The Journal of Dermatology Vol. 19: 644-647, 1992
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Structure, Function and Possible Clinical Application of Transforming Growth Factor-S Kohei Miyazono and Carl-Henrik Heldin Abstract
Transforming growth factor-S (TGF-J3) is a family of multifunctional 25 kDa proteins. TGF-J3 was originally identified because of its ability to induce the growth of normal rodent fibroblasts in soft agar, but is now known as a potent growth inhibitor for many different cell types. In addition, TGF-J3 is known to regulate the differentiation of cells, induce chemotaxis ofcells, and to induce the accumulation of extracellular matrix proteins. In vivo, TGF-J3 stimulates the repair of soft as well as hard tissues. It also acts as a potent immunosuppressant TGF-J3 is produced as latent high molecular weight complexes; since it is produced by many different cell types, and most cells have receptors for TGF-J3, the activation of latent TGF-J3 is likely to be an important step in the regulation of its action. TGF-J3 exerts its effects by binding to specific cell surface receptors. The type I and type II TGF-J3 receptors are suggested to be the most important for signal transduction; a recent report has disclosed that the type II receptor has a serine/threonine kinase domain. Since TGF-J3 is a potent growth regulator with multifunctional activity, it may be useful in the treatment of certain clinical disorders. Local application ofTGF-J3 is shown to accelerate wound healing. Since an increase in TGF-J3 activity is often observed in various fibrotic disorders, antagonists for TGF-J3 might be valuable in the treatment of such diseases.
Key words: TGF-J3; growth factor; growth inhibitor; latent form; receptor; wound healing TGF-J3 Superfamily Transforming growth factor-J3 (TGF-J3) is a family of structurally related proteins with molecular weights of about 25,000. So far, five different TGF-J3 isofonns have been identified which have 66-82% amino acid sequence identity with each other (1). TGF-J31 was first purified from human platelets and cloned from a eDNA library from human placenta. TGF-J32 and -133 were subsequently identified in several mammalian cells and tissues. In contrast, TGF-J34 and -135 have been found only in the chicken and frog, respectively. TGF-J3s are synthesized as precursor forms of 390 to 412 amino acids. The N-tenninal parts are cleaved off by proteolysis, and dimeric forms of the C-tenninal 112 amino acid peptides constitute the active TGF-J3 molecules. The structural similarity is less in the N-tenninal precursor parts, compared to the portions corresponding to the mature TGF-J3s. TGF-J31, -132, and -133 share most of their biological activities, although TGF-J31 and -133 are more potent than TGF-J32 on hematopoietic progenitor Ludwig Institute for Cancer Research, Box 595 Biomedical Center, 5-75124 Uppsala, Sweden.
cells and fetal bovine heart endothelial cells (2). The promotor regions of TGF-J31, -132, and -133 are different, and the productions of these proteins are differently regulated (3). Whether the TGF-J3 isoforms play distinct roles in vivo remains to be elucidated. There are also proteins that are more distantly related to TGF-J3s (4). These include inhibins and activins, Mullerian inhibitory substance, Drosophila decapentaplegic gene complex, Vg-l, and bone morphogenetic proteins. These molecules play important roles in the growth and differentiation of different cell types, e.g. during different stages of the development Activin A is a member of the TGF-J3 superfamily and one of the most well characterized factors in this category (5). It was originally identified as -a factor that regulated the production of follicle-stimulating hormone in pituitary cells. It was then shown to induce the differentiation of the erythroleukemia cells and to have a mesoderminducing activity in Xenopus embryos. Type II receptors for both activin and TGF-J3 possess serine/ threonine kinase domains in the cytoplasmic portions (see below).
