79

Blochtmlca et B,ophystca Acta, 1032 (1990) 79-87 Elsevier

BBACAN 87224

The cell biology of transforming growth factor fl J o h n A. B a r n a r d 2, R u s s e t t e M . L y o n s 1 a n d H a r o l d L. M o s e s 1 Departments of i Cell Btology and 2 Pedtatrtcs, Vanderbtlt Untoerstty School of Medtcme, Nashville, TN (U S A ) (Received 5 December 1989)

Contents I

Introduction

79

II

The TGFfl farmly of growth regulatory pepudes

80

III

Activation of latent TGF/3

80

IV

TGFfl receptors

81

V

TGF~ expression in normal cells, transformed cells, and dunng embryogenests

82

VI

Blologtc effects of TGFfl A Stimulation of cell growth B Stimulation of extracellular mamx formation C Inlubmon of cell growth by TGF~ D Effects of TGFfl on cell differentiation E Autoinductlon of TGFfl expression F Effects on immune function G Other biologic functions of TGFfl

83 83 83 83 84 85 85 85

VII Summary

85

References

85

I. Introduction

This decade has wttnessed a remarkable expansion m the understanding of the intricate mechamsms that govern control of cell growth and prohferatlon It is now apprecmted that most normal cells and tissues exist w~thm a complex rmheu of sUmulatory and mlubltory polypeptlde growth factors that are produced by the cells themselves or by adjacent cell types It has been tempting to speculate that various physiologic and pathophyslologlc alterations tn the rate of cell growth

AbbrevmUons TGFa, transforrmng growth factor a, TGFfl, transfornung growth factor fl, EGF, epldertnal growth factor, BMP, bone morphogenetic proteins, PDGF, platelet-denved growth factor, RGD, ammo acids, argmme, glycme, aspartic acid, HMBA, hexamethylene blsacetanude Correspondence H L Moses, Department of Cell Biology, Vanderbdt University School of Medicine, Nashwlle, TN 37232, U S A

result from a modtficaUon m a dehcate balance between the forces that stimulate growth and those that mbab~t growth As will be discussed below, substantml direct and redirect evidence has now accumulated m support of ttus speculation Two polypepttde growth factors have emerged as prototyp~cal 'autocnne' growth factors, the growth sumulator, transforrmng growth factor a (TGFa) and the growth mtubltor, transfornung growth factor /3 (TGF/3) These structurally and biologically distract molecules were originally named transforming growth factors because they reduced morphologlc transformation of hbroblastxc cells in monolayer culture and sumulated colony formaUon In soft agar [1-3] TGF~t, wtuch wdl not be further discussed m thas rewew, and epidermal growth factor (EGF) share 35% sequence homology, utilize the same cell surface membrane receptor, and possess a nearly ~dentlcal spectrum of btological acUvtty [4] TGF/3, the prototyplcal growth lnlubltor, exhtblts a remarkable dwerslty of biological

0304-419X/90/$03 50 © 1990 Elsevier Science Pubhshers B V (Biomedical Dwlslon)

80 activity m ad&t~on to ~ts effects on cellular proliferation and ~s now known to belong to a large farmly of related molecules with a wide range of regulatory actwlties

