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M i n i - R e v i e wSom e Recent Advances in the Chem istry and Bio logy ofTransforming Grow th Factor-BetaM i c h a e l B . S p o r n , A n i t a B . R o b e r t s , L a l a g e M. W akefield, a n d B e n o i t d e C r o m b r u g g h eNational Cancer Institute, Bethesda, Maryland 20 892

W ITHIN the past two years, there has be en an expo -nential increa se in research on transforming grow thfactor-beta (TGF-beta). ~ In this brie f minireview,we can not provide a de ta i led su rvey of this topic (see refer-enees 56 and 73 for other reviews) . Rather , we w i l l summa-rize so me new results, indicative of the importance o f thispeptid e as a multifunetional regulator o f cellular activity.The term ~n ul t ifunet ional" impl ies tha t TGF -beta may e i therstimulate cell proliferation and grow th, or inhibit ce ll prolif-era t ion and grow th, or have num erous oth er ac t ions havingl it tle re la t ionship to e i ther o f these two processes. We w i l ldevelop the theme tha t m any o f the ac t ions o f TGF-beta arere la ted to the response of ce l ls or t i ssues to st ress or injury,and to the repair o f resultant da mage . How ever, i t is clear thatthere i s no o ne pr inc ipal ac t ion fo r TGF-beta; moreover , thea lmost universa l ce l lular distr ibut ion of i t s receptor encom -passes a very broad spect rum o f ta rget t i ssues.Chemical Structure of TGF-betaDe spite a com mo n nomenclature, th e purification, cloning,and sequencing of TGF-alpha and TGF-beta have made i tclear that these are tw o entirely distinct peptides, each actingthrough i t s own unique receptor system. W e wi ll not discussTGF-alpha in this minireview. TG F-beta was originally puri-f ied to homog enei ty f rom human plate le ts (5) , hum an pla-centa (24) , and bovine kidney (58) and identi fied as a hom o-dimeric pept ide wi th a molecular mass of 25,000 D. Thehuman eD NA clon e sequence indica tes tha t the monom er issynthesized as the CO OH -terminal I12 amino ac ids of a 390-amino ac id precursor 05) . Recent eDN A cloning in our lab-ora tory has shown that there i s tota l sequ ence ident ity be-tween the respect ive human, bovine , and porc ine maturemo nom er sequences (Van Obbergh en, E. , and P. Kondaiah,personal comm unicat ion) ; there i s a s ingle amino ac id sub-sti tution in the mouse peptide (14).The original purification of TGF-beta use d the stimulationof anchornge-independent grow th of norm al fibroblast indi-cator cells as a functional assay (5, 24, 57, 58), and the nam eof the peptide is based on this activity. Since then, TGF-beta- l ike m olecules have been puri fied to homogenei ty f rom1 . A b b r e v i a t i o n s u s e d i n t h i s p a p e r : EGE epidermal growth factor; TGF-beta, transforming growth factor beta.

a variety of sources using other assays, reflecting the mul-tifunctional nature of this set of molec ules. Thus, two bovin ecar t i lage- inducing p ept ides isola ted f rom bone and a hum animmunosuppressive peptide isolated from glioblastoma cells,when purif ied and sequen ced, were each found to be one orthe other of two molecular forms o f TGF-beta . These twodistinct form s were identified first in bovine bone (66), fromwhich they were i sola ted using an assay tha t m easures induc-tion of extracellular matrix p roteins characteristic of carti-lage, and hence wer e nam ed carti lage-inducing factors A andB (66). C artilage-inducing facto r A is identical (67 ) to theform o f TG F-beta that was originally isolated from hum anplatelets (5), no w known as TG F-beta 1 (11), wh ile cartilage-inducing factor B represents a novel mo lecular form o f TGF-beta (65) , no w cal led TG F-beta 2 (11). Both form s have sub-seque ntly been fo und in po rcine platelets (11), and in bothbovine bone and porc ine pla te lets TGF-beta 2 i s less abun-dant than TGF -beta 1, constituting only ,o15-2 0% of the totalrecovered "l 'GF-beta. This second type of TGF-beta alsoshows remarkab le sequ ence conservation, in that no differ-ences have yet been found in the respect ive bovine and por-c ine TG F-beta 2 sequences (11, 65) , in spi te o f the fac t tha tboth have only a 69% hom olog y wi th the f irst 36 residuesof type I TGF-beta . The recent discovery of TGF-beta 2 inthe conditioned me dium o f a human gliohlastoma cell line(80) makes i t apparent tha t both types of TGF-heta are a lsorepresented in the human genom e. Interestingly, the hum antype 2 pep t ide was isola ted and charac terized by i t s immun o-suppressive ac t ivi ty (many gl ioblastomas are st rongly imm u-nosuppressive to the pa t ients wi th such tumors) , and thepurification was monitored w ith assays that meas ured the in-hibition of mitogenesis in T-lymphoeytes (80), another knownactivity of TGF-beta 1 (34). T he exact chem ical identit ies oftwo other pept ides c lear ly re lated to TGF-beta , nam ely thegrowth inhibi tor secre ted by monkey BS C-1 kidney ce l ls inculture (29, 74), and th e my oblast differentiation inhibitorsecreted by rat l iver cells in culture (21, 45, 50 ), st il l needto b e established.In m ost assays, the tw o forms o f TGF-beta are funct ionallyindistinguishable. However, the data at hand ind icate thatthere may be separa te receptors for TGF-beta 1 and TG F-beta2, som e of which are cross-react ive (11, 64) . Al l of these newfindings suggest that there may be some unique function for

