in Gn~wrh Fuc for Kesrarch. Vol. 4, PP. 321-335, 1992 Prlnled III Great Britain. All righIs reserved.

0955+?135’9? s15.0(1 , 1993 Perpamon Press Ltd

Ptyqrra.\

CONTROL

OF TRANSFORMING GROWTH FACTOR-/3 ACTIVITY: LATENCY VS. ACTIVATION

John G. Harpel, Christine N. Metz, Soichi Kojima and Daniel B. Rif’kin* New York

University

Department of Cell Biology and Kaplan Center Medical Center and the Raymond and Beverly Sackler 550 First Avenue, New York, NY 10016, U.S.A.

Foundation

Laboratory

Tratwfkminggrowthfactor-8 is u pluripotent regulutor ~j’cellgrowth und diflerentiution. The growth factor is expressed as u latent complex thut must be converted to cm uctive,form before interucting with its ubiquitous high afJinity receptors. This conversion itwolves the release of the muture growth,fuctor through disruption of the non-covulent i~~teractions with its pro-peptide or latency ussociuted peptide. The mechanisms.for this releuse in vivo huve not heen,fully characterized but appear to he cell spec$ic and might bwolve processes such us ucidljieution or proteolysis. Although severu1,filctor.s including trunscriptionul regulation, receptor modulation und scavenging of the uctive growth jtrctor huve been implicated, the critical step controlling the biological qffkts r?f transf~wming growth,factor-p mu?3 he the activation of’ the lutent molecule.

Transforming growth factors. transforming growth factor-/?. latent complexes,activation. Keywords:

INTRODUCTION Transforming growth factor-p (TGF$) was originally defined by its ability to induce anchorage-independent growth of normal rat kidney fibroblasts in soft agar [l-3]. Since that time, five isoforms of TGF-/3 (TGF+S)t have been identified and determined to be members of a family of regulators of cell growth and differentiation including Miillerian inhibiting substance, inhibins, activins, the Drosophikt decapentaplegic complex and bone morphogenic proteins (reviewed in [4-61). Among other activities, TGF-/I is a potent stimulator of monocyte chemotaxis [7], inducer of embryonic mesodermal tissue[S], mitogen for osteoblastsand chondrocytes [9, IO], an inhibitor of lymphocyte function [1 11, endothelial cell replication and migration [ 12,131and adipogenesisand steroidogenic capacity in certain cells [ 14.151.

.4ck,lorr/~,dKem~nl.F--This work was supported by grants (CNM). and CA 23753 from the National Institutes of Health. Society (DBR). *To whom correspondence should be addressed. i-Unless stated otherwise, TGF-/I will refer to TGF+‘I. 321

T32GM07308 NIGMS (JGH). CA 09 Ihl and grant CB 18 from the American Cancel

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Treatment of cells with TGF-P enhances the production and secretion of extracellular matrix (ECM) proteins [9,14,16] and increases expression of cell surface ECM receptors [ 171.Accompanying these effects, there is a decrease in the synthesis of several proteolytic enzymes, and an increase in the release of protease inhibitors that results in decreased ECM turnover [l&19]. These diverse actions of TGF-8 depend on the cell type, state of differentiation and cell culture conditions [6]. TGF-Pexerts its many effects via binding to high affinity receptors that are expressed on virtually all cell types [20], and as TGF-/I production is ubiquitous, there must be strict regulation of its activity. TGF-/I is released from cells in a latent form (latent TGF-j?) that does not bind to TGF-P receptors [5, 21, 221. Therefore, activation appears to be a critical step in regulating its biological effects. This review will summarize what is known about the latency and structure of TGF-/J and will discuss current theories on its activation. STRUCTURE OF LATENT

TGF-/iI

Several different configurations of latent TGF-/? have been identified, including small and large latent complexes, as well as a TGF-p-a>-macroglobulin (Q~M) form. All of these are biologically inactive. The small and large latent TGF-@ complexes are secreted from many cell types and can be activated to release mature TGF-j?. In contrast, mature TGF-P found in plasma is bound to a?M. Small Latent