Structure and Function ofTGF-p
645
Bioactivity ofTGF-p TGF-p was originally identified as a growth promoting activity for rodent fibroblasts in soft agar. However, a growth promoting activity of TGF-p is only observed for certain cell types and during certain culture conditions. It has been suggested that TGF-p induces the growth of certain mesenchymal cells in monolayer cultures through the production of platelet-derived growth factor (PDGF), which then acts as an autocrine growth stimulator (6-8). TGF-ps are potent growth inhibitors for many different cell types in vitro, such as endothelial cells, epithelial cells, including hepatocytes and keratinocytes, hematopoietic progenitor cells, and lymphocytes. TGF-p induces the chemotaxis of various cell types such as neutrophils, monocytes and fibroblasts, as determined by the Boyden chamber method. In contrast, migration of endothelial cells is dramatically inhibited by TGF-p when it is added after endothelial cell wounding (9). Other important functions of TGF-p are its effects on the extracellular matrix (4). TGF-p induces the accumulation of extracellular matrix by the increase of matrix protein production, such as fibronectin and collagen, and by the inhibition of the activity of enzymes that degrade matrix proteins. Moreover, the production of certain receptors for matrix proteins is also stimulated by TGF-p; thus the interaction between cells and the matrix may be promoted. A 'potent immunosuppressing activity has also been observed for TGF-p. It inhibits the proliferation of thymocytes, T cells, B cells, and natural killer cells (10, 11). In most cases, the functional activities of immune cells, such as the production of IgG and IgM, are also downregulated. TGF-j3 induces angiogenesis in vivo. The mechanism for this effect is unknown; TGF-j3 is a potent growth inhibitor for endothelial cells (12), but stimulates migration of other cells from surrounding tissues, which may be important for the angiogenic process induced by TGF-j3 (13). TGF-j3 also affects the differentiation of endothelial cells and promotes tube formation (14).
of receptors are important for understanding the regulation ofTGF-j3 action in vitro and in vivo (15). TGF-ps are produced as latent forms in most cell types. Latent TGF-p in human platelets is composed of three different components, i.e. activeTGF-pl dimer, TGF-j31 latency associated peptide (PI-LAP), and the latent TGF-pl binding protein (LTBP) (16). 131LAP is the N-terminal remnant of the TGF-j31 precursor; LTBP is produced by a different gene and associates with j31-LAP by a disulphide linkage. PI-LAP is most important for the latency ofTGF-j3, since TGF-pl produced by the transfection of TGF-j31 precursor cDNA into Chinese hamster ovary cells is composed only ofTGF-j31 and j31-LAP, but is still latent (17). The latent TGF-ps can be activated by treatments with acid, alkali, or heat Activation can also be achieved by certain enzymes such as plasmin and glycosidases (18). However, the mechanisms that control TGF-j3 activation in vivo are still not fully elucidated. TGF-j3s are produced as latent complexes with or without LTBP. For instance, the latent TGF-j31 observed in human platelets is associated with LTBP, whereas TGF-j32 observed in epidermal keratinocytes in vivo is not associated with LTBP (our unpublished observations). The role ofLTBP in the latent TGF-j3 complex is not known. Two thirds of the molecule are composed of two different types of repeat sequences; 16 copies of epidermal growth factor (EGF)-like repeats and 3 copies of another motif containing eight cysteine residues (19). EGF-like repeats are observed in several different proteins and may playa role in protein-protein interactions (20). Certain EGF-like domains have been found to bind Ca". The eightcysteine repeat was first found in LTBP; then a similar structure was also observed in fibrillin, the protein that is deficient in Marfan's syndrome (21). Fibrillin is a component of the extracellular matrix, and its deficiency results in such symptoms as skeletal abnormalities, aortic aneurysm, and aortic dissection. Whether LTBP is also a matrix component or can interact with the matrix is an interesting possibility which remains to be determined.
Latent Forms ofTGF.p Many different cell types produce TGF-ps, and most cells possess receptors for TGF-j3.However, in the action of TGF-j3, there are several critical steps regulating its activity. Among those, the regulation of the production and the activation of latent forms ofTGF-j3, as well as interactions with and activation
Signal Transduction by TGF·p Receptors for growth factors are divided into several different families based on their structures (22). Most of the receptors for growth factors, such as EGF, PDGF, insulin-like growth factor-I and fibroblast growth factors, have tyrosine kinase domains in their intracellular portions. Receptors
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for cytokines, on the other hand, do not have protein kinase structures in their cytoplasmic domains. However, the {3 chain of the interleukin-2 (IL-2) receptor has been shown to interact with a soluble tyrosine kinase, Lck (23). In contrast, the receptors for the TGF-{3 superfamily appear to be structurally and functionally different from the growth factor receptors and the cytokine receptors. Three different types of TGF-{3 receptors are observed in most cell types. The type I and type II TGF-{3 receptors are glycoproteins of 53 kDa and 60-70 kDa, respectively. The type III TGF-{3 receptor is a 280 kDa proteoglycan and, therefore, has also been denoted betaglycan. Type I and type II receptors are most important for the signal transduction ofTGF-{3, whereas the type III receptor may not be directly involved. cDNA cloning of the type III (24, 25) and type II TGF-{3 receptors (26) have recently been reported. The type III receptor is synthesized as a 120 kDa core protein. The cytoplasmic domain of the type III receptor is composed of only 43 amino acids with a high content (42%) of serine and threonine residues; no protein kinase domain was observed, but a 63% sequence identity with endoglin, a possible cell adhesion protein observed in endothelial cells and hematopoietic cells, could be found (27). The intracellular portion of the type II TGF-{3 receptor has a serine/threonine kinase domain. Autophosphorylation on serine and threonine residues has also been demonstrated for the TGF-{3 type II receptor (26). Transmembrane serine/ threonine kinases have previously been found only in the dafl protein (28), which is involved in larval development of nematodes, and in the activin type II (29) and type lIB (30) receptors. These observations suggest that transmembrane serine/threonine kinases may form a new receptor family, which possibly includes the receptors for proteins in the TGF-{3 superfamily. TGF-{3 regulates the expression of nuclear transcription factors; e.g. in murine lung epithelial cells, the amount ofJun-B is increased and the amount of c-Myc is decreased after TGF-{3 stimulation. TGF-{3 inhibits cell cycle progression at the late G1 phase. The retinoblastoma (RB) gene product has been shown to be involved in the antiproliferative effect ofTGF-{3 (31, 32).
