A dominant-negative receptor for type beta transforming growth factors ...

Communication

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268,No. 16, Issue of June 5,pp. 11500-11503, 1993 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U S A .

A Dominant-negative Receptor for Type f? Transforming Growth Factors Created by Deletion of the Kinase Domain*

(ll), and to control the expression of at least six cardiac-restricted genes (12, 13). Unlike the global suppression of differentiated gene expression by TGFp in skeletal muscle (14-161, neonatal cardiac myocytes possess a continuum of responses to TGFp1: up-regulation of a gene ensemble, including skeletal a-actin (SkA),expressed preferentially in fetal myocardium, concurrent with down-regulation of genes including a-myosin that are associated with adult ventricular (Received for publication, March 8, 1993) heavy chain (aMHC) muscle (12,13), dichotomous responses which correspond to the Thomas Brand$, W.Robb MacLellans, and generalized “fetal” phenotype produced by mechanical load (4, Michael D. Schneidefl 17). Positive and negative control of developmentally regulated From the Molecular Cardiology Unit, Departments of genes thus coexist i n this system, making the cardiac myocyte Medicine, Cell Biology, and Molecular Physiology and particularly intriguing as a model for studies of TGFO signal Biophysics, Baylor College of Medicine, transduction. Investigations of pluripotent cell lines (18), amHouston, Texas 77030 phibian cardiac progenitor cells (191, and avian cardiac endoTo prove the postulated role of type P transforming thelium (20) also suggest that TGFP-related peptides might growth factors (TGFP) in cardiac development and regulatecardiacorganogenesisitself.However,mechanistic other events, specific inhibitors of TGFPsignal trans- tests of this hypothesis requirea suitable inhibitorof the TGFp duction are needed. We truncated the type I1 TGFP signaling cascade and TGFP-dependent gene expression. receptor cDNA(AkTPRII),to delete the predicted Neonatal cardiac myocytes possess all three of the characserinekhreonine kinase cytoplasmic domain. teristic cell surface receptors for TGFp (TOR) visualized by AkTPRII was co-transfected into neonatalcardiac receptor cross-linking (21). Expression cloning proved the type myocytes, together with reporter constructs for two I1 TPR, a 75-kDa glycoprotein, to possess an intracellular docardiac-restrictedgenes that are regulated antithet- main distinct from the four classes of tyrosine kinase foundin ically by TGFP. AkTPRII impaired activation of the the receptors for platelet-derived, epidermal, insulin-like, and skeletal a-actin promoter by TGFP1, -2, and -3 and, fibroblast growth factors (22). Instead, TpRII resembles the conversely,impaired TGFP inhibition of a-myosin type I1 receptor for activin, a distant member of the TGFp heavy chain transcription. Thus, a kinase-defective superfamily, andDaf-1, a protein controlling larva formation in TPRII blocks signaling by all three mammalian TGFP Caenorhabditis elegans; all three constitute a novel class of isoforms, and can disrupt both positive and negative transmembrane protein with a consensus serinehhreonine kicontrol of transcription by TGFP. nase as the predicted cytoplasmic signaling domain (22-25). TpRII is fully functional in the absence of the type I11 receptor @-glycan), illustratedby the absence of t h i s proteoglycan from The biological importance of TGFP’ has largely been inferred L6 myoblasts (14, 26,27). TpRII is competent to bind TGFp in from the intricate