THE PHYSIOLOGY OF TRANSFORMING GROWTH FACTOR-(.11 Rik Derynck Program of Cell Biology, Departments of Growth and Development and of Anatomy, University of California, San Francisco, San Francisco, California 94143
11. TGF-a Is a Member of a Growth Factor Family 111. The Structure of TGF-a and Its Precursor
I v. V. VI. VII. VIII.
Interactions of TGF-a with the EGF/TGF-a Receptor The Transmembrane TGF-a Precursor A Role for TGF-a in the Physiology of Normal Cells TGF-a in Normal Development A Role for TGF-a in Tumor Development? References
1. Introduction Animal cells are normally exposed to a variety of extracellular factors that influence and determine their proliferative behavior. Many of these factors are polypeptides that have been secreted by the target cells themselves or by other cell populations. The polypeptide factors that either stimulate or inhibit cell proliferation are collectively called growth factors. The effects of growth factors on cells depend on a variety of factors, such as the nature of the growth factor, the cell type, and the physiological condition of the responding cell and its environment. In addition, the presence and the nature of other factors, including matrix proteins and other growth factors, profoundly influence the growth modulatory activities, frequently resulting in synergistic or antagonistic effects. Thus, individual factors may stimulate some cell types and inhibit others, depending on the conditions (Sporn and Roberts, 1988). In addition, growth factors often have a variety of activities, only some of which seem to be directly related to their effect on proliferation. Thus a great deal of apparent complexity accompanies the action of growth factors on cells. The effect of these growth factors on responsive cells is of major importance in all processes in which the modification or maintenance of a proliferative state of the cells is affected. It is thus expected that growth factors have profound effects in wound healing, in tissue formation, and in development as well as in formation and maintenance of tumors. We 27 ADVANCES IN CANCER RESEARCH, VOL. 58
Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
currently know of the existence of a largc variety of growth factors for mammalian cells, most of which can be classified in families of structurally related polypeptides. One of these factors is transforming growth factor-a (TGF-a), which is the subject of this review. We discuss here our current knowledge of the role of TGF-a in the proliferation of normal cells and its potential importance in tumor development. II. TGF-a Is a Member of a Growth Factor Family
TGF-a is a member of a small family of structurally related growth factors, of which epidermal growth factor (EGF) was the first member to be isolated and biochemically characterized (Savage et al., 1972, 1973). Besides EGF and TGF-a (Marquardt et al., 1984; Derynck et al., 1984; Lee et al., 1985), this family also contains the recently isolated amphiregulin (Shoyab et al., 1988) and heparin-binding EGF-like growth factor (HB-EGF) (Higashiyama et al., 1991). Another recently cloned growth factor derived from schwannoma cells is presumably the murine homologue of human amphiregulin (Kimura et al., 1990). In addition, there are three virally encoded polypeptide factors that also belong to this same family. These are vaccinia virus growth factor (VGF) (Venkatesan et al., 1982; Stroobant et al., 1985), myxomavirus growth factor (MGF) (Upton et al., 1987), and Shope fibroma growth factor (SFGF) (Chang et at., 1987), all encoded by members of the poxvirus family. T h e basis of the structural relationship is the “EGF unit,” a sequence of about 45-50 amino acids containing six characteristically spaced cysteines (CX, CX,-,CX,,- ,,CXCX,C), which are linked in a defined configuration in three disulfide bridges. T h e fully processed forms of EGF, TGF-a, and VGF correspond to this EGF unit and share a sequence identity of about 35%, including the six cysteines. The structural conservation and disulfide bond configuration in the core sequences of the different family members are the basis for the ability of these factors to interact with the same receptor, usually referred to as the EGF receptor (MassaguC, 1983; Stroobant et al., 1985; Lin et al., 1988; Shoyab et al., 1988; Higashiyama et al., 1991). This does, however, not preclude the possibility that these factors also may interact with other related receptors. TGF-a, EGF, amphiregulin, HB-EGF, and VGF are all made as larger precursors with a hydrophobic transmembrane domain, suggesting their existence as transmembrane, cell surface proteins (Fig. 1). T h e EGF domain in each of these growth factor precursors is localized in the extracellular segment of the precursor. In contrast to TGF-a (Derynck et al., 1984; Lee et al., 1985), amphiregulin (Plowman et al., 1990), HB-EGF (Higashiyama et al., 1991), and VGF (Venkatesan et al., 1982) precursors, the EGF
PHYSIOLOGY OF TGF-CX NH2
& + COOH
FIG. 1. Schematic representation of the precursors of the members of the EGF family. The precursors of EGF, TGF-a, amphiregulin (AR), HB-EGF, and VGF are represented as transmembrane proteins, whereas the precursors for MGF and SFGF are secreted. The black box in each precursor represents the fully processed form containing the EGF unit. The striped segments in the EGF precursor correspond to EGF-like sequences. All precursors have an NHz-terminal signal peptide (dotted). The branched structures represent Nlinked carbohydrate moieties.