TGF-/3 Complex

Recombinant TGF-P is expressed in mammalian cells as a precursor known as the small latent complex or pro TGF-j? [23, 241. This latent TGF$ complex (105 kDa) is derived from a single gene product [25]. The amino terminal portion of the small latent complex (residues 30-278) constitutes the pro-peptide or latency associated peptide (LAP; 75-80 kDa), whereas the mature growth factor consists of the carboxy terminal 112 amino acids (residues 279-390; 25 kDa). Each of these components exists as separate disulfide linked homodimers [23]. The signal sequence (residues l-29) is removed from the pre-pro form during its biosynthesis, and the mature TGF-8 peptide is proteolytically cleaved from LAP at dibasic residues (Arg 278Arg 279) in a postgolgi compartment to yield a complex in which LAP is associated with the mature TGF-j3 by non-covalent interactions [26] (Fig. 1). The cDNA sequences of both TGF/I2 [27] and TGF-/33 [28] encode isoforms structurally similar to TGF-/?I. The small latent TGF-pcomplex has been identified in the culture media of many cell types including murine calvarial bone cultures [29, 301, BSC-40 monkey kidney cells (TGF-/?2) [31], a human erythroleukemia (HEL) cell line [32], and a human glioblastoma cell line (TGF-/IIf 82 and p3) [33]. Similar to recombinant small latent TGF-j?, these forms consist of non-covalently associated homodimers of LAP and mature TGF-j3. Purified LAP can reassociate with mature TGF-j? and inactivate it [23], suggesting LAP is sufficient to confer latency. The precise mechanism by which LAP confers latency to TGF-p is not clear. Because less than 1% of recombinant TGF-B associated with LAP is recognized by antibodies raised against mature TGF-j? [22,24], it has been postulated that LAP might mask the mature TGF-P molecule and prevent its association with specific receptors.

Small latent TGF-l3 complex

Large latent TGF-8 complex

FIGURE 1. Structures of the small and large latent TGF-B complexes based on 1331. The electrostatic interactions 1351 and the exact number and precise location of intra- and intermolecular disulfide bonds are not known. The carbohydrate residues of LTBP are predicted by the cDNA sequence 1421.

Independently expressed mature TGF-/3 (without LAP) is not secreted [34]. indicating that LAP is required for secretion of the small latent complex. The regions of LAP involved in the processing, folding, structural stability and secretion of the small complex have been identified using site-directed mutagenesis [3.5]. The amino terminal region of LAP is rich in basic residues and may associate with mature TGF-jI through electrostatic interactions. Deletions in the largest conserved continuous sequence of LAP (amino terminal residues 40-60) prevent the association of mature TGF-8 with LAP [35]. Previous work demonstrates that activation of the recombinant small latent complex occurs after plasmin-mediated proteolysis of the amino terminal region ot LAP [36], further supporting the role of this region in stabilizing the small latent complex. Together, these observations suggest that interactions between the amino terminal region of LAP and mature TGF-/I stabilize and maintain the latency of the small latent TGF-P complex. Based on the analysis of the TGF-/I cDNA, three N-glycosylation sites were predicted in LAP [25]; all of these sites (amino acids 82, I36 and 176) are glycosylated in the expressed recombinant polypeptide. Recombinant LAP also contains mannosephosphate residues at the first two N-linked oligosaccharide chains, as well as several sialic acid residues [37, 381. N-glycosylation and mannose-6-phosphate residues are necessary for secretion of the small latent complex [39]. These carbohydrate residues.

31’4

.I. G. Hurpel ct rd.

as will be discussed later, may play a role in the activation complex.