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the secon d form of TGF-beta, b ut as yet, none has beendefinitively shown. Is it possibly a protein with a un ique em -bryo nic or tissue-specific function? Wh y is it found in por -cine platelets, but not human? I t is conceivable that some ofthe intrinsic cellular activities that have bee n attributed in thepast to 'q'GF-beta" are a reflection of the endogenous prod uc-tion of TGF-beta 2, as well as TGF-beta 1. Although greateramounts of TGF-beta 2 , com pared to TGF-beta 1, have notyet been reported in any tissue, this may be a possibility.It is now also clear that both these forms o f TGF-beta be-long to a much larger ge ne family, of which most membe rshave growth-regulatory functio ns (11, 51). This fam ily in-eludes two forms of inhibin (a gonadal protein that sup-presses pituitary sec retion of follicle stimulating hormon e),three forms of activin (another gonadal protein that stimu-lates follicle stimulating hormone secretion), Miillerian in-hibitory substance (a protein causing regression of the fe-male rudiments in the developing ma le reprodu ctive system),and a transcript from the decapentaplegic gene comp lex ofDrosophila, which acts to control morphogenesis in the flyembryo. A lthough all of these peptides show strong ho mol-ogy with TGF-beta with respect to the position of seven ofnine total cysteine residues, no cross- reac tivity with TGF-beta with respect to receptor binding has been shown fo r anyof these peptides.Latent TGF-BetaMany studies have shown that TGF-beta is se creted by virtu-ally all cell types in a biologically inactive form (35, 37, 39,40, 77). The biolog ical latency appears to be due to an inabil-ity to bind to the TGF-beta re ceptor (77). In the laboratory,this latent form has generally been activated by transientacidification, although alkali or chaotropie agents can alsoactivate, suggesting the process may involve disruption of anoncovalent complex (37, 39, 40). Recen t studies have begunto identify the chem ical nature of this complex and possiblephysiological mechanisms of activation (35, 76). The com-plex itself appears to be formed from the association of ma-ture, dimeric TGF-beta, together with the "precursor remain-der; which results from the proteolytic cleavage of matureTGF-beta from its precth-sor (at the Arg-Ala bon d in position278), plus a third compo nent (76). By analogy with the epi-dermal growth factor- and nerve growth factor-binding pro-teins, the third component might be a processing protease,involved in cleavage of the TGF-beta precursor.While the latent com plex might be activated in vivo by ex-posure to acidic microenvironments such as are found in thevicinity of the osteoclast or in healing w ounds, it seems mo relikely that it is activated by the action of exogenous prote asesthat disrupt the quaternary structure of the complex. Thus,plasm in and cathepsin D can activate latent TGF-beta in vitro(35). The exact chemical structure o f latent TGF-beta and thephysiological mechanism of activation of the complex are o fgreat importance. Since the cellular receptor for TGF-betaappe ars to be essen tially universally a nd constitutively ex-presse d (77), the target specificity of TGF-beta action maybe determined by the ability of a cell to activate the latentcomplex, and activation may be a critical regulatory step inTGF-beta action. Thus, for example, platelets release TGF-beta in the latent form (54, 76). This may provide an idealmechanism to allow sustained action of TGF-beta during