precursor is considerably larger and contains a number of additional EGF-like repeats (Gray et al., 1983; Scott et al., 1983). Its large size, some distinct structural features with homology to segments of the low-density lipoprotein receptor (Russell et al., 1984; Sudhof et al., 1985), and the observation that the EGF precursor is often not processed into mature EGF (Rall et al., 1985) have led to the suggestion that the EGF precursor may be a receptor for an unknown ligand (Pfeffer and Ullrich, 1985). There is as yet no experimental confirmation of this hypothesis. A large variety of other polypeptides share one or more EGF-related sequences. One of these proteins is a small peptide “Cripto,”which has a cysteine configuration that is reminiscent of but different from the EGF theme and which may function as a growth factor (Ciccodicola et al., 1989). None of the other polypeptides with EGF-related sequences that
have been described are known to be mitogenic. This large family can be subdivided into several functional groups. One group consists of proteases and protease cofactors, whereas another group contains various polypeptides that play a role in cell-cell or cell-matrix interactions and may be important in development. The possible roles of the EGF-like domains of these proteins have been discussed elsewhere (Carpenter and Wahl, 1990; Davis, 1990). 111. The Structure of TGF-a and Its Precursor
cDNA cloning has revealed that the 50-amino acid TGF-a peptide is synthesized as an internal segment of a 160-amino acid precursor (Derynck et al., 1984; Lee et al., 1985). The sequence coding for this precursor is contained within a mRNA of 4.5-4.8 kb long. The precursor polypeptide sequence starts with an N-terminal signal sequence of 22 amino acids long, which is removed from the rest of the precursor between the residues Ala and Leu (Brachmann et d.,1989). Following the signal peptide cleavage site and preceding the N-terminus of the 50amino acid TGF-a is a sequence of 17 amino acids, referred to as the pro-TGF-a sequence. This short sequence is N-glycosylated at the Asn (position 24) and also carries O-linked carbohydrates (Bringman et al., 1987; Teixid6 et aZ., 1987; Teixido and Massague, 1988).The proteolytic cleavage at the N-terminus of the 50-amino acid TGF-a is between an Ala and Val-Val and is localized in a hydrophobic sequence context. The same cleavage recognition site (Ala-Val-Val)also marks the boundary of the C-terminus of the 50-amino acid TGF-a in the precursor (Derynck et al., 1984; Lee et al., 1985). These specific cleavage sites indicate the involvement of a highly specific protease with elastase-like properties in the excision process of the short TGF-a peptide from the extracellular domain of the TGF-a precursor (Ignotz et al., 1986; Luetteke et al., 1988; Teixido et al., 1900; Massaguk, 1990; F’andiella and Massague, 1991). The C-terminus of the 50 amino acids is followed nine residues downstream by a long, hydrophobic sequence. This hydrophobic sequence is immediately preceded by a dibasic peptide sequence (Lys-Lys),which is the target for another type of proteolytic cleavage (Derynck et al., 1984; Lee et al., 1985; Bringman et al., 1987).The protease responsible for this cleavage is more generally used because many polypeptide precursors undergo proteolytic processing at dibasic residues. The hydrophobic sequence spans the cell membrane and thus defines the extracellular domain and the C-terminal cytoplasmic domain in the precursor (Bringman et al., 1987; Gentry et al., 1987; Teixid6 et al., 1987) (Fig. 2). The cytoplasmic domain downstream of the transmembrane sequence is
PHYSIOLOGY OF TGF-CL
Fig. 2. Schematic representation of the TGF-a precursor. An NHP-terminal 22-amino acid sequence is cleaved from the precursor and precedes the short proregion, which contains an N-linked carbohydrate group. The 50-amino acid TGF-a peptide, shown as the bold line segment, has three disulfide-linked cysteine (C) bridges and is flanked by cleavage sites for the Ala-Val-Val-specificprotease (bold arrows). Another cleavage site (light arrow) is at the dibasic peptide immediately preceding the transmembrane sequence (black box). The cysteine residues are marked with C.
39 amino acids long and is rich in cysteines. This same precursor segment has palmitate covalently attached to it. This linkage of the fatty acid occurs at the cysteine residues, but it is not known how many cysteines and which ones have undergone this modification. The function of the palmitoylation of the precursor is unknown, but this modification could be indicative of a close association of this cytoplasmic domain with the membrane or with cytoskeletal elements (Bringman et al., 1987). Characterization of the TGF-a released in conditioned medium of cells overexpressing this growth factor (Bringman et al., 1987; Gentry et al., 1987) and the presence of several proteolytic cleavage sites in the transmembrane precursor (Bringman et al., 1987; Brachmann et al., 1989; Wong et al., 1989) thus indicate that, following removal of the
signal peptide, there are at least two different proteases involved in the release of soluble TGF-a from the transmembrane precursor. As a result of these cleavages, several soluble TGF-a species can be generated (Bringman et al., 1987) (Fig. 3). One possible form is the short, 50-amino acid species, but two larger forms that have retained the propeptide and thus are N-glycosylated can also be generated. Analyses of the TGF-a naturally secreted by various tumor cells suggest that these glycosylated TGF-a forms are far more common than the 50-amino acid form (De Larco and Todaro, 1978; Todaro et al., 1980; Marquardt and Todaro, 1982; Ignotz et al., 1986). It is attractive to postulate that the cleavage of the precursor by the Ala-Val-Val-specific protease is differentially controlled, thus regulating the relative ratios of soluble, diffusible TGF-a and uncleaved, immobilized TGF-a precursor and the relative levels of soluble glycosylated TGF-a and 50 amino acid TGF-a species. A conversion of the cell surface precursor into diffusible TGF-a would then affect a much larger number of target cells and could thus be much more effective, but could also result in different physiological effects. Such posttranslational regulation of the levels of soluble TGF-a and the nature of the soluble TGF-a species would be meaningful in the context of the transition from normal cells toward malignant tumor cells and in wound healing. It is possible that activation of the protein kinase C pathway enhances this posttranslational cleavage, because treatment of the cells with the tumor promoter 12-O-tetradecanoyl phorbol- 13-acetate (TPA), a protein kinase activator, enhances the proteolytic cleavage of the precursor (Pandiella and Massague, 1991). Cell surface immunofluorescence and biochemical characterization have indicated that TGF-a-synthesizingcells usually, if not always, have cell surface TGF-a pecursor molecules that are not cleaved to release soluble TGF-a. In addition, many TGF-a-synthesizing cells do not secrete soluble TGF-a into the medium (Derynck et al., 1987; Brachmann et al., 1989). Two forms of transmembrane TGF-a can exist, according to the model, with different proteolytic cleavages (Fig. 3). One form has undergone cleavage at the Ala-Val-Val at the N-terminal boundary of the 50amino acid TGF-a sequence, whereas the other form has retained the prosequence and is thus N-glycosylated (Brachmann et al., 1989; Massaguk, 1990). An analysis of TGF-a-overproducing cell lines suggests the likelihood that cells contain a mixture of both forms (Bringman et al., 1987; Gentry et al., 1987; Pandiella and Massague, 1991), although the relative ratios of these forms may depend on the cell line and on the physiological conditions. In any case, it appears that the presence of the transmembrane TGF-a at the cell surface is a normal consequence of TGF-a synthesis (Brachmann et al., 1989; Pandiella and Massague, 1991)
't FIG. 3. Schematic representation of the different forms of TGF-a, derived from the precursor in Fig. 2. Three forms, two of which are Nglycosylated (branched structures), are released into the medium. The two other forms, one with and one without N-glycosylation, are transmembrane forms and remain cell associated. The arrows show the proteolytic cleavage sites, whereas the cysteine residues are marked with C.