II. The TGFfl family of growth regulatory peptides The TGFfl-related family of polypeptldes includes a group of closely homologous dlmenc proteins in which all nine cysteine residues are conserved, and a group of more distantly related polypept~des that nonetheless share sigmficant sequence identity and sinular blologxcal actlwtxes The three &stinct molecular forms of TGFfl wtuch have been ldentxfied in mammals have been designated TGFfll, TGFfl2 (also called BSC-1 cell growth mlubltor or polyergln) and TGFfl3 [5-8] These genes have been localized in humans to chromosomes 19, 1 and 14, respectively [9-10] The three closely related TGFfl molecules are first synthesized as larger precursor polypept~des that are then processed to yield 12 5 k D a monomers [5] The exact sequence of events m the processing of TGFfll is uncertam, but appears to include cleavage of a 29 anuno acid signal sequence, glycosylatlon and mannose-6-phosphorylation of the precursor, cleavage of the carboxy-terrmnal 112 amino acid (12 5 kDa) m o n o m e r from a 390 amino acid precursor, and disulfide lsomenzaUon [11-14] The biologically active, mature 25 k D a polypeptlde consists of two identical dlsulfide-hnked monomers [5] In the mature sequences, human TGFfl2 and TGFfl3 genes are approx 80% slmalar to TGFfll, while TGFfl3 is 72% slnular to TGFfl2 [15] By contrast, precursor sequences for the three TGFfls are markedly &sslmdar, nevertheless, selected structures within the precursor are preserved, e g , the R G D (cell adhesion receptor recognition sequence) residues in TGFfll and TGFfl3 Among mammals, the sequence for TGFfll is haghly conserved, m fact, it is identical m man [8], pig [16], cow [17] and monkey [18], and differs by only one amino acid in mace [19] Recently, a new member of the TGFfl family has been cloned from chicken cells and has been designated TGFfl4, although no m a m m a h a n homolog has yet been identified [20] As an increasing number of TGFfl farmly members are described, the nomenclature will become more cumbersome In the present revaew, specific reference to the various lsoforms of TGFfl, that is TGFfll, TGFfl2 and TGFfl3, Is made only when done so in the cited manuscript Otherwise, the more generic designation, TGFfl, is used The proteins that are d~stantly related to TGFfl include Mullenan mhtbitmg substance, a protein that reduces regression of the Mullenan duct system m the developing male genitourinary system [21], the mtubins and actlvms, molecules that modulate follicle stimulating hormone secretion [22], the Drosophtla decapentapleglc gene which directs dorsoventral orientation during embryogenesls [23], several bone morphogenetlc pro-

teins (BMP-2A, BMP-2B and BMP-3) which may regulate cartilage and bone formation [24], Vg-1, a protein produced in Xenopus that may be revolved in induction of mesoderm [25], and the product of the recently described vgr-1 gene [26] These molecules share with TGFfl 25-40% sequence identity and conservation of at least seven of the nine cysteine residues

III Activation of latent TGFfl TGFfl is secreted by cultured cells m an inactive or latent form that wdl not brad to cell surface m e m b r a n e receptors specific for TGFfl [27] Several physlcochermcal treatments wall activate latent TGFfl, thereby permitting receptor binding These include transient acidification (below p H of 4) or alkahmzatlon (above p H of 9), and treatment with chaotroplc agents hke sodium dodecyl sulfate and urea [28] The exact pathophyslolog~cal significance of these observations is not known, although it is possible that acidic rmcrocompartments within wounds or remodehng bone rmght be sufficiently acidic to activate TGF/3 More recent studles have shown that treatment of c o n d m o n e d cell culture me&urn with protemases such as plasmm and cathepsm D will activate a s~gnlficant proportion of the latent TGFB secreted by cells in culture [29] A series of interesting experiments by Sato and Rafkln [30] further support proteolytic cleavage of TGFfl by the serme proteinase plasrmn as a relevant physxologlc mechanism of activatxon In these experiments, TGFfl activity was measured by its lntubitory effect on endothelial cell rnlgratlon TGFfl was activated (and rmgrauon was inhabited) when bovine aortic endothelial cells were co-cultured with pencytes, but not when the cell hnes were cultured separately Local activation of TGFB at the cell surface appeared to be mediated by plasmm, as addition of plasmln inhabltors to the co-cultures blocked TGFfl mbabmon of cell migration The structure of the latent TGFfl molecule may be a function of the source from whach the latent complex is isolated The latent complex released by cells m culture simply consists of the N-terminal precursor glycopeptxde noncovalently associated with the mature TGFfll polypeptlde [31] Purchao and co-workers [32] have recently shown by site-dwected mutagenesls that substitution of serine at cysteme residues 223 or 225 within the precursor molecule results in the synthesis of monomeric precursors and secretion of a biologically active TGFfll molecule These observations suggest that dlmerlzation of the precursor may be necessary for latency A molecular model for activation of TGFfll based on the aforementioned observahons is shown m Fig 1 Latent TGFfll purified from platelets contains an additional large molecular weight 'binding protein' m assocmt~on wxth the precursor and mature polypeptlde [33,34] Carbohydrate moieties within the pre-

81 M u t a t e d 3ene

Normal gene p r o d u c t

product

CHO CHO CHO I

i

0J..~J..A I 4--

L a t e n t TGF/~I

(110 kDa)

I

L(-~

t i

i ] ~

All s e c r e t e d TGF(?I is a c t i v e

Disultlde bonds

Non-covolenl bonds

I

CHO

Y///////////A

/// I

CHO

CHO

Active T GF/~I

br//////////A

V///////////A

~.)..I.~I.....A c t , v e TGF~I

//|

( 25 k D a )

_COo c.