of the small latent TGF$

Lurge Lutent TGF-/3 Comples In platelets, TGF-Poccurs as a high molecular weight form known as the large latent TGF-8 complex [21, 221. This large latent complex has also been identified in the culture media from the HEL cell line [32], human glioblastoma cell lines [33], bone cultures [29] and cultures of bovine smooth muscle and endothelial cells [40]. Recently. TGF-/?2 and /I’3 have also been observed as large latent complexes [33]. The large latent TGF-a complex has been purified from human platelets; it is approximately 235 kDa and is composed of two distinct gene products-the small latent TGF-/I complex and an additional 125-160 kDa protein called latent TGF-/I? binding protein (LTBP). LTBP is disulfide-linked to a LAP monomer of the small complex [21,41] (Fig. 1). Similar to the recombinant or small latent TGF-P complex, the LAP polypeptide portion of the large latent complex contains N-linked carbohydrates and mannose-6phosphate residues [41]. These carbohydrate residues appear to be involved in the activation of the large latent TGF-/? complex (see below). The LTBP cDNA cloned from a human foreskin fibroblast cDNA library predicts a highly structured protein consisting of 16 epidermal growth factor (EGF)-like repeat sequences and three unique motifs rich in cysteine residues. Two of the EGF-like sequences of human platelet-derived LTBP protein contain /J-hydroxylated asparagine residues, an unusual post-translational modification that might serve to bind calcium. In addition, an RGD sequence is present in one of the EGF-like repeats and an eight amino acid sequence identical to the proposed cell binding domain of the laminin B2 chain is found in one of the unique cysteine-rich repeats [42]. Studies using the HEL cell line suggest a role for LTBP in the assembly and secretion of the large latent complex. By itself, the small latent TGF-8 complex is not efficiently secreted. It appears to be improperly folded and contains anomalous intermolecular disulfide bonds between LAP and mature TGF-/? that are absent from the readily secreted large latent complex [32]. It is interesting to note that the aberrant disulfide bonds between LAP and mature TGF-D yield an inactivatable complex [26, 391. Recently, our laboratory has explored the additional possibility that LTBP might play a role in the activation of the latent TGF-/? complex (see below) [40].

In plasma, essentially all mature TGF-Breleased from the latent complex is bound to sr,M [43]. cw,M is an abundant serum glycoprotein (approximately 2 mg/ml) with a nonspecific binding site for several serum proteases (reviewed in [44]). Serum u?M is synthesized and secreted by the liver, as well as other cell types including macrophages and adrenocortical cells. In addition to binding proteases and TGF-PI, cr,M interacts with additional cytokines, including TGF-82, basic fibroblast growth factor (bFGF), platelet-derived growth factor and interleukins IL-t& IL-2 and IL-6 (reviewed in ]451). ol,M binds only mature TGF-8. However, the precise nature of this interaction is not known. Because antibodies raised against mature TGF-/I do not recognize TGF-/3

bound to .r,M [43], a ‘trap’ model similar to that postulated for sr,M-protease interactions (reviewed in [44]) has been hypothesized for the cr,M-TGF-/?complex. The interaction between sr,M and the entrapped protease induces a change in the conformation of a,M that allows the complex to bind to aZM receptors expressed on hepatocytes. as well as other cell types. Once the cr,M complex is bound to the cell surface. it is internalized by receptor-mediated endocytosis and cleared from circulation. The functional consequences of the binding of TGF-b to cr,M are not known. Similar 10 z>w,M-protease complexes, E:M-TGF-P interactions might serve to clear active TGF-P from sites of production. For example, aZM secreted by macrophages during inflammation might sequester excess mature TGF-8 and thus prevent uncontrolled binding to cell surface receptors [44]. Using a murine model, LaMarre and co-workers [46] analyzed the clearance of radiolabeled TGF-/I bound to protease or methylamine treated r:M. Over 90% of the radioactivity was recovered in the liver, indicating that the cr,M-TGF$ complex binds preferentially to specific receptors on hepatocytes. These observations support the role of a,M in scavenging active TGF-b. Alternatively, cl,M might function to deliver active TGF-P to the liver and other tissues that lack TGF-/I receptors. As aZM does not always irreversibly inactivate bound proteases but rather masks their enzymatic activity [44]. it is possible that TGF-8 in complex with a>M retains its biological activity. Whereas the majority of or,M-TGF-/I complexes are covalent, a minor portion are formed by non-covalent interactions [43, 471. These complexes may be dissociated by heparin (I 100 ,ug/ml) [48]. Although several cell lines, including endothelial cells and confluent smooth muscle cells secrete high levels of heparin-like glycosaminoglycans. rhe biological relevance of the release of mature TGF-/? from r,M by heparin is not clear. ACTIVATION