and is far more common than the actual release of soluble TGF-a into the medium. IV. Interactions of TGF-a with the EGF/TGF-a Receptor
Competition studies have revealed that EGF and TGF-a compete for binding to the same receptor, usually referred to as the EGF receptor (Todaro et al., 1980; MassaguC, 1983). Whereas it is possible that there might be a different unique receptor for TGF-a, it is currently widely accepted that all TGF-a effects are mediated through the common EGF/TGF-a receptor. The existence of multiple forms of soluble and transmembrane TGF-a species raises the possibility that there might be differences between how the individual TGF-a species interact with the receptor. However, the biological effects of TGF-a and especially comparisons between TGF-a and EGF have been evaluated until now using only the secreted 50-amino acid form of TGF-a. In order to address the question whether TGF-a and EGF are functionally equivalent, highly purified, recombinant 50-amino acid TGF-a and 53-amino acid EGF were compared in several biological assays. In some assays, TGF-a and EGF induce very similar activities. This is apparent in the stimulation of DNA synthesis in several cell lines (Schreiber et al., 1986), in the induction of anchorage independence of some rodent fibroblasts (Anzano et al., 1983), and in the acceleration of eyelid opening in newborn mice (Smith et al., 1985). On the other hand, both ligands exert quantitatively different responses in a variety of other systems, although some qualitatively different aspects cannot be excluded. These differences usually result in a higher potency for TGF-a than for EGF. One of these assays measures the effect of TGF-a and EGF on ruffling of the cell membrane. At high doses, the TGF-a-induced response is higher than that of EGF, and pretreatment of cells with TGF-P extends the duration of the TGF-a-induced response, but antagonizes the EGF-induced cell ruffling (Myrdal et al., 1986). TGF-a is also more potent than EGF in several proliferation-dependent assay systems. One example of these differences between EGF and TGF-a is provided by human keratinocytes. TGF-a is more active than EGF in inducing colony formation in monolayers, an event that results from a combination of cell migration and proliferation (Barrandon and Green, 1987; Pittelkow et al., 1989). TGF-a is also stronger than EGF in inducing a mitogenic response in hepatocytes (Brenner et al., 1989). Finally, TGF-a and EGF exert quantitatively different results on several pancreatic carcinoma cell lines. Using these cells, TGF-a is at least 10-fold more potent than EGF in
PHYSIOLOGY OF TGF-CX
inducing anchorage-independent colony formation in soft agar (Smith et al., 1987). TGF-a and EGF both have the ability to induce neovascularization in uiuo, but again TGF-a is much more potent than EGF (Schreiber et al., 1986). A similar result is also seen with an organ culture assay in uitro, in which Ca2 release from bones in culture is measured. TGF-a is considerably more potent than EGF in this assay, which is thought to correlate with bone resorption and hypercalcemia in uiuo (Stern et al., 1985; Ibbotson et al., 1986). Yet another example is provided by an arterial blood flow assay. Both factors are vasoactive and increase blood flow in this system, but TGF-a is again more active than EGF. In addition, treatment with EGF induces a refractory period, during which administration of another dose of EGF is without apparent biological effect. I n contrast, TGF-a does not induce such a refractory period and can overcome the EGF-induced refractory period (Gan et al., 1987). In conclusion, TGF-a, which interacts with the same receptor as EGF, is very frequently a superagonist of EGF. As yet very few comparative data on the nature of the interaction of EGF and TGF-a with the EGF/TGF-a receptor are available. At least two studies have shown that the dissociation constants of both ligands with the receptor are very similar (Lax et al., 1988; Ebner and Derynck, 1991). This, however, does not necessarily guarantee that the ligands interact in a similar fashion with the receptor, because it does not give any information about the ligand-receptor interactions during and following internalization and about the trafficking and fate of the ligands and receptors. T h e major quantitative difference in biological activity could be related to the fact that TGF-a dissociates from the receptor at a considerably higher pH than does EGF. This difference is presumably due to differences in PI of both growth factors, which is much higher for TGF-a than for EGF. This difference in pH dependence of dissociation of the receptor-ligand complex makes it likely that TGF-a, but not EGF, dissociates from the receptor immediately following internalization of the receptor-ligand complexes in the gradually acidifying endosomes. In contrast, EGF remains associated with the receptor until its delivery into lysosomes, which have a pH of around 4.8. Such a difference in pHdependent dissociation could be of determining importance to the fate of the receptors and the ligands following internalization and could profoundly affect the degree of down-regulation of the receptors and, thus, the availability at the cell surface of the receptors to new ligands. As a consequence, a large fraction of the total number of EGFITGF-a receptors could remain continuously available, when the cells are in the presence of TGF-a, whereas addition of EGF results in a virtually +
complete down-regulation of the receptor within a short time. This down-regulation of the receptor will then give rise to a period during which the cells are unresponsive to any additional ligand. An evaluation of potential differences in the intracellular trafficking of the receptors and ligands is currently underway (Ebner and Derynck, 1991). V. The Transmembrane TGF-a Precursor