Dissociated amino - t e r m i n a l g l y c o p e p t l d e

Cleaved amino-terminal

glycopeptlde

A m i n o - t e r m m o l 91ycopeptlde monomers

Fig 1 Producuon of biologically acUve TGFfll The left panel dlustrates cell-secreted latent TGF/31 as a complex between the TGFfll precursor regton (open bar) and the mature region (cross-hatched bar) TGFfl can be released from the latent complex by dissocmtlon from the armno-ternunal glycopept]de with acid or by proteolytlc cleavage with plasnun [29,31] The n g h t panel shows that a mutation at cysteme residues 223 or 225 m the precursor results m secreUon of the active homodlmer w]thout treatment with extremes of pH, chaotroplc agents or protemases [32]

cursor sequence appear to be necessary for the latency of this platelet-denved complex [35] The latent form of TGFfl m serum or plasma may be a complex of TGFfl and a2-macroglobulin [36] Inasmuch as TGFfl is synthesized by most cell types and almost all cells have TGFfl receptors, dehmuve deterrmnauon of the nature of the latent TGFfl complex and a more complete understanding of the physiologic mechanisms by whach latent TGFfl is activated may be central to understandmg regulat]on of TGFfl acuvlty IV. TGF,O receptors All normal cells and most all neoplastic cells have cell surface membrane receptors or binding proteins for TGFfl [37,38] It is not clear that these proteins are classic cell surface membrane receptors or simply bindlng proteins, nevertheless, hereto, the term 'receptor' will be used Three distract, glycosylated receptors with molecular masses of 50-80 kDa (type I), 115-140 kDa (type II) and 280-330 kDa (type III) have been identified by photoaffimty and cross-hnklng expenments [39,40] The hagh molecular mass receptor is a proteo-

glycan of which nearly 50% of the molecular mass is heparan sulfate and chondroltm sulfate [41] TGFfll, TGFfl2 and TGFfl3 brad to all three receptors, while the more distantly related TGFfl-hke molecules do not [22,42] The type of receptor(s) on a given cell may depend to some extent on the type of cell and also whether the cell comes from a primary, early passage culture or an estabhshed culture hne For example, studies by Segarlnl and co-workers [43] showed that early passage hbroblasts, chondroblasts and osteoblasts brad TGFfll and TGF/32 to all three receptors By contrast, early passage epithelial, lymphoid, and endothehal cells that are responsive to the biological effects of TGFfl are capable of binding TGFfll and TGFfl2 only to the lower molecular mass receptors Ttus selecttve pattern of binding was not seen m cells maintained m continuous culture [43] These studies imply that binding to the tugh molecular mass proteoglycan is not required for TGFfl responsweness Currently, specific cellular responses cannot be confidently assigned to any of the three receptor types, however, mutant mink lung epithelial cell clones resistant to the growth mlubitory effects of TGFfl have lost the type I receptor [44]

82 Taken together, these studies suggest that the type I receptor may mediate most of the biologic effects of TGFfl in epathehal and perhaps other cell types All three receptors bind all three hgands with high affinity ( K d = 10-200 pM), but the type I and II receptors band TGFfl2 with an order of magnitude less affinity than TGFfll (Ref 39, and Lyons, R M , personal communication) Scatchard analysis of 125I-TGFfl binding to various cell types indicate that typically there are between 10000 and 50000 receptors per cell [37], however, no relationship between receptor number and sensmvlty to growth inhibition has been found in several cancer cell lanes [45,46] Retlnoblastoma cell lines are unique in that they have no identifiable TGFfl receptors and are not growth inhabited by TGFfl, whereas their nontransformed counterpart, fetal retinal cells, possess all three receptor types and are growth inhabited [47] Nonetheless, a loss or absence of TGFfl receptors appears to be a rare and unusual mechanism by which neoplastic cells escape growth regulation Mechanisms for regulation of TGFfl binding have not been extensively studied, but phenomena hke increased cell density and differentiation appear to down-regulate TGFfl receptor numbers [48,49] The lntracellular signaling events lmtlated by TGFfl receptor-hgand interaction are not fully understood The growth stimulatory effect of TGFfll on AKR-2B fibroblasUc cells is coupled to a pertussis-toxan-sensmve guanine nucleotade regulatory protein (G-protein) [50,51] Muldoon and co-workers [52] found that the growth stlmulatory effect of TGFfl on Rat-1 ceils was accompamed by activation of phosphoinositide turnover and that this effect was potentmted by E G F In fibroblastic cells that are growth inhabited by TGFfl, alterataons in intracellular pH, protean klnase C activity, protean phosphorylataon and phosphomosltlde metabohsm induced by fibroblast growth factor and thrombm were not affected by TGFfl [53] Similarly, no effect of TGFfl on N a + / H + exchanger activity in renal tubular epithelial cells was found [54]