Clinical Application of TGF-{3: Perspectives TGF-{3s are multifunctional proteins which may be of clinical use in certain different disorders. Since TGF-{3 is a potent immunosuppressant, sys-
temic administration of TGF-{3 could be useful in the treatment of autoimmune diseases and allograft rejection (10, 11). TGF-{3 stimulates wound healing (33). Defect in wound healing is often observed in aged persons and in patients with diabetes or undergoing glucocorticoid treatment, radiation, or cancer chemotherapy. Local application of TGF-{3 may accelerate the healing of the wounds in these patients. Furthermore, TGF-{3 plays an important role in bone remodeling and thus could be useful in the treatment of bone fractures. Some fibrotic diseases may occur through excess and/or inappropriate wound healing, such as liver cirrhosis, scleroderma, keloid formation, pulmonary fibrosis, glomerulonephritis, and others. Increased amounts of TGF-{3 have often been observed in diseased tissues with an excess of matrix proteins (34, 35). Use of TGF-{3 antagonists such as TGF-{3 neutralizing antibodies have been shown to prevent the progress of certain of these disorders (34, 36). So far, clinically useful TGF-{3 antagonists are not available. The development of substances that prevent the activation of the latent TGF-{3s or that compete for binding to TGF-{3 receptors might provide valuable tools in the control of these disorders. References 1) Roberts AB, Sporn MB: The transforming growth factor-Sa, in Sporn MB, Roberts AB (eds): Peptide Growth Factors and Their Receptors I, Springer-Verlag, Berlin, 1990, pp 419-472. 2) Cheifetz S, Hernandez H, Laiho M, et al: Distinct transforming growth factor-S (TGF-fi) receptor subsets as determinants of cellular responsiveness to three TGF-~ isofonns,] Biol Chern, 265: 20533-20538, 1990. 3) Roberts AB, Kim S], Noma T, et al: Multiple forrns of TGF-~: Distinct promoters and differential expression, Ciba Found Symp, 157: 7-28, 1991. 4) Massague J: The transforming growth factor-S family, Annu Rev Cell Biol, 6: 597-641, 1991. 5) Vale W, Hsueh A, Rivier C, et al: The inhibin;activin family of hormones and growth factors, in Sporn MB, Roberts AB (eds): Peptide Growth Factors and Their Receptors II, Springer-Verlag, Berlin, 1990, pp 211-248. 6) Leof BE, Proper JA, Goustin AS, et al: Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor ~: A proposed model for indirect mitogenesis involving autocrine activity, Proc Natl Acad Sci USA, 83: 24532457,1986. 7) Soma Y, Grotendorst GR: TGF-~ stimulates primary human skin fibroblast DNA synthesis via an autocrine production of PDGF-related peptides,] Cell Physiol, 140: 246-253, 1989.