As mentioned before, many TGF-a-synthesizing cells do not release soluble TGF-a peptides into the medium and all TGF-a-expressing cells examined exhibit transmembrane TGF-a precursors at their cell surface. The TGF-a precursors should thus be considered as normal physiological forms of TGF-a. Two independent studies have demonstrated that these TGF-ol forms interact with the EGF/TGF-a receptors on neighboring cells, without actual release of the growth factor (Brachmann et al., 1989; Wong et al., 1989).Thus cell-to-cell contact is sufficient to induce a mitogenic response in a neighboring cell. The relative activities of the two different transmembrane forms, i.e., the unglycosylated and the glycosylated forms, are not known. We also do not know the relative affinity or potency of the interaction of the receptor with this type of ligand, in comparison with the soluble TGF-a. The solubilized transmembrane TGF-a form is about 100-fold less active than the 50amino acid form (Brachmann et al., 1989),but this is presumably a result of the solubilization per se. The major difference between this interaction between the two cell surface molecules and the interaction of the receptor with the soluble TGF-a is that the former interaction remains very localized. Thus only cells in direct contact with the TGF-a-producing cells are stimulated. Obviously an autocrine interaction of this TGF-a form with the receptors can also take place in the same cell. In contrast, the soluble TGF-a is diffusible and can reach a much larger number of target cells. In addition, it is possible that there are qualitative differences between these two types of interactions. Interaction between the receptor and the cell surface TGF-a precludes an internalization of the ligand-receptor complex, in contrast with the interaction of the receptor with soluble TGF-a and EGF. It is conceivable, but not yet demonstrated, that the former interaction results in a more prolonged effect. In addition, this interaction between cell surface ligand and receptor may result in an enhanced adhesion between cells. Published results have demonstrated a low level of adhesion between cell surface TGF-a and EGF/TGF-a receptor-producing cells (Anklesaria et al., 1990), but the low efficiency of this adhesive interaction indicates that
PHYSIOLOGY OF TGF-OL
the TGF-a precursor should not be considered as an adhesion protein similar to cadherins and cell adhesion molecules (CAMS). The observation that a cell surface growth factor can productively interact with its receptor has important implications not only for the action of TGF-a, but also for a variety of other growth factors. Indeed, TGF-a is but one member of the family of EGF-like growth factors. Many of the growth factors in this family are thought to be synthesized as transmembrane precursors. This has been shown not only for TGF-a but also for the large EGF precursor that often remains uncleaved as a large cell surface protein (Rall et al., 1985). It is thus likely that the EGF, VGF, amphiregulin, and HB-EGF precursors are all able to interact productively with their receptors on neighboring cells, without actual release of the soluble growth factor. Some evidence supports this notion in the case of the large EGF precursor (Mroczkowski et al., 1989). In addition, there are at least three other growth factors that are made as transmembrane cell surface polypeptides: colony-stimulating factor-1 (CSF-1) (Rettenmier et al., 1987; Rettenmier and Roussel, 1988), tumor necrosis factor (TNF) (Kriegler et al., 1988),and the c-kit-encoded ligand (Anderson et al., 1990). The proteolytic cleavage of these transmembrane forms into soluble growth factors may be subject to regulation. Also in these cases it is likely that the cell surface-linked forms of the precursors can interact with the cell surface receptors on the same or on neighboring cells, thus resulting in a highly localized mitogenic stimulation of the target cells. The interaction of the transmembrane form of TGF-a and the receptor is thus an interaction between two cell surface proteins, during which there is signal transduction going through the receptor into the receptor-bearing cell. However, it is also possible that there is signal transduction through the TGF-a precursor into the TGF-a-producing cell. Thus the EGF/TGF-a receptor could represent a ligand for the TGF-a precursor as a receptor. Whereas such function is as yet still unproved, a possible physiological role associated with the cytoplasmic domain of the TGF-a precursor is substantiated by its extreme sequence conservation among animal species (Derynck et al., 1984; Lee et al., 1985), which is indicative of a conserved biological function. In addition, the spacing of the cysteine residues in the intracellular TGF-a precursor domain is reminiscent of the cysteine patterns that mediate the interactions between the cytoplasmic domains of the CD4 and CD8 cell surface receptors and the cytoplasmic lck protooncogene product, which functions as a tyrosine protein kinase (Shaw et al., 1989, 1990; Turner et al., 1990). It is possible that the somewhat similar sequence in the cytoplasmic domain
of the TGF-a precursor is indicative of and responsible for an interaction with an as yet to be identified cytoplasmic protein. If there is indeed signal transduction through the TGF-a precursor, then the interaction between both cell surface proteins, the precursor, and the EGF/TGF-a receptor would result in a two-directional signal transduction and cellcell communication and in an activation of cellular functions in both cells. If so, this interaction of the transmembrane TGF-a with the receptor would be qualitatively considerably different from the effect of soluble TGF-a on cells. A characterization of a receptor-like function of the TGF-a precursor would then also lead to a definition of a role for the soluble form of the EGFITGF-a receptor, which appears to be secreted by various cells, such as in the brain (Nieto-Sampedro, 1988) and the liver (Petch et al., 1990). This soluble domain could then function as a ligand that would modify biochemical pathways in cells that have the transmembrane TGF-a form.