BY CHEMICAL

AND PHYSICAL

MEANS

Latent TGF-/I can be activated by a variety of chemical and physical treatments including acidification, alkalinization, detergents, urea or temperature, but not 5 M NaCl [49-511. These findings imply that latency is maintained by non-covalent. electrostatic forces. Interestingly. large latent TGF-p from platelets is more resistant to activation by heat and low pH than the small latent TGF-/I complex [50], suggesting that the LTBP may enhance its stability. Although physicochemical activation of TGF-a functions well in vitro, there is little evidence that these mechanisms are utilized in viva, with one possible exceptionacidification. Bone resorption, a process that activates latent TGF-/I, begins when osteoclasts create an enclosed acidic (pH < 3) area beneath their plasma membranes. This results in the demineralization of the bone matrix and the release of activated TGF-fi [52-551. The proton pump responsible for this acidification is believed to originate in lysosomal membranes. This implies fusion between plasma and lysosomal membranes and concurrent release of lysosomal hydrolases into the acidified extracellular compartment. Although the existence of hydrolases (such as cathepsin D) at the site of resorption has not been conclusively demonstrated, their presence might provide an additional method of activation of latent TGF-8 as will be discussed below CW.

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M6P Receptor

FIGURE 2. uPA activates TGF-p from LTBP during LTBP 1115.

A model for the surface activation of latent TGF-jl by the PA/plasmin system. Receptor bound receptor bound plasminogen (plgn) to plasmin (plm). Plasmin proteolytically liberates mature the latent complex bound to the cell surface via the M6P receptor (36, 62-69, 771. The role of activation is unknown. It is possible that unidentified cell surface or ECM binding sites exist for 1161.

Enzymatic

Activation

Besidesphysicochemical activation, latent TGF-/I can be enzymatically activated. Treatment with high levels of endoglycosidase F (IO-20 U/ml), sialidase (15 U/ml), neuraminidase (1 U/ml), or N-glycanase (1 U/ml), as well as mannose-6-phosphate (M6P; 50 mM) or sialic acid (15 mM) can activate latent TGF-/? in vitro [50, 571. Although activated macrophages can secretesialidasesand can activate latent TGF-p [S-60], it is not known whether these enzymes participate in the activation process. Additionally, the high concentration of either monosaccharides or enzymes necessary for activation make these unlikely activators in viva. A potentially more physiological mechanism for activation is proteolysis. Plasmin or cathepsin D treatment of cell culture conditioned medium activates latent TGF-p by cleavage of LAP in its amino terminal region [36,56]. The cleavage apparently disrupts the interactions between LAP and TGF-P, releasing the mature growth factor. In solution, the large latent complex, possibly due to LTBP, is lesssusceptibleto enzymemediated activation than the small latent complex [36.57,61]. This observation hasled some to question the physiological relevance of activation by this method. Additionally, plasmin activates only a fraction (approximately 20-30%) of the acidactivatable pool, implying the existence of various populations of latent TGF-p that are differentially susceptible to proteases[SO,561.The amount of latent TGF-8 that is activated by plasmin, however, is sufficient to induce a variety of biological responses such as macrophage chemotaxis, inhibition of endothelial cell migration and division, and stimulation of extracellular matrix deposition [7, 13. 16. 621. Because of the