Structure and Function of TGF-p
iI) Battegay E], Raines EW, Seifert RA, et al: TGF-,8
9)
10)
II)
12)
13)
14)
15)
16)
17)
18)
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induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop, Cell, 63: 515-524, 1990. Sato Y, Rifkin DB: Inhibition of endothelial cell movement by pericytes and smooth muscle cells: Activation of a latent transforming growth factor-,81like molecule by plasmin during co-culture,] Cell Bioi, 109: 309-315, 1989. Kehrl JH: Transforming growth factor-S: An important mediator ofimmunoregulation, Int] Cell Cloning, 9: 438-450,1991. Ruscetti FW, Palladino MA: Transforming growth factor-S and the immune system, Prog Growth Factor Res, 3: 159-175, 1991. Takehara K, LeRoy EC, Grotendorst GR: TGF-,8 inhibition of endothelial cell proliferation: Alteration of EGF binding and EGF-induced growthregulatory (competence) gene expression, Cell, 49: 415-422,1987. Yang EY, Moses HL: Transforming growth factor ,81induced changes in cell migration, proliferation, and angiogenesis in the chick chorioallantoic membrane, ] Cell Bioi, 111: 731-742, 1990. Madri JA, Pratt BM, Tucker A: Phenotypic modulation of endothelial cells by transforming growth factor-S depends upon the composition and organization of the extracellular matrix,] Cell Bioi, 106: 1375-1384, 198B. Wakefield LM, Smith DM, Masui T, et al: Distribution and modulation of the cellular receptor for transforming growth factor-beta,] Cell Bioi, 105: 965-975, 1987. Miyazono K, Hellman U, Wernstedt C, et al: Latent high molecular weight complex of transforming growth factor ,81: Purification from human platelets and structural characterization,] Bioi Chern, 263: 6407-6415, 198B. Gentry LE, Webb NR, Lim GJ, et al: Type 1 transforming growth factor beta: Amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells, Mol Cell Bioi, 7: 3418-3427,1987. Miyazono K, Heldin C-H: Latent forms of TGF-,8: Molecular structure and mechanisms of activation, CibaFound Symp, 157: 81-92, 1991. Kanzaki 1', Olofsson A, Moren A, et al: TGF-,81 binding protein: A component of the large latent complex of TGF-,81 with multiple repeat sequences, Cell, 61: 1051-1061, 1991. Apella E, Weber IT, Blasi F: Structure and function of epidermal growth factor-like regions in proteins, FEBS Lett, 231: 1-4, 1988. Maslen CL, Corson GM, Maddox BK, et al: Partial sequence of a candidate gene for Marfan syndrome, Nature, 352: 334-337, 1991.
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22) Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity, Cell, 61: 203212,1990. 23) Hatakeyama M, Kono T, Kobayashi N, et al: Interaction of the IL-2 receptor with the src-family kinase p561Ck : Identification of novel intermolecular association, Science, 252: 1523-1528, 1991. 24) Lopez-Casillas F, Cheifetz S, Doody J, et al: Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-,8 receptor system, Cell, 67: 787-795, 1991. 25) Wang X-F, Lin HY, Ng-Eaton E, et al: Expression cloning and"'Characterization of the TGF-,8 type III receptor, Cell, 67: 797-805, 1991. 26) Lin HY, Wang X-F, Ng-Eaton E, et al: Expression cloning of the TGF-,8 type II receptor, a functional transmembrane serine/threonine kinase, Cell, 68: 775-785, 1992. 27) Gougos A, Letarte M: Primary structure of endoglin, an RGD-containing glycoprotein of human endothelial cells,] Bioi Chem, 265: 8361-8364, 1990. 28) Georgi LL, Albert PS, Riddle DL: daf-I, a C. elegans gene controlling dauer larva development, encodes a novel receptor kinase, Cell, 61: 635-645,1990. 29) Mathews 15, Vale WW: Expression cloning of an activin receptor, a predicted transmembrane serine kinase, Cell, 65: 973-982, 1991. 30) Attisano L, WranaJL, Cheifetz S, et al: Novel activin receptors: Distinct genes and alternative mRNA splicing generate a reportoire of serine/threonine kinase receptors, Cell, 68: 97-108, 1992. 31) Pietenpol JA, Stein RW, Moran E, et al: TGF-,81 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains, Cell, 61: 777785,1990. 32) Laiho M, DeCaprio, Ludlow JW, et al: Growth inhibition by TGF-,8 linked suppression of retinoblastoma protein phosphorylation, Cell, 62: 175-185, 1990. 33) Mustoe TA, Pierce GF, Thomason A, et al: Accelerated healing of incisional wounds in rats induced by transforming growth factor-S, Science, 237: 1333-1336, 1987. 34) Border WA, Okuda S, Languino LR, et al: Suppression of experimental glomerulonephritis by antiserum against transforming growth factor ,81, Nature, 345: 371-373,1990. 35) Catilla A, Prieto J, Fausto N: Transforming growth factor ,81 and a in chronic liver disease: Effects on interferon alfa therapy, N Engl] Med, 324: 933-940, 1991. 36) Shah M, Foreman DM, Ferguson MW: Control of scarring in adult wounds by neutralizing antibody to transforming growth factor ,8, Lancet, 339: 213-214, 1992.