VI. A Role for TGF-a in the Physiology of Normal Cells TGF-a was originally discovered in the medium of tumor cells (De Larco and Todaro, 1978; Todaro et al., 1980) and a possible function of TGF-a was considered only in the context of tumor cells. However, research during the last few years has now made it evident that TGF-a plays a role in the physiology of normal cells and tissues. Epithelial cells are the major source of TGF-a synthesis under normal conditions. TGF-a synthesis has been demonstrated by Northern hybridization, in situ hybridization, or immunochemical methods in a variety of normal epithelial cells (Valverius et al., 1989), and gastric and intestinal mucosa cells (Beauchamp et al., 1989). It is thus likely that most, if not all, types of normal epithelial cells synthesize TGF-a. These same cells also have EGF/TGF-a receptors (Carpenter and Wahl, 1990), thus making them responsive to the action of TGF-a in an autocrine fashion. Even though there is as yet no direct proof in uivo, it is likely that a normal role of the endogenous TGF-a synthesis in these epithelia is to drive their proliferation. There is certainly plenty of experimental evidence that epithelial cells in culture are responsive to TGF-a or EGF and that TGF-a or EGF addition results in their increased mitogenic activity (Carpenter and Wahl, 1990). Perhaps the best demonstration of this activity has been obtained using keratinocytes. Normal keratinocytes are dependent on exogenous EGF or TGF-a for their proliferation in culture. Following starvation, the proliferation of these cells is stimulated by TGF-a and EGF at subpicomolar levels. The effect of TGF-a and EGF on substrate-
PHYSIOLOGY OF TGF-OL
dependent colony formation of human keratinocytes has been studied in some detail. Starting from a single keratinocyte, a circular colony can only be formed efficiently when either TGF-a or EGF is present in the medium. The colonies of cells are considerably larger when grown in the presence of TGF-a than when using EGF. This colony formation is the result of cell proliferation and migration, but it is not known what the effects of TGF-a are on keratinocyte migration only (Barrandon and Green, 1987). T h e effects of EGF, TGF-a, and VGF on reepithelialization in pig skin due to keratinocyte proliferation in vivo have also been examined. TGF-a has the ability to induce reepithelialization, supporting the notion that it can induce keratinocyte proliferation in vivo (Schultz et al., 1987). It is as yet unknown if natural wound healing of the skin is accompanied by an enhanced TGF-a synthesis by the keratinocytes at the site of the wound. In vivo studies have also evaluated the expression levels of TGF-a in psoriasis. Psoriasis results in a local inflammation and topical proliferation and incomplete differentiation of skin keratinocytes. Analyses by immunohistochemistry, RNA hybridization, and immunological measurements of the TGF-a protein indicate that the levels of TGF-a expression by keratinocytes in the psoriatic lesions are enhanced in comparison with normal skin at unaffected sites o r in normal volunteers (Elder et al., 1989). These results, together with the responsiveness of keratinocytes to low levels of TGF-a, suggest that enhanced TGF-a expression could effectively contribute to the overproliferation of the skin in psoriasis. It is not known whether there is a direct link between the inflammation and the TGF-a synthesis, but recent data have shown that interferon-? can induce TGF-a synthesis (Kumar and Mendelsohn, 1990). Besides a presumed major role of TGF-a in proliferation of epithelia, TGF-a may also play a role in several other tissues. TGF-a expression has been detected in several structures in the brain and appears to be relatively highly expressed in the olfactory locus and in the pituitary (Kobrin et al., 1988; Wilcox and Derynck, 1988a). It is as yet unclear how the distribution of TGF-a expression in the brain compares with the localization of the EGF/TGF-a receptor expression. Because the detection of this growth factor has been mainly based on in situ hybridization of the TGF-a mRNA, it is hard to predict where exactly TGF-a exerts its activity. By analogy with many other peptides, there may indeed by axonal transport of growth factors and receptors in the neurons. No localization studies of both ligand and receptor at the protein level have been reported. We currently do not know the function of TGF-a in the context of the neurons and brain. There is, however, evidence that EGF, and thus presumably also TGF-a, have a potent neurotrophic activity
(Morrison et al., 1987). The role of the endogenous production of TGF-a and many other growth factors in the brain still needs to be established. TGF-a synthesis has also been reported in activated macrophages (Madtes et al., 1988; Rappolee et al., 1988a).Macrophages have the ability to synthesize a large variety of growth factors (Rappolee and Werb, 1991). The abundant presence of macrophages at sites of inflammation and wound healing and the effects of growth factors on cell proliferation strongly suggest that the role of the macrophage-released TGF-a is to participate in the wound-healing process and to stimulate the proliferation of epithelial cells. In addition, TGF-a is also able to induce proliferation of other cell types that have the EGF/TGF-a receptors, such as fibroblasts or endothelial cells. The abundance of the various growth factors released by macrophages is presumably essential for normal wound repair. Relatively little is as yet known about the regulation of TGF-a synthesis in epithelial cells or other cell types. Estrogen-responsivemammary epithelial cells have been shown to increase their level of TGF-a mRNA and protein synthesis following treatment with estrogen. This same treatment also results in an increased proliferation of such cells, suggesting that this could be due in part to an autocrine action of the higher levels of TGF-a (Liu et al., 1987). However, these cells presumably make several other growth factors, some of which may also be induced following estrogen treatment. Another factor that appears to modulate the synthesis of TGF-a by cells is the phorbol ester TPA. Treatment of cells with TPA, an activator of protein kinase C, induces a strong but transient increase of the level of TGF-a synthesis in keratinocytes and other cells (Pittelkowet al., 1989; Mueller et al., 1989; Bjorge et al., 1989). This increase has been shown to be due only to the activation of the protein kinase C pathway and is not due to any other effect of TPA. This leads to the possibility that exposure of cells to physiological levels of hormones or growth factors that activate the kinase C pathway may lead to an increase of TGF-a synthesis in the target cell. As mentioned above, treatment with TPA also results in an increased cleavage of the cell surface TGF-a form, thus releasing higher levels of soluble TGF-a (Pandiella and Massagut, 1991). Finally, TGF-a synthesis in keratinocytes and other cells is also enhanced following treatment with TGF-a or EGF, although not to the same extent as TPA-induced synthesis (Coffey et al., 1987; Bjorge et al., 1989; Mueller et al., 1989). Thus, there is an autostimulation of TGF-a production in these cells. A molecular analysis of this phenomenon could provide us with insight not only in the regulation of TGF-a synthesis, but also in the activation of expression of other genes that are the
PHYSIOLOGY OF TGF-OL
target of TGF-a action. It is as yet unclear what the biological meaning of this autoinduction of the TGF-a is. It could represent a simple mechanism to quickly amplify the effect of local TGF-a synthesis. Thus a low concentration of TGF-a released, e.g., by invading macrophages could result in a drastic increase of TGF-a synthesis by keratinocytes or other epithelial cells at a site of wound healing. Another possible consequence of this autoinduction is that such amplification of the TGF-a synthesis and the resulting response give rise to a more or less synchronized induction of cell proliferation. Finally, an autostimulatory mechanism may represent a relatively simple mechanism to maintain the TGF-a synthesis. Following an initial TGF-a induction, this autostimulatory mechanism may then be independent of the much more complex actions of regulators that were responsible for the initial induction of the TGF-a synthesis. Because this autostimulation of TGF-a synthesis has been reported for a few cell types only, it is unclear to what extent this also takes place in other types of normal cells o r tumor cells.