spatial and temporal programthat governs the absenceof the type I TpR, a 53-kDa protein whose structure the impact of this familyof growth factors during development, has notyetbeendefined,butsignalgenerationapparently exogenous TGFp on gene expression or growth (11, and, rerequires a heteromeric protein complex involving both recepcently, the use of neutralizing antibodies (2) or decorin, a TGFptors I and 11 (28). binding proteoglycan (3), to substantiatein vivo effects. Arole Kinase-defective mutations of receptor tyrosine kinases are for TGFp in differentiation and diseaseof the heart is deemed known to inhibit the function of wild-type receptors, possibly by likely (4, 5 ) , given the abundance of TGFp in myocardium (6), a block to the intermolecular autophosphorylationthat follows its up-regulation by infarction (7), mechanical load (8), or adligand-induced dimerization(29-31). Although the correspondrenergic agonists (9), and its ability to protect myocardium ing initial aspects of TGFp signal transduction are less comfrom ischemic injury (lo), to sustain contractility in culture pletely understood, we reasoned that a truncated TpRII, lacking the serinekhreonine kinase domain, would function as a * This work was supported by National Institutes of Health Grants R01 HL39341,R01 HL47567,and T32 HL07706and by American Heart dominant inhibitorof TGFP-dependent transcription.We have Association Grant 91-009790 (to M. D. S.), The costs of publication of used the cardiac myocyte model to demonstratethat the trunthis article were defrayed in part by the payment of page charges. This cated TpRII confers resistance to TGFp control of developmenarticle must therefore be hereby marked “aduertisement”in accordance tally regulated cardiac genes. with 18 U.S.C. Section 1734 solely to indicate this fact. $ Fellow of the Deutsche Forschungsgemeinschaft. EXPERIMENTALPROCEDURES 5 Fellow of the Medical Research Council of Canada and the AmeriPlasmids-To generate the truncated human TPRII by PCR amplican Heart Association-Bugher Foundation Center for Molecular Biology fication, each 100-pl reaction mixture contained 10 ng of TPRII clone of the Cardiovascular System. H2-3FF (22),600 ng of the primers shown, 200 VM of each dNTP, 50mM f Established Investigator of the American Heart Association. To whom correspondence should be addressed: Molecular CardiologyUnit, KCl, 1.5 mM MgCl,, 10 mM Tris-HC1, pH 8.0, and 5 units of Taq polymOne Baylor Plaza, Rm. 506C, Baylor Collegeof Medicine, Houston, TX erase (Promega).Amplification comprised5 min of initial denaturation at 94 “C,then 30 cycles (1 min at 94 “C,1.5 min at 72 “C, and 1min at 77030. Tel.: 713-798-6683;Fax: 713-798-7437. The abbreviations used are: TGFP, type p transforming growth 60 “C) using a Perkin-Elmer Cetus DNA thermal cycler. The final exfactor; aMHC, a-myosin heavy chain; CMV, cytomegalovirus; AkAcRII, tension reaction was for7 min at 72 ”C.The resulting PCR product was analyzed on an 8% polyacrylamide geland had the expected sizeof 883 truncated type I1 activin receptor; AkTPRII, truncated type I1 TGFP receptor; SkA,skeletal muscle a-actin;TPR, TGFp receptor;PCR, polym- nucleotides. For directional subcloning, the products of three PCR reerase chain reaction; SRF, serum response factor; SRE, SRF-binding actions werecombined, purified with a Centricon 100 spin column, element; CDTA, trans-1, 2-diaminocyclohexane-N,N,N’,N’-tetraacetic digested with EcoRI and HindIII, and loaded on a 1.2%agarose gel. The DNA band was excised, and DNA was isolated with the Quiaex gel acid.