stress or injury, since the duration o f action of many peptidehormones is very short i f they are not protected by a bindingprotein. In another situation, unregulated epithelial ce llgrowth ma y be a re sult of failure to activate th e latent formof autocrine TGF-beta. Thus, the human A549 lung carci-noma cell, which has abundant receptors for TGF-beta andis strongly inhibited in its growth by exogenous active TGF -beta , sec retes large amounts o f TGF-beta in the latent form.However, this tum or cell appears to have lost the ability toactivate latent TGF-beta and hence continues to proliferatein the presence of high concentrations of autocrine latentTGF-beta (77); in contrast, the parent normal cell type ap-pears to be inhibited by the TGF-beta that it secretes(Wakefield, L., and T. Masui, unpublished data).Proliferative Effects of TGF-Beta In VitroIn most respects, the intrinsic role of TGF-beta in the in vivophysiology of the organism is an unknown. On the otherhand, there are now many different actions that have beenshown in various cell culture systems, which c an arbitrarilybe categorized as either proliferative, antiproliferative, orunrela ted to proliferation. The original disco very of TGF-beta in an assay that measured promotion of anchorage-in-depende nt growth of fibroblasts clearly establishes TGF-betaas a bona fide growth factor, and there are many examplesof cells of mesenchymal origin, in which proliferation is en-hance d by TGF-beta. O ne such cell type that is the focus ofintense interest at present is the osteoblast, because of theimportance o f this cell for the formation of new bone duringfracture healing, as well as fo r the maintenance of existingbone to prevent osteopo rosis. Two gro ups (10, 61) have re-cently shown that no t only do cultured osteoblasts have high-affinity receptors for TGF-beta and respond to exogenousTGF-beta w ith a mitogenic response, they also produ ce andsecrete TGF-beta, implying that there may be some autocrinegrowth c ontrol in bone, possibly related to bon e remodeling;the highly acid microenvironme nt of the osteoclast may pro-vide a me chanism to activate latent TGF-beta released b y os-teoblasts. The impo rtance of TGF-beta for bone function isfurther emphasized by its key role in controlling formationof proteins of extracellular matrix, as will be discussedbelow.Another cell type that has recently been shown to have arnitogenic response to TG F-beta is the Schwann cell o f theperipheral nervous system, for which there are few knownmitogens (Rather, N., personal communication). Th e Schwanncell makes myelin and is involved in the repair of peripheralnerves after injury, although no role for TGF-beta in theseprocesses is known at present. Like the osteoblast, the Schwanncell is highly specialized f or synthesis o f extracellular matrix(although the set of matrix proteins formed b y Schwann cellsis different from those forme d by osteoblasts), and it will beof interest to determ ine w hether "l'GF-beta has any uniquerole in promoting matrix formation in this particular cell.Antiprolifera tive Effects o f TGF-Beta In VitroTGF-beta is a po tent inhibitor of the proliferation of manycells in vitro, particularly of epithelial cells (46, 48, 74).Moreover, th ese antiproliferative actions are not confined toepithelial cells, and strong antimitogenic effects are seen inmesenchym al cells such as em bryonic fibroblasts (1), en-