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Structure, Function and Possible Clinical Application of Transforming Growth Factor-S Kohei Miyazono and Carl-Henrik Heldin Abstract
Transforming growth factor-S (TGF-J3) is a family of multifunctional 25 kDa proteins. TGF-J3 was originally identified because of its ability to induce the growth of normal rodent fibroblasts in soft agar, but is now known as a potent growth inhibitor for many different cell types. In addition, TGF-J3 is known to regulate the differentiation of cells, induce chemotaxis ofcells, and to induce the accumulation of extracellular matrix proteins. In vivo, TGF-J3 stimulates the repair of soft as well as hard tissues. It also acts as a potent immunosuppressant TGF-J3 is produced as latent high molecular weight complexes; since it is produced by many different cell types, and most cells have receptors for TGF-J3, the activation of latent TGF-J3 is likely to be an important step in the regulation of its action. TGF-J3 exerts its effects by binding to specific cell surface receptors. The type I and type II TGF-J3 receptors are suggested to be the most important for signal transduction; a recent report has disclosed that the type II receptor has a serine/threonine kinase domain. Since TGF-J3 is a potent growth regulator with multifunctional activity, it may be useful in the treatment of certain clinical disorders. Local application ofTGF-J3 is shown to accelerate wound healing. Since an increase in TGF-J3 activity is often observed in various fibrotic disorders, antagonists for TGF-J3 might be valuable in the treatment of such diseases.
Key words: TGF-J3; growth factor; growth inhibitor; latent form; receptor; wound healing TGF-J3 Superfamily Transforming growth factor-J3 (TGF-J3) is a family of structurally related proteins with molecular weights of about 25,000. So far, five different TGF-J3 isofonns have been identified which have 66-82% amino acid sequence identity with each other (1). TGF-J31 was first purified from human platelets and cloned from a eDNA library from human placenta. TGF-J32 and -133 were subsequently identified in several mammalian cells and tissues. In contrast, TGF-J34 and -135 have been found only in the chicken and frog, respectively. TGF-J3s are synthesized as precursor forms of 390 to 412 amino acids. The N-tenninal parts are cleaved off by proteolysis, and dimeric forms of the C-tenninal 112 amino acid peptides constitute the active TGF-J3 molecules. The structural similarity is less in the N-tenninal precursor parts, compared to the portions corresponding to the mature TGF-J3s. TGF-J31, -132, and -133 share most of their biological activities, although TGF-J31 and -133 are more potent than TGF-J32 on hematopoietic progenitor Ludwig Institute for Cancer Research, Box 595 Biomedical Center, 5-75124 Uppsala, Sweden.
cells and fetal bovine heart endothelial cells (2). The promotor regions of TGF-J31, -132, and -133 are different, and the productions of these proteins are differently regulated (3). Whether the TGF-J3 isoforms play distinct roles in vivo remains to be elucidated. There are also proteins that are more distantly related to TGF-J3s (4). These include inhibins and activins, Mullerian inhibitory substance, Drosophila decapentaplegic gene complex, Vg-l, and bone morphogenetic proteins. These molecules play important roles in the growth and differentiation of different cell types, e.g. during different stages of the development Activin A is a member of the TGF-J3 superfamily and one of the most well characterized factors in this category (5). It was originally identified as -a factor that regulated the production of follicle-stimulating hormone in pituitary cells. It was then shown to induce the differentiation of the erythroleukemia cells and to have a mesoderminducing activity in Xenopus embryos. Type II receptors for both activin and TGF-J3 possess serine/ threonine kinase domains in the cytoplasmic portions (see below).
Structure and Function ofTGF-p
645
Bioactivity ofTGF-p TGF-p was originally identified as a growth promoting activity for rodent fibroblasts in soft agar. However, a growth promoting activity of TGF-p is only observed for certain cell types and during certain culture conditions. It has been suggested that TGF-p induces the growth of certain mesenchymal cells in monolayer cultures through the production of platelet-derived growth factor (PDGF), which then acts as an autocrine growth stimulator (6-8). TGF-ps are potent growth inhibitors for many different cell types in vitro, such as endothelial cells, epithelial cells, including hepatocytes and keratinocytes, hematopoietic progenitor cells, and lymphocytes. TGF-p induces the chemotaxis of various cell types such as neutrophils, monocytes and fibroblasts, as determined by the Boyden chamber method. In contrast, migration of endothelial cells is dramatically inhibited by TGF-p when it is added after endothelial cell wounding (9). Other important functions of TGF-p are its effects on the extracellular matrix (4). TGF-p induces the accumulation of extracellular matrix by the increase of matrix protein production, such as fibronectin and collagen, and by the inhibition of the activity of enzymes that degrade matrix proteins. Moreover, the production of certain receptors for matrix proteins is also stimulated by TGF-p; thus the interaction between cells and the matrix may be promoted. A 'potent immunosuppressing activity has also been observed for TGF-p. It inhibits the proliferation of thymocytes, T cells, B cells, and natural killer cells (10, 11). In most cases, the functional activities of immune cells, such as the production of IgG and IgM, are also downregulated. TGF-j3 induces angiogenesis in vivo. The mechanism for this effect is unknown; TGF-j3 is a potent growth inhibitor for endothelial cells (12), but stimulates migration of other cells from surrounding tissues, which may be important for the angiogenic process induced by TGF-j3 (13). TGF-j3 also affects the differentiation of endothelial cells and promotes tube formation (14).