Tiru Activrrtion c?fLatent TGF-fi

127

presenceof plasmin inhibitors, it is unlikely that the high concentration of plasmin required to activate TGF-his present in solution in viva. These observations have led to the model of cell surface mediated activation of latent TGF-@ by plasmin (Fig. 2). Plasminogen can be converted to plasmin by plasminogen activators (PA) on a variety of biological surfaces such as platelets, endothelial cells and ECM. When bound to the surface along with PA, plasminogen activation is enhanced and its product, plasmin. is protected from plasmin inhibitors [63-691. Surface bound plasmin is required for latent TGF-/? activation in three cell culture systems-co-cultures of bovine aortic endothelial (BAEs) and smooth muscle cells (BSMs) or pericytes. and retinoid or bFGF-treated BAEs [62, 70, 711. The addition of plasmin inhibitors or antibodies to urokinase type plasminogen activator (uPA) inhibits activation of latent TGF-/j in these systems. Surface bound plasmin alone, however, is not sufficient for activation, asdemonstrated by the inability of the human fibrosarcoma line HT- 1080 to activate latent TGF-B [70]. although it produces uPA and can activate plasminogen to plasmin on the cell surface [72, 731. Activation oflatent TGF-j?in co-cultures of BAEsand BSMs (or pericytes) has been studied extensively. Homotypic cultures of both cell types secretepredominantly large latent TGF-P. Activation occurs rapidly, however upon co-culturing [40. 74. 751. Besidesplasmin and uPA, it appears that latent TGF-B must also be surface bound for activation. Recombinant small latent complex and large latent TGF-@ bind to the cell surface mannose-6-phosphate/insulin-like growth factor II (M6P/IGF-II) receptor through LAP’s M6P moieties [38, 761.Addition of M6P (100 pM) or antibodies to the M6P/IGF-II receptor inhibits activation [77], possibly by blocking surface localization of the latent TGF-p. Recently. the role of LTBP in the activation of large latent TGF-P in co-cultures has been explored. Anti-LTBP antibodies, as well as purified soluble LTBP. inhibit activation [40]. Although the precise role of LTBP in activation is still under investigation, domains in the molecule possibly involved in protein--protein interactions-namely. EGF-like repeats, RGD or B2-laminin like regions-might direct the large latent TGF-Pcomplex to activation siteson the cell surface or the ECM Fibrillin, a component of the microfibrils present in the connective tissue space. is structurally similar to LTBP with multiple EGF-like repeats and several cysteine-rich motifs [78], further suggestingpossible interactions between LTBP and the ECM. Studies in our laboratory have implicated an additional molecule involved in latent TGF-/j activation. Antibodies to or inhibitors of tissue type II transglutaminase (TGase) block the activation oflatent TGF-Pin both co-culture and retinoid-activated RAE cell cultures [79]. The role of TGase in these systemshas not been fully elucidated. It has been recently reported, however, that plasminogen can be cross-linked LO iibronectin by Factor XIIIa or TGase [80]. Thus. TGase may play a similar role in localizing the activation complex to the cell surface or the ECM. The two other plasmin-dependent culture systemsfor TGF-Bformation, transient treatment of BAEs with hFGF or retinoids, demonstrate similar if not identical requirements as the coculture system (unpublished data). In addition to these plasmin-based latent TGF-P activation reactions. a number of other largely uncharacterized systemshave been described. These include y-interferon treated macrophages [60], retinoid, bone. or glucocorticoid treated osteoclasts or osteohlasts[54,81], and, more recently, angiotensin II treated vascular smooth muscle cells [X2] and thrombospondin treated BAEs. Interestingly. thrombospondin not only

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rt al.

stimulates BAEs to activate latent TGF-b, but also reversibly binds the mature growth factor which remains active [83, 841. Malignant cell lines can also secrete and activate latent TGF-8. These include the HEL cell line [85], phorbol ester treated lymphoma lines [86] and glioblastoma cell lines [33]. In addition, both hormone-dependent and independent breast cancer lines secrete and activate latent TGF-/I [87-891. Treatment of hormone-dependent lines with antiestrogens such as tamoxifen, increase both latent and active levels of TGF-p [89]. Hormone-independent lines treated with progestins, such as the synthetic progestin gestodene, demonstrate 3394-fold increases in total TGF-b levels, with a major proportion being active [87]. Activation of latent TGF-p by thesetumor lines is not well characterized. There is evidence, at least for the glioblastoma lines, that proteases might be involved inasmuch as protease inhibitors abrogate activation. Unexpectedly, a wide variety of inhibitors of exopeptidases, as well as serine, cysteine and aspartyl proteases[90] inhibit conversion of latent TGF-/J. Whether proteasesof all theseclasses are involved is not clear. Some of these inhibitors may alter intracellular processingof the latent molecules resulting in the secretion of an inactivatable complex. Alternatively, theseinhibitors may prevent activation of regulatory proteasesdirectly involved in TGF-/I activation. CONCLUDING