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A Dominant-negative TGFP Receptor

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extraction kit (Qiagen). For expression in eukaryotic cells, AkTpRII was subcloned betweenthe EcoRI and HindIII sites of pSV-Sport1 (GIBCO/ BRL), under the controlof the SV40 early promoter and enhancer. The truncatedactivinreceptor cDNA comprisednucleotides 36-787 of pmActR2 (23). Clones were sequenced by the dideoxy method using Sequenase 2.0 (U.S. Biochemical Corp.). The skeletal a-actin reporter, -39#+24SkALuc, was constructed by subcloning nucleotides -394 to +24 of the chicken SkA gene as an RsaI-Hind111 fragment between the SmaI and HindIII sites of the firefly luciferasereporterexpressionvector pXPl (32).TheaMHC-luvehlcle TGFPl TGFK TGFB3 ciferase reporter, 5500aMHCLuc, was prepared by subcloning the intergenic region between the murine pMHC and aMHC gene loci as a FIG.2. AkTmII suppresses up-regulation of the SkA promoter 5.5-kilobase pair Kpn I-Hind111fragment (33)into pXPl (Fig.2 B ) . This by TGFB in cardiac muscle cells. Transfected ventricular myocytes fragment was selected because previous studies had proven its fidelitywere cultured in absence or presenceof TGFps for 36 h and then were t o tissue-restricted, stage-specific, and thyroid hormone-dependentex- assayed for activity of the SkA-luciferase andCMV-lac2 reporter genes. pression of endogenous aMHC (33), and because TGFP-inhibited eleLevels of luciferase expression, correctedfor transfection efficiency, are ments cannot not yet be ascribed to smaller portions of the gene. The expressed relative to that of the SkA promoter in the vehicle-treated, constitutive P-galactosidase expression vector, pCMVp, places the Es- vector-transfected cells. Mean values t S.E. are shown. Open bar, pSVcherichia coli lac2 gene under the transcriptional control of the cytome- Sportl; solid bar,AkTPRII; gray bar, AkAcRII. galovirus (CMV) immediate-early promoter (34). Cell Culture a n d Zhnsfection-Neonatal cardiac myocytes were iso- amino acids in the serinehhreonine kinase motif. For expreslated as previously described from 1-2-day-old rats (12, 13). Myocytes sion in eukaryotic cells, AkTPRII was inserted between the were purified by density centrifugation through aPercoll step gradient (1.050 g.ml", 1.060 g.ml-' and 1.082 gml" Percoll in 116 mM NaCl, EcoRI and HindIII sites of pSV-Sportl, under the transcrip406 p~ MgCl,, 11mM NaH2P04,5.5 mM glucose, 39 mM HEPES, pH 7.3, tional control of the SV40 early promoter and enhancer, up0.002% phenolred) and were plated a t a density of 1 X lo6 cells/35-mm stream from a modified SV40 small t intron and polyadenyladish (Primaria, Falcon). Cells were cultured overnight in Dulbecco's tion signal. modified Eagle's m e d i u d a m ' s n u t r i e n t mixture F-12 (l:l), 17 mM To determine whether AkTPRII could prevent up-regulation HEPES, 3 mM NaHCO,, 2 mM L-glutamine, 50 pg.ml-l gentamicin, 10% of a "fetal" cardiac geneby TGFp1, we co-transfected neonatal horse serum, and 5% fetal bovine serum. Cells were transfected 24 h after plating by a diethylaminoethyl-dextran sulfate method (10 pg of rat cardiac myocytes with (i)AkTpRII or the equivalentvector AkTpRII or pSV-Sportl, 7.5 pg of a luciferase reporter construct, 2.5 pg lacking insert, to control for promoter competition bySV40 of CMV-ZacZ).Cells were incubatedwith the DNA-DEAE-dextran comsequences; (ii) the SkA-luciferase reporter, -394/+24SkALuc; plex for 3 h, then for 60 s with 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium. Cells were cultured overnight in the mediumand (iii) the constitutive lac2 gene, pCMVp, a to correct for described above, which was replaced on the following day by serum-free transfection efficiency, cellrecovery, and potentialglobal effects mediumcontaining 1 pgml-'insulin, 5 pg.ml-' transferrin, 1 nM of the growth factors (Fig. 2). Numerical values for expression Na,Se04, 1 nM LiCl, 25 pg,ml-' ascorbic acid, and 0-1.0 nM thyroxine myosin reporter genesshown below (luciferase, (12, 13). Porcine TGFpl and -2 and chicken TGFp3 (R&D Systems) of the actin and corrected for lacZ) are normalized to that in simultaneous culwere added at final concentration of 1 ngm-', and the medium and growth factor were replaced at 16 h. tures in the absence of TGFP and AkTpRII. In overall agreeLuciferase a n d p-Galactosidase Assays-Cells were harvested after ment with previous results using a related SkA reporter (13), 36 h in the presence or absence of TGFp, in 150 pl of 25 mM Tris phosphate, pH 7.8, 2mM dithiothreitol, 2 mM CDTA, 10% glycerol, 0.1% the -394/+24SkA-luc construct was expressed at levels at least 100-fold greater than in parallel cultures of cardiac fibroblasts' Triton X-100. Luciferase activity was monitored as the oxidation of luciferin in the presence of coenzyme A (35), using an Analytical Lumi- and was up-regulated by 1 n g m - ' T G F p l (2.748 5 0.382, nescence model 2010 luminometer. For lac2 determinations, extracts compared to thevehicle control; p = 0.0005; Fig. 2.4). AkTpRII were incubated with 4.85 m g m - ' chlorophenol red-P-D-galactosidase (Boehringer Mannheim),62.3 mM Na,HPO,, 1mM MgCl,, 45 mM p-mer- reduced SkA expression in thepresence of TGFpl to the basal level found in vehicle-treated cells ( p = 0.0005, uersus the captoethanol for 1 4 h at 37 "C (36) and activity was measured as absorbance at 575 nm. Results were compared by Scheffe's multiple vector control). By contrast to expression triggered by exogecomparison test for analysis of variance and the unpaired two-tailed t nous TGFpl, whose suppression by the truncated TGFpreceptest, using a significance level o f p < 0.05.