V. TGFfl expression in normal cells, transformed cells, and during embryogenesis Most transformed and nontransformed fibroblastic and eplthehal cells maintained in continuous cell culture express all three TGFfl mRNAs and secrete TGFfl in a latent form into conditioned culture medium [15,27] It is not known to what extent the nearly uhaqmtous expression of TGFfl in cell culture systems reflects the distribution of expression In vivo Miller et al [55,56] have described the distnbuUon of TGFfll, TGFfl2 and TGFfl3 mRNA in a variety of normal adult mouse tissues inchadmg spleen, testis, kidney, liver, lung, brain, heart, adipose tissue, submaxillary gland and placenta Expression of at least one of the three mRNAs was

found in all of these tissues but, m some instances, the predominantly expressed TGFfl m R N A was variable For example, only TGFfl3 was detected an mouse tests (otherwise the loci for TGF/32 and TGFfl3 expression were similar) TGFfll expression was high in spleen, lung and placenta TGFfl2 and TGFfl3 m R N A was also abundant in the lung and placenta, but no expression was detected in the spleen The significance of this comphcated pattern of TGFfl expression is uncertain, although the lmmunohlstochemical and in SltU hybn&zatlon studies outhned below have begun to unravel some of the complexities The biologic effects of TGFfl on cell proliferation, extracellular matrix formation, differentiation, cell migration and angxogenesis, in conjunction with the aforementioned sequence identity between the TGFBs and other members of the TGFfl family that appear to be ~mportant m morphogenesls, suggest that the TGFBs may be involved in embryonic development TGFB2, but not TGFfll, induced the mesoderm associated gene, a-actln, m Xenopus blastulae [57] In a related study, Klmelman and Karschner [58] showed that TGFfll and basic fibroblast growth factor synergistically induced actm expression in Xenopus Several studies have attempted to define the role of the TGFBs in mammalian embryogenesls In one study, TGFfll m R N A was locallzed by in SltU hybridization in day 9-16 mouse embryos [59] Hybridization was detected in hematopoletic cells and in the laver, a hematopoietic organ in fetal life Other investigators have described a much more extensive distribution of TGFB1 m R N A in mouse embryos [60] In this study, high levels of TGFfll m R N A expression were found in megakaryocytes and osteoclasts Expression was also detected an mesenchymal tissues of the intestine, lung and lodney an various eplthehal structures during intervals of morphogenesls TGFfl lmmunoreactivity was detected by Heine and co-workers [61] in a large number of mesoderm derived structures in 11-18-day-old mouse embryos These tissues and structures included the palate, larynx, teeth, cardiac valves, hmr follicles, bones and cartilage, amongst others The lmmunostalnxng appeared most intense dunng periods of active tissue modeling and remodeling These marked differences an the description of TGFB1 m R N A and protein distribution during m u n n e embryogenesis remain to be reconciled TGFB2 m R N A levels an whole mouse embryos progressively increase from day 10 5 17 5 post coitum [62] Exarmnatlon of the temporal and spatial distribution of TGFB2 m R N A expression in mouse embryos and newborn mouse pups by in situ hybridization showed expression in bone, cartilage, tendon, gastrointestinal submucosa, tracheal submucosa, blood vessels, skin and placenta [62] TGFfl2 expression in the skin occurred in a particularly interesting pattern, that is, no expression on post coital day 13 5-14 5, followed by prominent hybridization in the

83 dermis on day 15 5, dissipation of signal m the derrms on day 17 5, and h y b n d l z a u o n m the suprabasal layer of the epidermis for the first time on day 18 5 These orderly, sequentml changes in TGFfl2 expression imply that tins polypepUde may regulate mesenchymal-eplthehal interactions d u n n g embryogenesls