VII. TGF-a in Normal Development Growth factors play major roles in morphogenesis and organogenesis during development. The pattern of TGF-a expression during the development of the mouse has been studied mostly using various RNA detection techniques. Polymerase chain reaction (PCR)-based analysis has detected the presence of TGF-a mRNA in the unfertilized egg (Rappolee et al., 1988b). The TGF-a mRNA is then rapidly destroyed following fertilization and during the initial development of the mammalian embryo, but reappears again in the preimplantation embryo as early as the four-cell stage. The presence of TGF-a protein in virtually all cells of the blastocyst has also been observed by immunostaining (Rappolee et al., 1988b). T h e highest levels of expression occur around days 9-1 1 post coitum. TGF-a is then synthesized in the decidua (Han et al., 1987) and in several structures of the developing fetus (Wilcox and Derynck, 1988b). The levels of TGF-a transcripts in the decidua are highest at day 8 of gestation and decline as the decidua is resorbed. There is no detectable TGF-a mRNA in the nonpregnant uterus or in the pregnant uterus before decidualization (Han et al., 1987). In situ hybridization has revealed that TGF-a transcripts are also present in the developing fetus and that their levels are highest in several structures of ectodermal origin, such as in the branchial arches, the oral and nasopharyngeal epithelia, the otic vesicle, and the developing mesonephric tubules of the kidneys. The levels of TGF-a expression are maximal around days 9 and 10 and decrease at later stages in the fetal development (Wilcox and
Derynck, 1988b). There is, however, no doubt that a more detailed and sensitive analysis would reveal TGF-a synthesis at additional sites and at other stages of the fetal development. Little is known about the development expression of the EGF/TGF-a receptor. Northern hybridization suggests that the receptor mRNA level in the fetus is increased between days 11 and 14. In addition, EGFbinding activity is present in various fetal tissues and in blastocyst outgrowths. Unfortunately, no parallel studies localizing the synthesis of both the ligand and the receptor are available. Results from in uivo experiments or organ cultures using EGF (Carpenter and Wahl, 1990) and from cell culture experiments using TGF-a suggest that the major role of TGF-a in development is to drive the proliferation of various cell populations, especially epithelial cells. The coexpression of the TGF-a and the EGF/TGF-a receptor genes by many epithelial cells likely results in the exertion of the activities of TGF-a in an autocrine manner. Thus TGF-a synthesis could be of major importance in the development of the epithelia and of structures of ectoderma1 origin. A variety of studies, especially by Pratt and colleagues, have examined the effects of exogenous EGF on craniofacial development, particularly of the palate. These results suggest that the function of TGF-a, synthesized by the oral epithelia, is to promote the proliferation of the medial epithelium until the fusion of the palate shelves takes place (Lee and Han, 1990). Several other studies have evaluated the effects of EGF on the development and maturation of intestinal and gastric mucosa and suggest a role for the endogenous TGF-a synthesis in the proliferation and functional maturation of these epithelia (Lee and Han, 1990). A function of TGF-a in the development of epithelia is furthermore also suggested by the histological analysis of epithelia from transgenic mice overexpressing TGF-a (Jhappan et al., 1990; Matsui et al., 1990; Sandgren et al., 1990). Several organs of these mice, developed in three independent studies, had epithelial hyperplasia. This was most striking in the liver, coagulation gland, and intestines. Both colon and duodenum displayed a considerable mucosal hyperplasia. These effects of overexpression of TGF-a on different epithelia in transgenic mice appeared to be most influential during postnatal development, because overexpression of TGF-a had no major influence on the fetal development. Another possible target of the function of TGF-a during development is the mammary gland. Not only are mammary epithelial cells in culture stimulated in their proliferation, but application of TGF-a in a slow-release form to mammary glands of 5-week-old mice results in local alveolar and ductal growth (Vonderhaar, 1987). In accordance, TGF-aoverexpressing transgenic mice exhibit an increased penetration of an
PHYSIOLOGY OF TGF-O1
abnormally dense network of mammary epithelial ducts into the mammary fat pad. Thus the TGF-a synthesized in mammary epithelial cells could play an important role in the morphogenesis of the mammary gland. No effects of the overexpression of TGF-a on the mammary epithelial duct system were apparent during the first 4 weeks after birth, suggesting that some additional event or a hormonal stimulus related to puberty is required (Jhappan et al., 1990; Matsui et al., 1990; Sandgren et al., 1990). A further exploration of the role of endogenous TGF-ol synthesis in fetal and postnatal development will eventually require the generation of mice that are defective in TGF-a synthesis. As yet, no mice have been developed in which TGF-a synthesis was abolished following targeted gene disruption.