I

RESULTS

To construct the truncated TPRII (AkTpRII), we subjected the human TORI1cDNA H2-3FF (22) to PCR amplification, usingprimersthat correspond to nucleotides 306-326 and 1153-1172 and incorporated asymmetric linkersfor directional cloning (Fig. 1).The resultant fragment,encoding the nominal extracellular and transmembrane domains of TORI1 and 272 amino acids of the cytoplasmic domain, was identical to the corrected sequence of Lin et al. (22) and excludes 264 of 298

TPRll 1

336 E+ I

816 902

1172

1967 2036 2090 (nr)

4-H II

FIG.1. Schematic representation of the AkTpRII construct. Predicted domains of the wild-type and truncated human TORI1 are shown. Numbers represent the corrected nucleotide sequences of Lin et al. (22). Arrowheads denote the positionsof the PCR amplimers.

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tor was virtually complete, AkTPRII had little effect on basal, tissue-specific transcription of the SkA promoter. The modest inhibition which was observed was reproducibleand significant (0.670 2 0 . 0 7 2 ; ~= 0.0156), consistent with findingsby Roberts et al. (11)that TGFp is secreted in culture by neonatal rat cardiac myocytes and acts inan autocrine fashion on the cells. The block to SkA induction was specific to AkTPRII; no inhibition of basal or TGFp-induced transcriptionresulted from AkAcRII, a corresponding truncation of the murine typeI1 activin receptor. By contrast, as shown in Fig. 3, exogenous fulllength TPRII amplifies the induction of SkA transcription by TGFp1. Thus, the block to TGFP-dependent transcription by AkTpRII is contingent on truncation of the cytoplasmic domain. One stringent test for the specificity of dominant-negative mutations is whether exogenous wild-type protein can rescue the mutant phenotype. Increasingtheamount of fulllength TpRII cDNA progressively restored the responsiveness of cardiac muscle cells to TGFP1, despite a constant amount of the expression vector encoding AkTpRII. As was true for the truncated activin receptor (37), complete rescue required less than stoichiometric amounts of the wild-type receptor (cf, Refs. 29-31).