Vl. Biologic effects of TGFfl The m vitro, biologic effects of TGFfl are remarkably dwerse Depending on the cell type, TGFfl may stimulate or minb~t prohferatlon, block or effect entry into a dlfferentmuon pathway, stimulate extracellular matrix formation, and promote or minb~t cell m~grat~on The molecular events that regulate these cell-type-speofic actions of TGFfl are currently the subject of intense mvest~gauon In addition, the m vwo experimental effects of TGFfl and the potentml contributions of TGFfl to a wide variety of pathophyslolog~cal processes have recewed increasing attention

TABLEI Ref

Increased synthes~sof ECM components Collagen (type I) F~bronectm Proteoglycan Tenascln

68 68, 69 70 71

Decreased synthesis of protelnases Collagenase Transm Cathepsm L

72 73 74

Increased synthesis of protemase mhlbltors Plasmmogen activator mlubltor Tissue specific metalloprotemase inhibitor Uroktnase

75, 76 72, 77 78

Increased synthesis of cell adhesion receptors Vttronectln receptor LFA-1 Flbronectln receptor Other mtegnns

79 79 80 81

VI-A Sttrnulatton of cell growth

Although TGFfl was discovered because of its ablhty to stimulate anchorage independent (soft agar) growth of nontransformed fibroblasUc cells [63], no eplthehal cell types and only a few mesenchymal cell types (fibroblasts and osteoblasts) actually prohferate in response to TGFfl treatment [64-66] The cell-cycle kanetlCS of TGFfl stlmulaUon of fibroblast prohferatlon are interesting m that Ga ~s prolonged by approx 12 h when compared to G~ m other growth factor-stimulated cell types Leof and co-workers [67] demonstrated that TGFfl first reduced c-sis m R N A and increased platelet derived growth factor (specifically P D G F B chaan, the polypeptlde product of the c-sis gene) competing matenal m AKR-2B fibroblast condmoned medium Presumably, P D G F , a known mitogen for AKR-2B cells, was then mitogemc by an autocnne pathway On the basis of these observations, TGFfl may be considered an 'indirect' rmtogen m fibroblasts

VI-B Stimulation of extracellular matrix formation A m o n g the most striking effects of TGF/3 on cellular funcUon m vitro ~s its promotion of extracellular matrix formation Tins effect occurs not only as a result of stimulation of extracellular m a t n x protein synthesis, but also by m i n b m o n of protelnase synthesis, stlmulaUon of protemase inhibitor synthesis and an increase m synthes~s of cell surface membrane proteins that brad extracellular m a t n x proteins These effects are summanzed m Table I The potent effect of TGFfl on extracellular matrix formation m vitro revokes excmng speculauon that TGFfl may function m the regulation of such processes as wound and tissue repmr, bone

formation and remodeling, and embryogenes~s Indeed, an expanding body of data supports a potent effect of TGFfl on the extracellular matrix m vlvo For example, subcutaneous injection of TGFfl in neonatal mace caused the formation of a lump of collagen-contalmng granulation t~ssue at the s~te of mjecUon within 48 h [82] A p p h c a u o n of TGFfl to experimentally mfl~cted slon wounds m rats resulted m an accelerated rate of heahng as measured by wound tensile strength [83] Hlstologlc examination of the TGFfl treated wounds showed an increase m synthesis and deposition of collagen In a related study, a single apphcation of TGFfl to a slon wound m rats fully reversed the impairment of wound heahng induced by systemic glucocortlcold treatment [84] The TGFfl treated wounds were characterized by increased numbers of procollagen type I synthesizing fibroblasts Several recent studies have described increased expression of TGFfl in assocmtxon with diseases characterized by excessive fibrosis TGFfll treatment of freshly isolated hepatocytes caused a marked elevation (13-fold) of a2(I) collagen m R N A expression [85] When TGFfll m R N A expression was then examined In two m vlvo models of hepatic fibrosis, increased levels of TGF/31 m R N A were seen just prior to the increase in collagen expression In patients with proliferaUve xqtreoretmopaty, TGFfl2 (but not TGFfll) protein levels were 3-fold elevated m mtraocular fluid when compared to controls [86]