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dothel ia l ce l ls (6 , 22, 26) , and T- and B-lymphocytes (33,34 ) . Bas ed on the s e in v i t ro expe r imen t s , i t appea rs tha tT GF-be ta migh t be an impor tan t nega t ive g rowth con t ro l fo rmany ce l l types . Whether " l 'GF-beta t ru ly funct ions as aphys io log ica l nega t ive au toc r ine o r pa rac r ine g rowth fac to rin v ivo i s s ti l l unp roven . E n hanced l eve l s o f mR NA fo r T GF-be ta have been s hown in the regene ra t ing l ive r a t a t im e wh enDN A s yn thes is beg ins to d imin i s h , and i t ha s been s ugges tedtha t T GF-be ta may ac t a s a regu la to ry s igna l to s top fu r the rce l l repl ica t ion as regen era t ion is comple te d (9 , 19) . Recen ts tud ie s have a l s o im p l i ca ted T GF-be ta a s a nega t ive g rowthcon t ro l fo r mam ma ry ep i the l ium in v ivo ; minu te p la s ti c im-p lan t s , con ta in ing T GF-be ta in a s us t a ined re l ea s e fo rm,w e r e p l a c e d i n t h e m a m m a r y g l a n d s o f m i c e a n d f o u n d tocaus e m arked s uppre s s ion o f the g rowth o f ep i the l ia l duc ta lend buds (D an ie l , C . , and G . S i lbe rs t ein , pe r s ona l com mun i -ca t ion ) . However , t h i s u s e o f exogenous T GF-be ta does no tdemons t ra t e tha t T GF-be ta i s an endogenous regu la to r inv ivo , and fu r the r s tud ie s , u s ing an t ibod ie s o r an tagon i s t s ,w i l l be requ i red to p rove th i s po in t .Dur ing ca rc inogenes is , t he pa ren t c e i l s o f the even tua lma l ign an t c lone may lo s e the i r s ens i t iv ity to g row th regu la -t ion by T GF-be ta ; m echan i s ms a s d ive rs e a s fa i lu re to s yn -thes ize , process , or re lease TG F-beta ; loss of receptors forTGF -beta ; loss of abi l i ty to ac t iva te la tent TGF-b eta ; or a fa i l -u re in the in t race l lu l a r T GF-be ta s igna l ing pa thway have a l lbeen s ugges ted a s con t r ibu t ing to ca rc inogenes is . T h e re i ss ome expe r imen ta l ev idence fo r s eve ra l o f the s e pos s ib i l it i e s(68; Wakefie ld , L . , unpubl ished data) . However, there areep i the l ia l t umo r ce l l s t ha t s t il l r e t ain s om e m eas u re o f g rowthregu la t ion by T GF-be ta , pa r t i cu la r ly the mu ch s tud ied ho r -mo na l ly re s pons ive M CF-7 hum an b rea s t c ance r ce ll . T hes ece l l s have func t iona l r ecep to rs fo r T GF-be ta and s ec re tes ma l l amou n t s o f b io log ica l ly ac t ive T GF-be ta unde r ba s a lcond i t ions and much l a rge r amoun t s o f a c t ive T GF-be tawh en t rea ted w i th g rowth - inh ib i to ry concen t ra t ions o f an t i -e s t rogens s uch a s t amox i fen o r i t s a c tive me tabo l i te , hydroxy-t amox i fen (36 ) . T hus , i n th i s s ys t em, T GF-be ta ha s beens hown to be a ho rmo na l ly regu la t ed g rowth inh ib ito r w i th anega t ive au toc r ine ac t ion on i t s p roduce r ce l l .Bio log ica l Ef fec t s o f TGF-Beta Unre la ted toPro l i f era t ionTGF -beta has effec ts on m any differe nt ce l l types , unre la tedto control o f prol i fera t ion. The se effec ts a re so varied tha tthey do no t appea r to con fo rm to any pa r t i cu la r pa t te rn , a l -though i t i s o f in t e re s t t ha t t he ac t ion o f T GF-be ta on m anyce l l s i s o f t en un ique ly re l a t ed to the regu la t ion o f the s pec ia l -i z ed , c r i t i c a l func t ion o f a pa r t i cu la r c e l l t ype .Pe rhaps the m os t s t r ik ing recen t advance tha t ha s occu r redin T G F-be ta s tud ie s in the pa s t two yea rs ha s been the e luc i -da t ion o f an ex tens ive ro le fo r T GF-be ta in enhanc ing the fo r -ma t ion o f ex t race l lu l a r ma t r ix , and th i s i s undoub ted ly o fma jo r im por tance w i th respect to embryog enes is and to repairo f t i s s ue in ju ry . T GF-be ta ha s m any d i rec t a c t ions on f ib ro -blas ts . Thus , i t i s a potent chemotact ic agent for these ce l ls(55). Furthe rmo re , i t s t imula tes ma tr ix forma t ion and inhibi tsma t r ix deg rada t ion , a s s how n in F ig . 1 . A p r imar y e f fec t o fT GF-be ta i s i ts s t rong enhan cemen t o f the fo rma t ion o f bo thco l l agen and f ib ronec t in in f ib rob la st s o f human , ra t , mous e ,and ch icken origin (30, 60) . W ithin as l i t t le as 6 h , s ignif ican t

TGF-I3 SSmulates TGF-/3 InhibitsMa tr ix Syn the s is COLLAGEN. Ma tr ix Degrada t ionFIBRONECTIN.OTHER TGF-~DecreasesMA TRIX PROTEINS Secretionof Pr ote a s e s

' 7 ix *

I Genes or Collagen. .L [ Secine,Thiol, andFibronentin Metalloproteases [l / ~'--TGF -~lncroasesTGF-~]/ncr~ses ,~ Secretion of ProteaseInhibitx~s PA I,TIMP)Transcription AM INO ACIDS ,SMA LL PEPTIDES

Figure . TGF-beta stimulates the formation of r matrixand inhibits its degradation. See text for d etails.