VIII. A Role for TGF-a in Tumor Development? A possible link between the production of TGF-a and the transformed character was apparent from the initial discovery of this growth factor. TGF-a was indeed first detected in the medium of murine sarcoma virus-transformed cells and several other retrovirus-transformed fibroblasts. Its name originated from the observation that preparations of this growth factor, even though impure, had the ability to induce phenotypic transformation of normal rat kidney cells, an immortalized fibroblast cell line, in culture. This transforming activity, which was apparent from the acquisition of anchorage independence in soft agar and from a different appearance and loss of contact inhibition in monolayer culture, was phenotypic and reversible, because removal of the TGF-a preparations resulted in a reversal to the normal phenotype (De Larco and Todaro, 1978; Todaro et al., 1980). We now know that the full transforming effect of these preparations was due to a synergism between TGF-a and TGF-P, and that neither of these factors alone has this ability to its full extent (Anzano et al., 1983). The transforming activity and the initial finding that TGF-a was only made in transformed fibroblasts and not in their normal counterparts have resulted in the concept that TGF-a could significantly contribute to malignant transformation and tumor development. Examination of a variety of cell lines subsequently showed that TGF-a was not only synthesized by fibroblasts transformed by retroviruses, but also by SV40- and polyoma-transformed cells (Kaplan and Ozanne, 1982; Kaplan et al., 1981). In addition, transformation with the ras oncogene or polyoma middle T coding sequences was sufficient to induce TGF-ol expression in the host cell (Kaplan and Ozanne, 1980; Salomon et al., 1987; Ciardiello et al., 1988). More recent studies have shown that the
induction of endogenous TGF-a expression by immortalized fibroblasts can also be achieved by introduction of and concomitant transformation by a variety of other oncogenes (Ciardielloet al., 1990). However, not all oncogenes had the ability to induce TGF-a synthesis, even though all transfected cells were transformed, indicating that TGF-a expression is not the result of transformation per se, but of a biochemical pathway triggered by selected oncogenes. Although there has not been a systematic study, the current data suggest that expression of oncogenes with gene products localized in the nucleus and presumably directly involved in transcription, does not induce TGF-a expression. In contrast, expression of ras and oncogenes that induce tyrosine phosphorylation or are tyrosine kinases themselves results in TGF-a expression. Examination of human tumor cell lines and biopsies revealed that TGF-a expression is fairly common among tumor types (Derynck et al., 1987; Bates et al., 1988; Nistkr et al., 1988).Hematopoietic tumors apparently do not synthesize TGF-a, but a variety of solid tumors do. Among the latter, carcinomas are most likely to synthesize TGF-a. More than half of the mammary carcinomas and all squamous and renal carcinomas examined synthesize TGF-a. Also, most hepatomas, melanomas, and glioblastomas exhibit an endogenous TGF-a synthesis. The incidence of TGF-a synthesis among tumors of mesenchymal origin is less common, but is by no means rare. Thus, TGF-a synthesis is prevalent among epithelial and other ectodermally derived tumors (Derynck et al., 1987). It is apparent that expression of TGF-a is frequently accompanied by enhanced synthesis of the EGF/TGF-a receptor, at least as assessed by Northern hybridization of the mRNA. This is most striking in the case of squamous and renal carcinomas, which consistently synthesize high levels of TGF-a and EGFITGF-a receptor (Derynck et al., 1987). It is likely that high receptor levels result in an enhanced sensitivity of the cell to the autocrine stimulation of TGF-a, thus considerably amplifying the effects of the growth factor (Di Fiore et al., 1987; Di Marco et al., 1989). It is not known whether the transition from a TGF-asynthesizing epithelial cell to a fully transformed carcinoma results in an increase of TGF-a synthesis, although anecdotal data on unmatched samples suggest that this may be the case. The endogenous synthesis of TGF-a in tumor cells could be of importance in tumor development and maintenance. Unfortunately, results of direct experiments, e.g., aimed at an abolition of TGF-a synthesis in normally TGF-a-synthesizingtumor cells, have as yet not been reported. Thus our knowledge about the role of TGF-a in tumorigenesis is primarily based on a variety of observations from indirect experiments.
PHYSIOLOGY OF TGF-a
Because TGF-a is mitogenic for many cells, we assume that the TGF-a produced by tumor cells enhances proliferation of these same cells in an autocrine way, provided these cells have EGFITGF-a receptors. Indeed, most, if not all, TGF-a-producing tumor cells have EGF/TGF-a receptors, and, as mentioned above, an enhanced receptor expression level is frequently encountered. Ligand and receptor could interact in an autocrine fashion in several ways. The most common interaction takes place at the cell surface, but could in principle also occur in cytoplasmic vesicles, such as the exosomes, making these interactions oblivious to the neutralizing effects of antibodies or proteases. Internal ligand-receptor interactions are thus possible and have been unambiguously documented in the case of the platelet-derived growth factor B chain (Keating and Williams, 1988). T h e extracellular TGF-a could exert its activities either as a soluble secreted and diffusible ligand, which thus can interact with many cells in proximity to the producer cells, or as the uncleaved transmembrane form of TGF-a, which can interact only with the neighboring cells in contact with the producer cells. Various experiments strongly suggest that endogenous TGF-a synthesis provides the means for an increased proliferation rate to the tumor cells. A good example of this effect is provided by a study comparing the susceptibility to exogenous TGF-a of untransformed mammary epithelial cells and their polyoma middle T-transformed and rm-transformed counterparts (Salomon et al., 1987). The normal cells and the polyoma middle T-transformed cells are strongly stimulated in their proliferation, whereas the rm-transformed cells are not. T h e polyomatransformed cells have a low level of endogenous TGF-a synthesis and their proliferation rate is enhanced by exogenous TGF-a to a level very similar to that of the rm-transformed cells. The latter cells have a high level of endogenous TGF-a synthesis and are not very dependent on exogenous growth factors. The autocrine stimulation by a high level of TGF-a synthesis may be responsible in part for the very high proliferation rate of the rm-transformed cells, which cannot be further stimulated. Other experiments, comparing the rate of tumor formation in nude mice by papilloma cells that were or were not transfected by a TGF-a expression vector, clearly established an increased tumor size and proliferation rate as a consequence of the TGF-a production (Finzi et al., 1988). Considerable attention has been given to the question whether endogenous TGF-a synthesis by cells is able and sufficient to convert untransformed cells into transformed and tumorigenic cells. The basis for this hypothesis was the original observation that TGF-a preparations