W. R. MacLellan, T.-C. Lee, R. J. Schwartz, and M. D. Schneider, unpublished observations.

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Fp

5.5

lac2 gene was indistinguishable inAkTPRII- and vector-transfected cells.3

4.5

DISCUSSION

t 4

Identification of the cDNA sequence for TpRII hasprovided a critical opportunity to construct a truncated receptor variant, - 3.5 3 as a reagent to interdict TGFp signal transductionat thelevel - 2.5 of the receptor itself. These experiments indicate that deletion a 2 of the serindthreonine kinase domain generates a trans-dom1.5 inant inhibitor of TGFP signal transduction. In overall agree1 10 10 0 10 10 WgAkTBRII: 0 ment with this conclusion, Melton and colleagues (37) have 0 2.5 5 7.5 10 ~rg wlTBRII: 0 recently shown that a kinase-defective form of the homologous FIG.3. Rescue of cardiac myocytes’responsiveness to TGFB by type I1 activin receptor can block activin-dependent events in wild-type Tf3RII. Ventricular myocytes weretransfected with AkTPRII early Xenopus embryos. Thus, alteration of the cytoplasmic and full-length TpRII cDNA in the amounts shown and were analyzed for the activity of the SkA-luciferaseand CMV-lac2 reporter genes. For signaling domain may be a generic strategy for producing lossthis experiment, AkTBRII was subcloned into the CMV-driven expres- of-function mutations, notonly in receptor tyrosine kinases but sion vector, pcDNA, to ensure direct comparability with the wild-type also in those whose action depends on a serindthreonine kinase TpRII as provided by Lin and colleagues (22). Results are shown for domain. An additional inference to be drawn from both studies TGFpl-treated cells; full-length TpRII had no effect on basal transcripis that mutationof the respective type I1 receptors is sufficient tion of the SkA promoter in cardiac cells. Luciferase activity (mean ? S.E.) is expressed relative to that of the SkA construct in vehicle- to repress signal transduction with no need for concomitant treated, vector-transfected cells. mutation of other proteins in theligand-binding complex (25, 28). By analogy t o receptortyrosine kinases,mutations of TpRII and the type I1 activin receptor might be expected to act vehicle TGFBl through a block to autophosphorylation intrans. Recently, Massague and colleagues (28) have confirmed the prediction that kinaseactivity is essential for signaling butis superfluous for ligand binding by TPRII. Thus, othermechanisms are plausible to account for the dominant-negative action, including sequestration of TGFp and activin by the truncated receptors or, conceivably, impaired expression of normal typeI1 receptor. “mor AkT$Rll vector AkTpRII An additional caveat is the potential, for which credible support FIG.4. AkTmII suppresses down-regulation of aMHC tran- exists (381, that unexpected tyrosine kinase activity could also scription by TGFB in cardiac muscle cells. Transfected ventricular be inherent to thisclass of transmembrane protein. myocytes were cultured for 36 h in absence or presence of TGFp and The ability of all three isoforms of TGFp t o activate theSkA thyroid hormone and were assayed for activity of the aMHC-luciferase promoter in neonatal rat ventricular myocytes concurs with and CMV-lac2 reporter genes. Luciferase activity (mean S.E.) is expressed relative to that of the aMHC construct in vehicle-treated, their shared ability to antagonize depressive effects of interleukin-1B on beating rate and equivalence for binding to carvector-transfected cells. Solid bar, 1 nM T3; open bar, minus T3. diac cells (11). Analogously, the fact that AkTpRII blocks gene potency Biological actions of the TGFp isoforms, while often similar, activation by all three peptides agrees with their equal differ drastically in some systems. As one illustration, only for inhibition ofDNA synthesis in receptor-defective DR-27 TGFP3 is implicated in the epithelial-mesenchymal transfor- mink lung cells transfected with thefull-length human TpRII cormation required for creation of cardiac valves (20). Therefore, (28). Thus, our results with the dominant-negative TpRII to ascertain whether AkTPRII might disrupt signaling by more roborate the conclusion that TpRII acts as a receptor for all than one form of TGFP, we first tested if neonatal rat cardiac three mammalian TGFp isoforms (28). In contrast to other TGFP-regulated genes, activationof SkA myocytes in fact possess transcriptional responses to TGFp2 and 4 3 (Fig. 2). TGFP2 and $3 each induced the SkApromoter transcription by TGFpl appears to be mediated largely via a proximal serum response factor (SRF)-binding element (SRE) 2 0.506 and 3.910 to at least the same extent as TGFpl(3.882 and a potential TEF-1 site,which are indispensable aswell for ? 0.897, relative toluciferase activity in vehicle-treated cells; p basal, tissue-restrictedexpression.2 However, the 3‘ arm of this = 0.0001 and 0.0001). For both peptides, AkTPRII impaired up-regulation by at least 75% (1.473 ? 0.299 and 1.354 2 0.105; SREpossesses a n overlapping recognition site for a second p = 0.0149 and 0.0474 uersus vector-transfected cells). Thus, SRE-binding protein, thebifunctional transcription factor W1 the truncated TPRII successfully inhibits control of the SkA (39), a competitive antagonist for SRF at this location (40, 41). It is unknown whether TGFp acts through up-regulation of promoter by all three mammalian TGFpisoforms. To facilitate analysisof a TGFP-inhibitedpathway incardiac SRF, modification of SRF, or, conceivably, decreased W1 activmuscle cells, we generated ancYMHC-luciferase construct, since ity. cis-Acting sequences for TGFp repression of aMHC have aMHC is the cardiac gene whose expression, at the mRNA not yet been delineated, but candidate elements within the for cardiac-specific expreslevel, is repressed most completely by TGFPl (12,13). As 5”flanking region that was required sion in vivo include consensus sites both for SRF and the SRFshown in Fig. 4, cYMHC-luciferase activity was highly dependent on thyroid hormone (1.000 t 0.117 uersus 0.093 2 0.028 at related MADS box protein, MEF-2 (33, 42). Genetic methods to obtain a mechanistic understanding of 1 and 0 nM; p = 0.0017) and was inhibited nearly 70% by TGFPl (0.344 ? 0.088; p = 0.0110). AkTPRII specifically abol- growth factor signal transduction canbe confounded by ambigished down-regulation by TGFP1, with no effect on up-regula- uous of counterintuitive results from conventional techniques or loss-of-function mutations. For example, tion by T3. Thus, AkTpRII impairs TGFP-dependent signals for used to create gainboth negative and positive control of gene expression, without induction of endogenous Fos and Jun by mechanical stress is spurious effects on aTGFP-independent pathway. In agreement with this evidence that AkTPRII specifically disrupts T. Brand, W. R. MacLellan, and M.D. Schneider, unpublished obTGFP-dependent transcription, activity of the CMV-driven servations.