of receptors are important for understanding the regulation ofTGF-j3 action in vitro and in vivo (15). TGF-ps are produced as latent forms in most cell types. Latent TGF-p in human platelets is composed of three different components, i.e. activeTGF-pl dimer, TGF-j31 latency associated peptide (PI-LAP), and the latent TGF-pl binding protein (LTBP) (16). 131LAP is the N-terminal remnant of the TGF-j31 precursor; LTBP is produced by a different gene and associates with j31-LAP by a disulphide linkage. PI-LAP is most important for the latency ofTGF-j3, since TGF-pl produced by the transfection of TGF-j31 precursor cDNA into Chinese hamster ovary cells is composed only ofTGF-j31 and j31-LAP, but is still latent (17). The latent TGF-ps can be activated by treatments with acid, alkali, or heat Activation can also be achieved by certain enzymes such as plasmin and glycosidases (18). However, the mechanisms that control TGF-j3 activation in vivo are still not fully elucidated. TGF-j3s are produced as latent complexes with or without LTBP. For instance, the latent TGF-j31 observed in human platelets is associated with LTBP, whereas TGF-j32 observed in epidermal keratinocytes in vivo is not associated with LTBP (our unpublished observations). The role ofLTBP in the latent TGF-j3 complex is not known. Two thirds of the molecule are composed of two different types of repeat sequences; 16 copies of epidermal growth factor (EGF)-like repeats and 3 copies of another motif containing eight cysteine residues (19). EGF-like repeats are observed in several different proteins and may playa role in protein-protein interactions (20). Certain EGF-like domains have been found to bind Ca". The eightcysteine repeat was first found in LTBP; then a similar structure was also observed in fibrillin, the protein that is deficient in Marfan's syndrome (21). Fibrillin is a component of the extracellular matrix, and its deficiency results in such symptoms as skeletal abnormalities, aortic aneurysm, and aortic dissection. Whether LTBP is also a matrix component or can interact with the matrix is an interesting possibility which remains to be determined.
Latent Forms ofTGF.p Many different cell types produce TGF-ps, and most cells possess receptors for TGF-j3.However, in the action of TGF-j3, there are several critical steps regulating its activity. Among those, the regulation of the production and the activation of latent forms ofTGF-j3, as well as interactions with and activation
Signal Transduction by TGF·p Receptors for growth factors are divided into several different families based on their structures (22). Most of the receptors for growth factors, such as EGF, PDGF, insulin-like growth factor-I and fibroblast growth factors, have tyrosine kinase domains in their intracellular portions. Receptors
646
Miyazono and Heldin
for cytokines, on the other hand, do not have protein kinase structures in their cytoplasmic domains. However, the {3 chain of the interleukin-2 (IL-2) receptor has been shown to interact with a soluble tyrosine kinase, Lck (23). In contrast, the receptors for the TGF-{3 superfamily appear to be structurally and functionally different from the growth factor receptors and the cytokine receptors. Three different types of TGF-{3 receptors are observed in most cell types. The type I and type II TGF-{3 receptors are glycoproteins of 53 kDa and 60-70 kDa, respectively. The type III TGF-{3 receptor is a 280 kDa proteoglycan and, therefore, has also been denoted betaglycan. Type I and type II receptors are most important for the signal transduction ofTGF-{3, whereas the type III receptor may not be directly involved. cDNA cloning of the type III (24, 25) and type II TGF-{3 receptors (26) have recently been reported. The type III receptor is synthesized as a 120 kDa core protein. The cytoplasmic domain of the type III receptor is composed of only 43 amino acids with a high content (42%) of serine and threonine residues; no protein kinase domain was observed, but a 63% sequence identity with endoglin, a possible cell adhesion protein observed in endothelial cells and hematopoietic cells, could be found (27). The intracellular portion of the type II TGF-{3 receptor has a serine/threonine kinase domain. Autophosphorylation on serine and threonine residues has also been demonstrated for the TGF-{3 type II receptor (26). Transmembrane serine/ threonine kinases have previously been found only in the dafl protein (28), which is involved in larval development of nematodes, and in the activin type II (29) and type lIB (30) receptors. These observations suggest that transmembrane serine/threonine kinases may form a new receptor family, which possibly includes the receptors for proteins in the TGF-{3 superfamily. TGF-{3 regulates the expression of nuclear transcription factors; e.g. in murine lung epithelial cells, the amount ofJun-B is increased and the amount of c-Myc is decreased after TGF-{3 stimulation. TGF-{3 inhibits cell cycle progression at the late G1 phase. The retinoblastoma (RB) gene product has been shown to be involved in the antiproliferative effect ofTGF-{3 (31, 32).
