingsworth, L. R., and Seyedin, S. M. (1986) Exp. Cell Res. 165,127-130
R. A. Ignotz and J. Massagui., unpublished observations.
14. Masui, T., Wakefield, L.M., Lechner, J. F., LaVeck,M. A., Sporn, M. B., and Harris, C.C. (1986) Proc. Natl. Acad. Sci. U. S. A . 83,2438-2442 15. MassaguL, J., and Like, B. (1985) J. Bwl. Chem. 260,2636-2645 16. Cheifetz, S., Like, B., and Massague, J. (1986) J. Bwl. Chem. 261,9972-9978 17. Ling, N., Ying, S.-Y., Ueno, N., Shimasaki, S., Esch, F., Hotta, M., and Guillemin, R. (1986) Nature 321, 779-782 18. Cate, R. L., Mattaliano, R. J., Hession, C., Tizard, R., Farber, N. M., Cheung, A., Ninfa, E. G., Frey, A. Z., Gash, D. J., Chow, E. P., Fisher, R.A., Bertonis, J. M., Torres, G., Wallner, B. P., Ramachandran, K. L., Ragin, R. C., Manganaro, T. F., MacLaughlin, D. T., and Donahue, P. K. (1986) Cell 45,685-698 19. Padgett, R. W., St. Johnston, D., and Gelbart, W.M. (1987) Nature 3 2 5 , 8 1 4 4 20. Ellingsworth, L. R., Brennan, J. E., Fok, K., Rosen, D. M., Bentz, H., Piez, K. A., and Seyedin, S. M. (1986) J. Biol. Chem. 2 6 1 , 12362-12367 21. Ignotz, A.,R. and Massague, J. (1986) J. Bwl. Chem. 261,43374345 22. Roberts, A. B., Sporn, W. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H., and Fauci, A. S. (1986) Proc.Natl.Acad.Sci. U. S. A . 83,4167-4171 23. Sporn, M. B., Roberts, A. B., Shull, J. H., Smith, J. M., Ward, J. M., and Sodek, M. (1983) Science 219,1329-1331 24. Laiho, M., Saksela, O., and Keski-Oja, J. (1986) Exp. Cell Res. 164,400-407 25. Chua, C. C., Geiman, D. E., Keller, G. H., and Ladda, R. L. (1985) J. Biol. Chem. 260,5213-5216 26. Nilsen-Hamilton. M.. and Hollev, - . R. W. (1983) Proc. Natl. Acad. Sci. U. S. A . 8 0 , 5636-5640 27. Chirewin. J. M.. Przvbvla. " " . A. E.. MacDonald. R. J.. and Rutter. WTJ. (1979) Biochemistry 18,'5294-5299 28. Fagan, J. B., Sobel, M. E., Yamada, K. M., decrombrugghe, B., and Pastan, I. (1981) J. Biol. Chem. 2 6 6 , 520-525 29. Adam, S. L., Alwine, J. C., decrombrugghe, B., and Pastan, I. (1979) J. Biol. Chem. 254,4935-4938 30. Podleski, T. R., Greenberg, I., Schlessinger, J., and Yamada, K. M. (1979) Exp. Cell Res. 1 2 2 , 317-326 31. Leibovitch, S. A., Hillion, J., Leibovitch, M.-P., Guillier, M., Schmitz, A., and Harel, J. (1986) Exp. Cell Res. 166,526-531 32. Leof, E. B., Proper, J. A., Getz, M. J., and Moses, H. L. (1986) J. Cell Physwl. 127,83-88 33. Tyagi, J. S., Hirano, H., Merlino, G. T., and Pastan, I. (1983) J. Biol. Chem. 258,5787-5793 34. Sandmeyer, S., Gallis, B., and Burnstein, P. (1981) J. Biol. Chem. 256,5022-5028 35. Setoyama, C., Liau, G., and decrombrugghe, B. (1985) Cell 4 1 , 201-209 36. Olden, K., and Yamada, K. M. (1977) Cell 11,957-969 '