VI-C Inhtbmon of cell growth by TGFfl TGFfl is the most potent polypeptlde growth ininbltor known for a wide variety of cell types including selected cell types of mesenchymal and myelold ongm,

84 as well as nearly all eplthehal, lymphoid and endothehal cells [65,66,87-94] TGFfll, TGFfl2 and TGFfl3 are eqmpotent m the degree to which they lnl~b~t cell growth [88,95] The molecular mechanisms by winch the actwe TGFfl molecules mtublt cell growth have been the subject of intense interest Many of the studies designed to explore mechanisms of growth inhibition by TGFfl have been conducted using normal human keratmocytes and normal mouse keratmocytes ( B A L B / M K cells), cell hnes that reqmre T G F a / E G F for prohferaUon [96] Both cell types retain the capacity for differentmtlon m the presence of high calcmm concentrations [97] Early stu&es determined that TGFfl reversibly arrested keratmocytes in the G 1 phase of the cell cycle, but did not effect entry into a differentiation pathway [88,98] The TGFfl concentration reqmred for half-maximal lntubmon was approx 10 pM, thereby estabhshlng TGFfl as the most potent growth lnhlbxtor known for eplthehal cells Inasmuch as these cell hnes also have specific membrane receptors for TGFfl and synthesme and secrete actwatable TGFfl into the cell culture medmm [88,98], all the requisite elements exist for an lninbxtory autocnne pathway of growth control Thus, these lnltml stu&es described a useful model system for investigation of the molecular mechamsms by winch TGFfl suppresses cell growth The effects of TGFfl on the expression of protooncogenes associated with cellular proliferation have been exarmned in a variety of cell hnes, including keratmocytes TGFfll at a concentraUon of 10 n g / m l (40 pM) caused a 50-60% decrease in c-myc m R N A expression following restlmulatlon of qmescent B A L B / M K cells with E G F [99] Tins effect was most apparent 30 and 60 mln following restlmulatlon, although a second interval of attenuated c-myc expression was seen at 3 and 6 h after rest~mulatlon Similar observaUons were made when the expression of another prohferauon-assocmted gene, KC, was examined, however, the expression of other EGF-mduclble genes, such as c-fos and fl-actm, was not decreased by TGFfll Related observations have been made m endothelial cells [87] and colon carcinoma cells [100] In contrast to the aforementioned observations, the transcnpUonal factors junB and c-jun were upregulated by treatment with TGFfll and TGFfl2 in several cell hnes, some of which are exquisitely sensltwe to the growth inhibitory effects of TGFfl [101], these observations suggest that TGFfl may lninblt eplthehal cell growth by selectively decreasing expression of lmmedmte-early genes that are assocmted w~th prohferat~on Inasmuch as some EGF-lnduclble cellular responses are affected by TGFfl and others are not, an alternate interpretation of the aforementioned studies ~s that the growth minbltory effect of TGF/3 must occur as a result of an interaction downstream from hgand binding to receptor Tins appears to be true, at least m keratmo-

cytes and rmnk lung eplthehal cells [88,102] In other cell types, however, TGFfl may up or down-regulate EGF binding to xts receptor, either by altenng receptor density or affinity for E G F [103-105] Thus there does not appear to be a simple, umfymg mechanism by winch TGFfl affects cell prollferaUon by influencing E G F r e c e p t o r / h g a n d interaction A recent study has demonstrated marked, selective down-regulation of platelet-denved growth factor ( P D G F AA) binding and a decrease m the rmtogenlc response to P D G F AA m 3T3 cells following TGFfl treatment [106] Although ~t ~s tempting to speculate that neoplastic transformaUon may, in some instances, result from a loss of negative regulatory influences through mutation or deletion of the TGFfl gene, no specific examples are known to have occured Selected tumor cell hnes, partlcularly squamous carcinoma cell hnes, have lost the normal growth inhibitory response to exogenous TGFfl [98,107-108] While not a universal mechamsm by winch transformed cells grow m an unregulated fasinon, loss of normal autocrme or paracrlne negatwe regulatory control pathways may contribute to progression to mahgnant transformation Several stu&es suggest that TGFfl not only minb~ts cell growth m vitro, but also in v~vo Implantation of TGFfl impregnated pellets adjacent to mouse mammary epithelial end buds sxgmficantly inhibited mammary gland ductal growth, an effect that was fully reversible upon removal of the pellet [109] Intravenous rejection of TGFfll or TGFfl2 dramatically minblted the early phase of hepatocyte proliferation that occurs in response to partml hepatectomy in the rat [110] A role has been postulated for TGFfl in the regulation of cell turnover m a continuously regenerating ep~theha hke the epidermis and small intestinal eplthehum In mouse skin, phorbol ester-induced TGFfl expression was detected only in epidermal cells committed to terminal differentiation [111] In the rat jejunal epithelium, TGFfl expression was highest m terrmnally dffferentmted wllus-tlp cells and wrtually undetectable in rapidly prohferatlng crypt cells [89] These studies imply that TGFfl may be an important physiologic modulator of cell growth in wvo