i nc rea s e s in co l l agen can be m eas u red ; in no rm a l ra t k idneyce l l s concen t ra tions o f T GF-be ta be low 100 pg /ml (4 pM ) a res t imu la to ry . Recen t s tud ie s have s how n tha t T G F-be ta in -c rea s e s the l eve l o f mRN A fo r co l lagen ( types I , m , and V)and f ib ronec tin in no rma l ra t k idney ce l l s (31 ; Robe r t s , A . ,unpub l i s hed da ta ) . T h i s inc rea s e in co l l agen and f ib ronec t inmR NA s i s , a t l e a s t in pa r t , t he re s u l t o f a s t imu la t ion by T GF-be ta o f the p romote rs fo r the type I co l l agen gene and thef ib ronec tin gene , a s meas u red in expe r imen t s in wh ich therespect ive col lagen or f ibronect in prom oters (Rossi , P . , andB . de Crom brugghe , m anus c r ip t s ubmi t t ed fo r pub l i ca t ion ;Bourgeo i s , S . , pe r s ona l com mun ica t ion ) have been l inked tothe repor t e r gene fo r ch lo ramphen ico l a ce ty l t ransfe ra s e ; nu -c lea r RN A ru nof f expe r imen t s s t il l mu s t be pe r fo rm ed toconf i rm tha t T GF-be ta induces an ac tua l inc rea s e in genet rans c r ip t ion . T h e inc rea s ed p rom ote r a c t iv i ty fo r the co l l a -gen gene i s med ia ted by a b ind ing s i t e fo r nuc lea r fac to r I(a l s o known a s t r ans c r ip t ion fac to r CT F) , a DNA-b ind ingprote in tha t ac t iva tes eukaryot ic gene t ranscript ion (32, 69) .T hes e re s u lt s have been ob ta ined w i th s pec i f i c mu tan t s o f theprom oter o f the a lpha2(I) col lagen gene (63; Ross i , P. , andB . de Crom brugghe , m anus c r ip t s ubmi t t ed fo r pub l ica t ion ) ,wh ich ind ica te tha t t he re i s a func t iona l b ind ing s i te fo r nu -c lea r f ac to r I i n th i s p romote r , med ia t ing the s t imu la to rye f fect o f T GF-be ta .As s how n in F ig . 1 , a s econd ac t ion o f T GF-be ta d i rec tlyre l a t ed to enhanced fo rma t ion o f ex t race l lu l a r ma t r ix i s i t sab i l i t y to inh ib i t t he p ro teo ly t i c deg rada t ion o f newly fo rmedma t r ix p ro te ins . T h i s occu rs by two d i s t inc t mechan i s ms ,one o f wh ich invo lves an inc rea s e in the fo rma t ion and s ec re -t ion o f p ro tea s e inh ib i to r s , and the o the r o f wh ich invo lvesa decrease in the secre t ion o f proteases them selves . In sev-era l ce l l types , TGF-beta increases the synthes is and secre-t ion o f a p l a s minogen ac t ivato r inh ib ito r (38) (a mem ber o fthe c lass of ser ine protease inhibi tors , serpins) ; i t has beensugges ted tha t such inhihi tors funct ion effec t ive ly o s tabi l izenewly s yn thes ized ma t r ix p ro te ins by p ro tec t ing them f ro mproteolyt ic degradat ion. St i ll anoth er protease inhibi tor whosesynthes is and secre t ion is increased by TG F-beta is t is sue in-h ib i to r o f m e ta l lop ro te ina se s , a r ecen t ly c loned (16) po ten tinac t iva to r o f ye t ano the r s e t o f p ro tea s e s invo lved in m a t r ixdegra dat ion (Post le thwal te , A . , and G. S tr ic ldin , person alcom mu nicat io n) . In contras t to i ts s t imula tory effec ts on pro -tease inhibi tors , TGF -beta decreases the format io n or secre-t ion by f ib rob la s ts o f th ree d i f fe ren t types o f p ro tea s e s them-selves , inc luding a ser ine protease (plasminogen ac t iva tor)(38) , a th iol protease ("major excre ted prote in ' ) (12) , and a