REMARKS

The activity of this pluripotent molecule must be controlled. Secretion as a latent precursor allows adequate concentrations of the growth factor to be available for activation when necessary. However, several factors other than latency have been implicated in the biological control of TGF-8 activity. Most cells constitutively express TGF-/I mRNA. Unexpectedly, active TGF-/? increasesexpression of its own messagein a variety of normal and transformed cells which are growth inhibited by TGF-/I [88, 91, 921. Therefore, it is likely that posttranscriptional events such as activation, scavenging of mature growth factor by cc,M and down-modulation of receptors contribute to the regulation of TGF-p. The best described TGF-/I activation system, co-cultures of BAEs and BSMs, reveals several potential regulatory steps.Although blocking the binding of the latent complex to the cell surface should inhibit its activation, there is no evidence that this occurs in rive. Blocking of plasminogen’s binding to the cell surface, however, may occur. Lipoprotein (a) [Lp(a)], an atherogenic lipoprotein, may modulate TGF-p activation as it was found to inhibit, in a concentration dependent manner, the production of mature TGF-,!I in the co-culture system. Control studiesindicated that Lp(a) inhibited the formation of cell surface associated plasmin activity, suggestingcompetition for plasminogen binding to the cell surface or ECM. The ability of Lp(a) to impede the formation of an inhibitor of smooth muscle cell migration suggestsa mechanism for the atherogenic activity of this lipoprotein [93]. The activation of plasminogen by PA is another possible regulatory step. Total PA activity in a variety of cells, including the vascular cells in the co-culture system. is decreasedby TGF-/?, suggestinga self-regulated system. Besidesdecreasing PA levels, TGF$can increase PAI- expression, further inhibiting plasmin production [94-1021. The remaining surface bound plasmin is then inactivated by its inhibitor, cr,antiplasmin [ 1031(Fig. 3).

Tl7r .4ctivution

qf’L,utent

TGF-/Y

Mature TGF-I3 M

.j!,,. “PA

+pro.“pA q-antiplasmin

Plasminogen : j,,,. .-; i

40

Plasmin

LargeLatent TGF-I3

FlGURE 3. Self-regulationof the plasmin-based activation of latent TGF-/I]104]. Binding of mature TGF-/? to its receptor results in decreased pro-uPA and increased PAL1 levels that inhibit uPA ]94-102]. The overall decrease in UPA activity reduces the activation of plasminogen to plasmin. The remaining plasmin is inactivated by qantiplasmin

]103],

thereby

further

decreasing

activation

of latent

TGF-8.

When this model was examined in the co-culture (BAE/BSM) system, the generation of active TGF-B was prolonged by the inclusion of antibodies to PAI-1. Additional studies demonstrated that PAI- levels increased following co-culture, and that this increase could be abrogated by addition of neutralizing antibodies to TGF-,6’ [ 1041. Control of TGF-/I function after activation may be mediated by receptor downregulation or scavenging of the active molecule by cr,M. Prolonged exposure to mature TGF-jj’ results in partial down-regulation of its receptors [20]. The biological significance of this level of down-regulation ( < 50%) is not clear sinceeven occupation of a small number of receptors can result in biological effects. Besidesdown-regulation. receptors may play a role in controlling TGF-/J’ activity as a soluble form of betaglycan (type III TGF-fi receptor) has been described [105]. Through interactions with the ECM. the soluble receptor may provide a potential reservoir of growth factor, OI protect it from degradation. Several ECM molecules have been implicated in binding mature TGF-8 [IO&l lo]. Binding to these matrix molecules may remove excessgrowth factor aswell asprovide an available pool of TGF-P, similar to that described for bFGF [l I I]. In addition, binding of TGF:/l to ECM moleculesmay play a protective role in riro. Administration of decorin, a small chondroitin-dermatan sulfate proteoglycan. for example, blocked the accumulation of ECM in a murine mode1of experimental glomerulonephritis [I 131. Neutralizing antibodies to TGFj3 activity had a similar effect [I 131.The inhibitory function of decorin may be another example of negative feedback regulation since TGF-P stimulates decorin’s production [ 1091. Despite the increasing knowledge of TGF$s diverse biological functions, very little is known about the regulation of its formation. Because of its deleterious effects. activation of latent TGF-/I must be tightly controlled-both spatially and temporally. Inappropriate activation of latent TGF-/I has been implicated in a number 01 pathological conditions, including acute mesangial proliferative glomerulonephritis, diabetic nephropathy, immunosuppression, restenosisof vesselsafter angioplasty. as well as fibrosis of the skin, central nervous system, lungs, liver and heart (reviewed in [I 141). Understanding the mechanism(s) of latent TGF$ activation may lead to directed therapeutic interventions in casessuch as these where there is inappropriate control of mature TGF-fi formation.

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et al.

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i%e Activation

qf Latent

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33 I

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