V1-D Effects of TGFfl on cell dtfferenttatton Although the effects of TGFfl on cellular &fterentlatlon have been intensively examined, they are not predictable and s~mply appear to be a function of the cell type or marker of differentiation being studied For example, TGFfl induces selected markers of &fferentlat~on in human bronchtal eplthehal cells and colon carcinoma cells [46,112], but has no dxrect effect on other eplthehal cells such as mouse keratmocytes or rat small intestinal eplthehal cells [88,90] Terminal dlfferentlatxon and calcification of rabbit chondrocyte cul-

85 tures is inhabited by TGF/3 [113], but TGFfl promotes osteoblasUc dffferenUaUon m rat osteosarcoma cells [94] TGF/3 is a potent mhabltor of adlpocyte and myoblast dffferentmUon [114,115] It seems most plausible that TGFfl funcuons in concert with a variety of other factors such as extraceUular matrix proteins and other polypepttde growth factors to affect cellular differentiaUon In support of this, the cellular dlstnbuuon of TGFfl expression m sltu and in vavo frequently is associated with the differentiated phenotype [90,111] Also, differentiating agents such as HMBA and retlnoic acid induce TGFfl expression [116.117]

VI-E Automductton of TGFfl expresswn Several polypeptlde growth factors, inchidmg T G F a and PDGF, reduce thetr own expression in wtro [118,119] More recently, autoinducUon of TGFfl also has been described [90,120,121] Interestmgly, Bascom et al [121] demonstrated that the phenomenon of 'automductlon' occurs between the various lsoforms of TGFfl Two spectfic regions of the human TGFfll gene promoter appear to be important for autoinductxon, a 130-bp fragment located upstream of the 5'-most transcriptional start site and a sequence located between the two start sites [122] These sequences are distmct from the NF-1 binding site that mediates TGFfll induction of mouse a(2)I collagen gene expression [123] It has been postulated that autoinductxon represents a mechanism by which the biologic effects of a growth factor such as TGFfl maght be amplified

VI-F Effects on tmmune functton The effects of TGFfl on immune function are strikmg The growth of T- and B-cells is suppressed by TGFfl, as is production of lmmunoglobuhn by B-cells and cytotoxtcity of natural killer cells [124-126] Human TGFfl2 was first isolated from a cell hne derived from a human ghoblastoma, a tumor that ts frequently associated with an immunosuppressed host [127] Interestingly, overexpresslon of a transfected TGFfll gene in a highly immunogentc flbrosarcoma cell hne confers greater tumorigenicity in nude mace [128]

VI-G Other btologw functions of TGFfl TGFfl has a number of other potentmlly ~mportant biologic effects Increased quanUtles of TGFfl are secreted by activated macrophages, and thts secreted material is a potent chemoattractant for macrophages and flbroblasts, agaan suggesting a hkely role in the processes of inflammation and wound repair [129-131] In addition, both TGFfll and TGFfl2 markedly suppress hydrogen peroxade generation by macrophages [132] Potent ang~ogemc effects of TGFfl are observed

m cbacken chonoaUanto~c membranes (Yang, personal commumcatlon) and following subcutaneous rejection of TGFfl m mace [82]

VII. Summary The TGFfl family of polypeptide growth factors regulates a remarkable diversity of cellular functions, many of whtch are not directly assooated wtth cell growth The present review has summarized many of the recent studies that have just begun to conceptually integrate this expanding array of TGFfl functions into the context of a three-dtmenslonal, multtcellular organ or tissue, be tt normal or dtseased This fascinating research strongly implicates TGFfl as a key modulator of a wide variety of tmportant physiologic and pathophyslologlc processes

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