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metaUop rotease (transin /strom elysin) (47). In this last case,it has been shown that TGF-beta can block the inductio n byEG F of the mRNA for transin/stromelysin, which is a m ajorproteolytic enzyme of broad specificity, produced in largequantit ies by variou s fibroblasts (20, 23, 79). Th us, th e con-certed actions of TGF-beta, to increase synthesis of proteaseinhibitors and to decrease synthesis ofpro teases themselves,both serve to augment the accumulation of matrix proteinsby TGF-beta.Some of the effects of TGF -beta on extracellular matrix areindirect, and are mediated through an in termediate cell suchas the macrophage. Thus , TGF-beta has been found to be anextremely potent chem otactic agent for monocytes (75); peakeffects have been found at concentrations as low as 1 pg/ml,which makes TGF-beta the mo st potent known agent in thisregard. At higher concentrations, TGF -beta increases levelsof mR NA in these cells for a fibroblast mitogen, such as in-terleuldn 1 (75). Furtherm ore, macroph ages themselves arestimulated to secrete as much as lO-fold more TGF-betawhen they are activated by an exogenous agent such as lipo-polysaccharide (3). This increased secretion of TGF-beta byactivated macrophages could fu rther stim ulate fibroblasts tomake more m atr ix.All of the above actions of TGF -beta are highly relevantto the problems o f inflammation and tissue repair in responseto injury; presumably they may a lso be of major importancewith respect to the role o f extracellular matrix in controllingembryonic development (25).Mechanism of Action o f TGF-BetaAs is true for most growth factors, the overall molecularmechanism of ac tion o f TGF-beta is unknown at present .However, the recent discovery of a nuclear factor 1-bindingsite in the alpha2(l) collagen gene, activated by 'l'GF-beta(Rossi, P. , and B. de Crombrugghe, manuscript submittedfor publication) represents an im portant advance. This siteis found in the p romoter for man y genes (8, 13, 32, 41, 63,81), and its widespread occurrence m ay account, in part, forsom e aspects of the pleiotropic actions of TGF-beta. Severallaboratories have reported that there is no detectable tyrosinekinase activity associated w ith TG F-beta receptors (18, 42).However, TGF-beta can antagonize the mitogenic actions o fother growth factors that do act through a tyrosine kinasereceptor, such as EGF (43, 48, 59, 68, 74), platelet-derivedgrowth factor (1), fibroblast growth factor (6, 22), and insu-lin/ins ulin-lik e growt h factor-I (43, 48). This antag onistic ac-tion of TGF-beta does not appear to occur directly at therespective tyrosine kinase receptors or at some other locusclose to these receptors. Thus, it has been shown that al-though TGF-beta blocks the mitogenic effects of either E GFor insulin in mink lun g epithelial cells, it does not block theelevation of ribosom al $6 kinase activity induced by eithermitogen (43).In a related system, it has been fo und that although mito-genic stimulation of hamster lung fibroblasts by fibroblastgrowth factor or thrombin is completely blocked by TGF-beta, the various early signals induced by these mitogens arenot blocked; these include increases in phospholipid turn-over and activation of protein kinase C, in Na antiportactivity, in exp ression of myc and los in the nucleus, and inornithine deearboxylase activity (Chambard, J. , and J. Pouys-sdgur, personal com munication ). These data all suggest that

signals from the TGF-beta receptor probably do not involvepathways com mo n to receptors with tyros ine kinase activity,but rather novel pathways, yet to be discovered, that convergein the nucleus to block DN A synthesis at som e step distal tothose already known. The further characterization of thestructure and function of the TGF-beta receptor (or recep-tors) is thus one of the most critical problems in this entirefield at the p resent time.Anoth er important mechanistic consideration is that manyof the actons of TGF-beta on individual cell types may be in-direct, that is mediated through an other cell. This is particu-larly true for the effects of TGF-beta on angiogenesis: in vitroTGF-beta itself is a strong antimitogenie agent for capillaryendothelial cells (6, 22, 26), y et, by virtue o f its ability toact as a chem otactic agent for macrophages (75), and p re-sumably by its ability to stimu late macrophages to secrete an-giogenic peptides, it can act as a potent stimulator of anglo-genesis in vivo (60).