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A Dominant-negative TGFP Receptor associated with up-regulationof atrial natriureticfactor in ventricular muscle cells (171, not repression as seen with forced expression (43). Similarly, despite the importance of homologous recombination, an increasingly encountered shortcoming of this strategy is the risk of a misleading or false-negative outcome after disrupting only one member of a redundant multi-gene family. This may be the case for knock-out mutations in TGFpl (44); other recent examples include Fos (4.9, MyoD (461, and E2A proteins (47). Thus, dominant-inhibitory genes such asAkTPRII offer a crucial alternative to otherprocedures for generating loss-of-function mutations.Indeed, there exist corresponding dominant-negative forms of TGFP itself (48). The finitegrowth capacity of cardiac muscle cells in culture precludes stable transfection as a means to uniformly modify ventricular myocytes, which would be required to assess the impact of AkTPRII on other aspects of the cardiac phenotype such as endogenous genes and gene products, signaling intermediaries, or DNA synthesis. This limitation canbe overcome using replication-defective recombinantadenovirus to achieve efliciencies for gene transfer that approach 100% in neonatal and even adult ventricular muscle cells.4 However, cell culture model systems substitute only partially for investigations of cardiac organogenesis itself.By analogy to their use in Xenopus oocytes (31, 371, dominant-negativegenes like AkTPRII might serve as a generic approach, complementaryto gene ablation, to create loss-of-function mutations in transgenic mammals. Acknowledgments-We are grateful to H. Y. Lin, X.-F. Wang, R.A. Weinberg, and H. F. Lodish for the TpRII cDNA, J. Robbins for the aMHC genomic clone, L. S. Mathews and W. Vale for pmActR2, S. K. Nordeen for pXF'1,G. R. MacGregor and C.T. Caskey for pCMVp, F. Ervin for technical assistance, R. Schwartz andB. French for comments on the manuscript, and R. Roberts for encouragement and support. REFERENCES 1. Sporn, M. B., and Roberta, A. B. (1992) J. Cell Biol. 119, 1017-1021 2. Border, W. A., Okuda, S., Languino, L. R., Sporn, M.B., and Ruoslahti, E. (1990) Nature 346, 371374 3. Border, W. A,, Noble, N. A,, Yamamoto, T., Harper, J. R., Yamaguchi, Y., F'ierschbacher, M. D., and Ruoslahti, E. (1992)Nature 380, 361-364 4. Parker, T. G., and Schneider, M. D. (1991) Annu. Reu. Physiol. 53, 179-200 5. Schneider, M. D., and Parker, T. G. (1991)Progr: Growth Factor Res. 3, 1-26 6. Roberts, A. B., Anzano, M. A,, Lamb, L. C., Smith, J. M., and Sporn, M. B. (1981) Proc. Natl. Acad. Sci. U. S. A . 78,5339-5343 7. Thompson, N. L., Bazoberry, F., Speir, E. H., Casscells, W., Ferrans, V. J., Flanders, K. C., Kondaiah, P., Geiser,A. G., and Sporn,M. B. (1988)Growth Factors 1, 91-99 8. Villarreal, F. J., and Dillmann,W. H. (1992)Am. J . Physiol. 262, H1861-Hl866 9. Bhambi, B., and Eghbali, M. (1991)Am. J. Pathol. 139, 1131-1142 10. Lefer, A.M., Tsao, P., Aoki, N., and Palladino, M. A. J. (1990) Science 249, 61-64 11. Roberta, A. B., Roche, N. S., Winokur, T. S., Burmester, J. K., and Sporn,M. B. (1992) J. Clin. Znuest. 90, 2056-2062

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