were able to induce phenotypic transformation. In addition, the original autocrine hypothesis (Sporn and Todaro, 1980) proposed that endogenous expression of TGF-a and concomitant autocrine interaction and stimulation of the producer cell lines by these factors resulted in independence of exogenous growth factors, a basis for malignant transformation. Thus cells were transfected with a TGF-a expression plasmid, and selected TGF-a-producing cell lines were examined for their acquisition of characteristics of malignant transformation. Results from various studies established that a high level of TGF-a synthesis can indeed result in transformation and tumorigenicity, but that this clearly depends on the choice of the cell line and on the assays used as criteria for the transformed phenotype. Some immortalized cell lines such as the Rat-1 (Rosenthalet al., 1986)and NRK (Watanabeet al., 1987) fibroblasts and the epithelial NOG-8 cell line (Shankar et al., 1989) clearly lost contact inhibition and anchorage independence when transfected with a TGF-a expression vector and thus were transformed. Also, expression of TGF-a in Rat-1 cells (Rosenthal et la., 1986) and of EGF in Fisher rat 3T3 cells (Stern et al., 1987) resulted in tumorigenicity in animals. However, TGF-a expression in early-passage NIH 3T3 cells resulted only in a higher cell density in the monolayer, but not in anchorage independence or tumorigenicity (Finzi et al., 1987), whereas TGF-a expression in cultured primary epidermal cells did not result in tumor formation either (Finzi et al., 1988). In addition, TGF-a overexpression in skin papillomas did not result in neoplastic progression, but only in increased size of the resulting benign tumors (Finzi et al., 1988). All these results suggest that TGF-a expression can induce transformation and tumorigenicity only when the cells have already evolved closely to the transformed character. A parameter that is very important for the effect of TGF-a expression on the acquisition and establishment of transformation and tumorigenicity is the number of EGF/TGF-a receptors. Indeed, overexpression of the receptors can result in ligand-dependent transformation and there is a minimal quantitative requirement of cell surface receptors in order to obtain the TGF-a-induced malignant phenotype (Di Fiore et al., 1987; Di Marco et al., 1989). It is therefore likely that a high level of TGF-a synthesis, combined with a high receptor expression level, as seen in squamous carcinomas or renal carcinomas, can be of considerable importance to the behavior, the phenotype, and the malignant character of some tumor cell types. Transgenic mice that overexpress TGF-a developed a variety of neoplastic lesions (Sandgren et al., 1990; Jhappan et al., 1990; Matsui et al., 1990). Depending on the mouse strain and the promoter that directs the
PHYSIOLOGY OF TGF-(Y
transcription of the TGF-a coding sequence, these mice displayed development of coagulation gland carcinomas, mammary adenocarcinomas, and hepatocellular carcinomas. Thus, expression of the TGF-a gene can be oncongenic in vivo. There is not necessarily a discrepancy between these findings and the result from in vitro transfection experiments, which suggested that the cells had to be already close to transformation in order to see a transforming effect of TGF-a expression. T h e tumors that develop in transgenic mice appear only after an extended period of postnatal development and are often found together with hyperplastic areas in the same tissues or organs. This suggests that the overexpression of TGF-a and the high autocrine responsiveness of the cells and tissues result in an extensive proliferation, before tumor development is initiated. Thus the high proliferation rate may considerably increase the probability of tumor development. The induction of TGF-a expression can thus be seen as a tumor-promoting effect or as a contributing step in the progression of the cell toward a fully transformed phenotype. It is not known to what extent these tumor cells in the transgenic mice are dependent on TGF-a for their neoplastic character and behavior. A recent study has established that a high level of TGF-a expression also influences motility and the capability of the cell to digest the extracellular matrix (Gavrilovic et al., 1990). Expression of a transfected TGF-a cDNA in the NBTII rat carcinoma cell line resulted in the conversion from an epithelial to a vimentin-positive fibroblastic phenotype. These cells also acquired a highly motile behavior and secreted significant levels of a 95-kDa gelatinolytic metalloproteinase, presumably corresponding to a type IV collagenase, which was virtually absent in the parent, untransfected cells. These changes, resulting from the expression of TGF-a, could contribute to a more invasive phenotype in vivo. No experiments have as yet been done to evaluate the role of TGF-a in the invasiveness in vivo and the metastasis of these TGF-aproducing tumor cells. T h e ability of TGF-a to induce neovascularization (Schreiber et al., 1986) could also provide an additional advantage to tumor formation. Solid tumors are indeed very dependent on vascularization, as soon as they reach a critical diameter, which does not allow sufficient access to oxygen and nutrients by diffusion. In order to grow beyond this critical size it is imperative that neovascularization takes place (Klagsbrun and Folkman, 1990). the endogenous synthesis of TGF-a can contribute to this process of angiogenesis, because it has been shown that this growth factor is a potent inducer of angiogenesis in vivo (Schreiber et al., 1986). We can assume that TGF-a may not be the only angiogenic factor released by these cells, because a variety of other growth factors can also
induce neovascularization. It is thus likely that the effect of TGF-a occurs in concert with other factors. Finally, TGF-a secreted by the tumor cells in vivo could also influence calcium metabolism and could contribute to a hypercalcemic state. Malignancy-associated hypercalcemia occurs relatively frequently in patients with renal carcinoma, squamous carcinoma, melanoma, or breast carcinoma (Mundy et al., 1985). Tumors of these types are very consistent producers of TGF-a. I n vitro studies have shown that TGF-a is able to induce Ca2+ release from bone cultures (Stern et al., 1985; Ibbotson et al., 1986),thus suggesting that the TGF-a synthesis and release by these tumors could contribute to the induction of malignancy-induced hypercalcemia. It is, however, important to recognize that TGF-a-induced Ca2+ release may only be one mechanism, because parathyroid hormone-related polypeptide synthesized by tumors could also play a role in this type of hypercalcemia (Mundy et al., 1985). The latter factor could maybe synergize with TGF-a, whereby TGF-a would exert its effects mostly at the local level, e.g., at the site of tumor development or at the site of growth of a metastatic nodule, and the parathyroid hormone-like peptide would represent a more systematic activator of Ca2+ release. As is evident from this review, we know as yet little about the biology and the physiological role of TGF-a. Our current knowledge clearly indicates that TGF-a expression is not restricted to tumors, but is very common in normal cells, especially epithelial cells. Thus TGF-a should be considered as a perfectly normal physiological ligand of the EGFITGF-a receptor, one that plays a role in cellular proliferation not only in the adult, but presumably even more importantly in organ and tissue development. Its role in normal tissues certainly does not exclude a role in the establishment and maintenance of the malignant character of tumor cells. In this context, TGF-a could play a role in and contribute to phenotypic transformation, and could certainly stimulate the proliferation of the tumor cells and of the tumor in vivo. In addition, TGF-a expression may influence the invasive behavior of the tumor cells and contribute to the induction of neovascularization of the tumors and to malignancy-induced hypercalcemia. REFERENCES Anderson, D. M., Lyman, S. D., Baird, A,, Wignall, J. M., Eisenmann, J., Rauch, C., March, C. J., Boswell, H. S., Gimpel, S. D., Cosman, D., and Williams, D. E. (1990). Cell 63, 235-243. Anklesaria, P., Teixid6, J., Laiho, M., Pierce, J. H., Greenberger, J. S., and Massague, J. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 3289-3293.
PHYSIOLOGY OF TGF-CL
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