Roles of TGF-Beta In VivoAt present there is relatively little information about the in-trinsic physiologic role of TGF-beta in vivo. ExogenousTGF-beta, prepared by acid-ethanol extraction of platelets(and therefore stripped of the protein that confers latency),has been used in a variety of in vivo studies, which havedemonstrated that TGF-beta can stimulate wound healingand can indu ce the formation of the typical granulation tissuefound in tissue repair (49, 60, 72). Two o ther situations inwhich TGF-beta has significant actions in cell culture, name-ly stim ulation of osteoblasts and suppression of't7- and B -lym-phocytes, also have potentially important in vivo applica-tions, but investigations along these lines are still in apreliminary state. Th e progress that has been made in woundhealing studies has benefited greatly from the ability to applyTGF-beta directly to a wound site; better methods for com -plexing TGF-beta with a binding o r a carrier protein will berequired for in vivo studies of its potential application for usein bone formation or as an immunosuppressive agent.At present, very little is known about the intrinsic physio-logical role of TGF-beta as an endog enous med iator of cellfunction in vivo. Immun ohistochem ical studies have begunto implicate TGF-beta as a mediato r that may be im portantin controlling proliferation and differentiation n the develop-ing em bryo (17, 27), particularly in the differentiation of tis-sues of mesenchym al origin such as bone, mu scle, blood ves-sels, and blood cells, as well as in the differentiation ofvarious epithelial tissues. There is no qu estion that TGF-betafunctions as a mediato r of inflammation and repair, since itis known to be released from platelets whe n they are degran-ulated with thromb in (2) or from activated macrophages (3)and T-lymph ocytes (34). Wh ether there are disease states inwhich TGF-beta contributes to pathogenesis by virtue ofprovoking an excessive inflammatory response (such as over-recruitmen t of macrophages) or an excessive repair response(overproduction of matrix proteins s uch as collagen, charac-teristic of many proliferative diseases) remains to b e deter-mined.The intrinsic roles of TGF-beta in the physiology of bone,the immune system, and many other tissues also remain tobe determined . It is not yet clear why bone has such a highconcentration of TGF-beta relative to mo st soft tissues (66),

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and the possible roles of TGF-beta in bone remodeling or re-pair are important problems for the future. It has recentlybeen found that agents that s t imulate bone resorption, suchas parathyroid hormo ne, 1,25Mihydroxyvitamin D3, and in-terleukin 1, all increase TG F-beta activity in organ culturesof fetal rat or neonatal mouse calvaria (53). Whether TGF-beta is a direct mediator of the action of these agents, orwheth er the increase in its activity is a comp ensatory re-sponse to their presence, is not yet known. With respect tothe im mun e system, the identification of TGF-beta 2 as aprincipal mediator responsible for the in vivo immunosup-pression in a patient with glioblastoma suggests that thismolec ule might have an important intrinsic function in con-trolling cell kinetics and function in lym phocytes and m ono-cytes (80). Finally, almost nothing is know n about the physi-ological role of TGF-beta in adult tissues such as brain,heart, and kidney, in whic h there is little mitotic activity. Inthe initial description of TGF-beta as a new typ e of molecule,it was noted that the specific activity of TGF-beta-like pep-tides in acid-ethanol extracts of brain, he art, and kidney wasalmost the same as in extracts of sarcoma cells (57).

Future DirectionsThe numerous actions of TGF-beta in regulating epithelialcell proliferation, the growth and activity of immune cells,and the synthesis and degrad ation of extracellular matrix, allsuggest that significant interactions will be found betweenthe TGF-beta system and two other ma jor classes of regula-tory molecules, na mely the glucocorticoids and the retinoids.There is a particularly impressive overlap in man y of the ac-tions of retinoids and TGF-beta, an d it is attractive to suggestthat the mechanisms o f act ion of these two dif ferent mole-cules mig ht be closely related (71). There are nume rous lociat which one might suggest interactions, but this would beentirely speculative at the present time. The TGF-beta systemcan poten tially be regulated by controlling synthesis of eitherthe peptide itself or its receptor, a nd as w e have noted above,regulation of the activation of the latent form also appears tobe an important physiological process.Lit t le is known about promoters or enhancers of the genesfor either TGF-beta 1 or 2, and w e would suggest that activa-tion of such promoters o r enhancers m ight be useful targetsfor development of new pharmacological agents. There isgood reason to believe that preneoplastic epithelial lesions,as compared with malignant cancers , should be more sus-ceptible to control by an inhibitory growth factor (70) suchas TGF-beta; therefore, an increase in TGF-beta gene tran-scription and translation in epithelial target cells might offera new approach to chemoprevention of epithelial cancer,which is characterized in general by a very prolonged,premalignant, latency period. In any event, the elucidationof the regulatory elements that control transcription of theTGF-beta gene is a problem of paramount importance.We have stressed the importance of TGF-beta in repair o ftissue damage, a nd this suggests that there may be significantinteractions between TGF-beta and another system that ap-pears to be intim ately involved in repair of cellular damage ,nam ely the fam ily of heat shock proteins (28, 52, 62). Atpresent no such relationships are know n; however, on e mightspeculate that TGF-beta exerts some transcriptional or trans-lational control over heat shock genes, as it is known to do