Clinical Application of TGF-{3: Perspectives TGF-{3s are multifunctional proteins which may be of clinical use in certain different disorders. Since TGF-{3 is a potent immunosuppressant, sys-
temic administration of TGF-{3 could be useful in the treatment of autoimmune diseases and allograft rejection (10, 11). TGF-{3 stimulates wound healing (33). Defect in wound healing is often observed in aged persons and in patients with diabetes or undergoing glucocorticoid treatment, radiation, or cancer chemotherapy. Local application of TGF-{3 may accelerate the healing of the wounds in these patients. Furthermore, TGF-{3 plays an important role in bone remodeling and thus could be useful in the treatment of bone fractures. Some fibrotic diseases may occur through excess and/or inappropriate wound healing, such as liver cirrhosis, scleroderma, keloid formation, pulmonary fibrosis, glomerulonephritis, and others. Increased amounts of TGF-{3 have often been observed in diseased tissues with an excess of matrix proteins (34, 35). Use of TGF-{3 antagonists such as TGF-{3 neutralizing antibodies have been shown to prevent the progress of certain of these disorders (34, 36). So far, clinically useful TGF-{3 antagonists are not available. The development of substances that prevent the activation of the latent TGF-{3s or that compete for binding to TGF-{3 receptors might provide valuable tools in the control of these disorders. References 1) Roberts AB, Sporn MB: The transforming growth factor-Sa, in Sporn MB, Roberts AB (eds): Peptide Growth Factors and Their Receptors I, Springer-Verlag, Berlin, 1990, pp 419-472. 2) Cheifetz S, Hernandez H, Laiho M, et al: Distinct transforming growth factor-S (TGF-fi) receptor subsets as determinants of cellular responsiveness to three TGF-~ isofonns,] Biol Chern, 265: 20533-20538, 1990. 3) Roberts AB, Kim S], Noma T, et al: Multiple forrns of TGF-~: Distinct promoters and differential expression, Ciba Found Symp, 157: 7-28, 1991. 4) Massague J: The transforming growth factor-S family, Annu Rev Cell Biol, 6: 597-641, 1991. 5) Vale W, Hsueh A, Rivier C, et al: The inhibin;activin family of hormones and growth factors, in Sporn MB, Roberts AB (eds): Peptide Growth Factors and Their Receptors II, Springer-Verlag, Berlin, 1990, pp 211-248. 6) Leof BE, Proper JA, Goustin AS, et al: Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor ~: A proposed model for indirect mitogenesis involving autocrine activity, Proc Natl Acad Sci USA, 83: 24532457,1986. 7) Soma Y, Grotendorst GR: TGF-~ stimulates primary human skin fibroblast DNA synthesis via an autocrine production of PDGF-related peptides,] Cell Physiol, 140: 246-253, 1989.