for collagen and f ibronectin genes. The occurrence of s imilarnuclea r factor I binding sites in the promoters fo r collagen,fibronectin, and heat sho ck genes (8, B, 41, 63, 81) suggeststhat this may be the case. Many of the roles of TGF-beta intissue repair are suggestive of roles that it mig ht also play inembryonic development, and studies in these two areasshould complement each other. Indeed, cellular and bio-chemical mechanisms involved in embryogenesis may bereiterated during tissue repair in the adult (25); collagen,f ibronectin , and heat shock gene products al l play majorroles in early embryonic development. In this regard, wewould like to suggest that TGF-beta is a morph ogenetic sub-stance, one that "gives form to things: TGF-beta is a mole-cule that somehow regulates and organizes the activities ofother growth factors (73), and at the cellular level even ap-pears to be able to organize cells into functional units. Recentexperiments showing that TGF-beta can indu ce disorganizedcapillary endothelial cells to form tubular structures in three-dimensional collagen gel cultures are germane to this con-cept (Madri , J . , personal communication) .With respect to embryonic developm ent, new studies haveshown specif ic immunohistochemical localization of TGF-beta in ear ly mouse em bryos in both the notochord and so-mites (27), critical structures in embryon ic morphogenesis.In a similar way, the product of the decapentaplegic genecomplex, related to TGF-beta, is important in morphogene-sis of the Drosophila embryo, particularly in establishingpositional information and dorso-ventral patterning of em-bryonic structures (51). It will be of interest to determinewhe ther TGF-beta might have a similar role, in the vertebrateembryo, in establishing the segmental pattern of somites,which in turn is responsible for the segmental nature of muchof the musculoskeletal system in the adult (7). Althoughthere is no known relat ionship between TGF-beta- l ike m ole-cules and the function of the products of other patterninggenes in Drosophila embryos, the known abil i ty of TGF-betato regulate the function of both EG F and its receptor (4, 44)in vertebrate cells and the known presence of EGF-like pro-teins in Drosophila (78), suggest that such relationshipsmight exist. In this regard it will be of great interest to seeif addit ional membe rs of the TGF-beta gene family, beyondthe decapentaplegic gene complex, will be discovered inDrosophila. There is no reason to believe that all membersof the TGF-beta gene family have already been discovered,either in Drosophilaor vertebrates. The e volutionary originof this entire family is unknown at present.

ConclusionsAt the rate that investigation of TGF-beta is proceeding, wehave only seen the tip of the iceberg, especially in terms o funderstanding its physiology in vivo, in both the dev elopingembryo and the adult animal. The discovery of a second formof TGF-beta offers a unique o pportunity to investigate manyproblem s relating to the specificity of the mech anisms of ac-tion of these substances. The unusual conservation of aminoacid sequences across many species suggests that the func-tions controlled by both form s of TGF-beta are of critical sur-vival value to the organism . T he finding that TGF-beta acti-vates a collagen gene promoter and that this activation ismediated by a functional binding site for the transcription

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factor, nuclear factor I, represents a new advance in under-standing the molecular mechanism of action of TGF-beta .The o pportunities to use both TGF-beta 1 and 2 for practicaltherapeutic purposes rem ain an exciting challenge.We dedicate this review to the memory of our colleague,George Khoury, who contributed so much to the knowledgeof promoters and enhancers.We thank many investigators, l isted in the text, for their kind permissionto cite their work prior to publication; and Victor Fried and LawrenceHightower for discussions of heat shock proteins. Susan Perdue hasprovided expert assistance with the manuscript.Received for publication 30 A pril 1987, and in revised form 21 May 1987.

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15. Derynck, R., J. A. Jarrett, E. Y. Cben, D. H. Eaton, J. R. Bell, R. K. As-soian, A. B. Roberts, M. B. Sporn, and D. V. Goeddel. 1985. Humantransforming growth factor-beta eDNA sequence and expression in tumorcell lines. Nature (Loud.). 316:701-705.16. Docherty, A. J. P., A. Lyons, B. J. Smith, E. M. Wright, P. E. Stephens,and T. J. R. H arris. 1985. Sequence of human tissue inhibitor o f metal-loproteinases and its identity to erythroid-potentiating activity. Nature(Land.). 318:66-69.17. Ellingsworth, L. R., J. E. B rennan, K. Fok, D. M. Rosen , H. Bentz, K. A.Piez, and S. M . Seyedin. 1986. Antibodies to the N-terminal portion ofcartilage-inducing factor A and tra nsforming growth fa ctor beta. J . BioLChem. 261:12362-12367.18. Fanger, B. O., L. M. W akefield, and M. B. Sporn. 1986. Structure andproperties of the cellular rec eptor for transforming growth factor type

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Sporn et al. Advances in TGF-Beta Chemistry and Biology 1045

Published September 1, 1987