Structure and Function of TGF-p
iI) Battegay E], Raines EW, Seifert RA, et al: TGF-,8
9)
10)
II)
12)
13)
14)
15)
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induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop, Cell, 63: 515-524, 1990. Sato Y, Rifkin DB: Inhibition of endothelial cell movement by pericytes and smooth muscle cells: Activation of a latent transforming growth factor-,81like molecule by plasmin during co-culture,] Cell Bioi, 109: 309-315, 1989. Kehrl JH: Transforming growth factor-S: An important mediator ofimmunoregulation, Int] Cell Cloning, 9: 438-450,1991. Ruscetti FW, Palladino MA: Transforming growth factor-S and the immune system, Prog Growth Factor Res, 3: 159-175, 1991. Takehara K, LeRoy EC, Grotendorst GR: TGF-,8 inhibition of endothelial cell proliferation: Alteration of EGF binding and EGF-induced growthregulatory (competence) gene expression, Cell, 49: 415-422,1987. Yang EY, Moses HL: Transforming growth factor ,81induced changes in cell migration, proliferation, and angiogenesis in the chick chorioallantoic membrane, ] Cell Bioi, 111: 731-742, 1990. Madri JA, Pratt BM, Tucker A: Phenotypic modulation of endothelial cells by transforming growth factor-S depends upon the composition and organization of the extracellular matrix,] Cell Bioi, 106: 1375-1384, 198B. Wakefield LM, Smith DM, Masui T, et al: Distribution and modulation of the cellular receptor for transforming growth factor-beta,] Cell Bioi, 105: 965-975, 1987. Miyazono K, Hellman U, Wernstedt C, et al: Latent high molecular weight complex of transforming growth factor ,81: Purification from human platelets and structural characterization,] Bioi Chern, 263: 6407-6415, 198B. Gentry LE, Webb NR, Lim GJ, et al: Type 1 transforming growth factor beta: Amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells, Mol Cell Bioi, 7: 3418-3427,1987. Miyazono K, Heldin C-H: Latent forms of TGF-,8: Molecular structure and mechanisms of activation, CibaFound Symp, 157: 81-92, 1991. Kanzaki 1', Olofsson A, Moren A, et al: TGF-,81 binding protein: A component of the large latent complex of TGF-,81 with multiple repeat sequences, Cell, 61: 1051-1061, 1991. Apella E, Weber IT, Blasi F: Structure and function of epidermal growth factor-like regions in proteins, FEBS Lett, 231: 1-4, 1988. Maslen CL, Corson GM, Maddox BK, et al: Partial sequence of a candidate gene for Marfan syndrome, Nature, 352: 334-337, 1991.
647
22) Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity, Cell, 61: 203212,1990. 23) Hatakeyama M, Kono T, Kobayashi N, et al: Interaction of the IL-2 receptor with the src-family kinase p561Ck : Identification of novel intermolecular association, Science, 252: 1523-1528, 1991. 24) Lopez-Casillas F, Cheifetz S, Doody J, et al: Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-,8 receptor system, Cell, 67: 787-795, 1991. 25) Wang X-F, Lin HY, Ng-Eaton E, et al: Expression cloning and"'Characterization of the TGF-,8 type III receptor, Cell, 67: 797-805, 1991. 26) Lin HY, Wang X-F, Ng-Eaton E, et al: Expression cloning of the TGF-,8 type II receptor, a functional transmembrane serine/threonine kinase, Cell, 68: 775-785, 1992. 27) Gougos A, Letarte M: Primary structure of endoglin, an RGD-containing glycoprotein of human endothelial cells,] Bioi Chem, 265: 8361-8364, 1990. 28) Georgi LL, Albert PS, Riddle DL: daf-I, a C. elegans gene controlling dauer larva development, encodes a novel receptor kinase, Cell, 61: 635-645,1990. 29) Mathews 15, Vale WW: Expression cloning of an activin receptor, a predicted transmembrane serine kinase, Cell, 65: 973-982, 1991. 30) Attisano L, WranaJL, Cheifetz S, et al: Novel activin receptors: Distinct genes and alternative mRNA splicing generate a reportoire of serine/threonine kinase receptors, Cell, 68: 97-108, 1992. 31) Pietenpol JA, Stein RW, Moran E, et al: TGF-,81 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains, Cell, 61: 777785,1990. 32) Laiho M, DeCaprio, Ludlow JW, et al: Growth inhibition by TGF-,8 linked suppression of retinoblastoma protein phosphorylation, Cell, 62: 175-185, 1990. 33) Mustoe TA, Pierce GF, Thomason A, et al: Accelerated healing of incisional wounds in rats induced by transforming growth factor-S, Science, 237: 1333-1336, 1987. 34) Border WA, Okuda S, Languino LR, et al: Suppression of experimental glomerulonephritis by antiserum against transforming growth factor ,81, Nature, 345: 371-373,1990. 35) Catilla A, Prieto J, Fausto N: Transforming growth factor ,81 and a in chronic liver disease: Effects on interferon alfa therapy, N Engl] Med, 324: 933-940, 1991. 36) Shah M, Foreman DM, Ferguson MW: Control of scarring in adult wounds by neutralizing antibody to transforming growth factor ,8, Lancet, 339: 213-214, 1992.
