The Beta-Type Transforming Growth Factor Mediators of Cell Regulation in the Lung 1 • 2 RON W. PELTON and HAROLD L. MOSES
TGF13 Family Chemical Structure and Sequence Similarity Transforming growth factor 131 (TGF13I) was first recognized and named as a factor that can cause normal rodent fibroblasts to behave in vitro as transformed cells (I, 2). Since the discovery oflGF13I, the prototypic TGF13 family member, cloning experiments and sequence analysis have revealed that this molecule is but one member of a much larger lGF13 family. This family includes the closely related molecules lGF13I, lGF132,lGF133,and lGF134 and the more distantly related TGF~like molecules Miillerian-inhibiting substance, the inhibins and activins, the bone morphogenetic proteins, Xenopus tissue culture mesoderminducing factor, Xenopus Vg-I, murine Vgr-I, and the Drosophila decapentaplegic protein (for a more complete review of the entire TGF13 family see [3]). Biologically active (mature) TGF131 is composed oftwo identicalI2.5-kD monomers that form cysteine-cysteine disulfide bonds to give rise to a 25-kD homodimeric molecule. Each monomer is synthesized in a pre-pro form as a 390 aminoacid protein (figure I); the first 29 amino acids of the monomer form the signal sequence, the next 249 amino acids make up the region known as the N-terminal glycopeptide, and the last 112 amino acids constitute the C-terminal mature region, which forms the 12.5-kD monomer. The 112 amino acid mature region is released from the N-terminal glycopeptide at a dibasic cleavage site and dimerizes to yield TGF13l. TGF131 is generally found to be secreted by the cell in a latent (inactive) form, which can be activated upon exposure to extremes of pH or by chaotropic agents, but it is more likely activated in vivo by molecules such as plasmin (3). The region of highest sequence similarity among the members of the TGF13 family is found in the C-terminal mature region. Within the closely related group, TGF13 I to 4 show amino acid sequence similarity of 70 to 800/0 in this 112 amino acid region, with nine of nine cysteine residues invariably conserved (3). The N-terminal glycopeptide regions of these molecules are much less conserved. Within the distantly related group, the proteins demonstrate amino acid similarity of 30 to 40% in the C-terminal region when compared with TGF131 and have only seven of 9 cysteines invariably conserved. Amazingly, sequence comparison of TGF131 from various species reveals that within the 112 amino acid mature region, human, simian, bovine, and porcine TGF131 demonstrate 1000/0 amino acid
SUMMARY An Increased Interest In the role of growth factors In the regulation of processes eencernlng normal and pathologic lung physiology has spurred a flurry of research In this area. Peptide growth factors are known to control not only cell prollfaratlon but other events such as differentiation, chemotaxis, and matrix deposition 88 well. The transforming growth factor j3 (TGFj3l family of regulatory peptldes serves as a prime exemple to Illustrate the multiplicity of effects elicited by peptide growth factors In varIous lung-derived cell types. At present, the TGFj3 family conslste of at least 17 proteins and, based on sequence analysis, they can be divided Into two groups: a cluster that shows very high sequence similarity to TGFj31, the closely related group, and a cluster that ahows weaker sequence similarity to TGFj31, the distantly related group. The purpose of this brief revl_ls to summarize the ullent faatures of TGFI3 structure and regulatory abilities of the closely related group. In addition, we will outline the evidence suggesting a role for TGFj3 In normal lung devalopment and physiology. Emphasis will be placed on studies with the closely related members TGFj31 and TGFj32 because, until recently, purified proAM REV RE8PIR DI8 1990; 142:831-835 tein was evallable only for these two proteins.
sequence conservation, and the murine lGF131 differs by only one amino acid (3). This remarkable level of sequence conservation would suggest that lGF131 has critical functions.
TGF13 Receptors Cross-linking studies with lGF131and lGF132 indicate that both of these molecules interact with the same cell surface binding molecules (presumed to be TGF13 receptors). These receptors, three glycosylated proteins numbering from 2 x l()3 to 4 x IQ4 per cell, are found on a wide rangeof normal and transformed cell types of mesenchymal as well as epithelial origin (4,5). They have been classified as type I (60 to 70 kD), type II (85 to 95 kD), and type III (280 to 330 kD) receptors and display dissociation constants on the orderof25 to 140pM. At present it is unclear what the exact role each of these molecules plays in TGF13 signal transduction; however, studies indicate that the type I receptor is most likely the mediator of the TGF13 signal (6). The type III receptor is thought to be a cell surface TGF~binding proteoglycan that, after binding, can be released from the cell (7). Equally unclear is the second messenger system employed by the receptor to transduce the lGF13 signal to the inside of the cell. It appears that the TGF13 receptors possess no intrinsic kinase activities or Na"/H+ activity (8, 9). Likewise, studies have found no fluctuations in inositol phosphate or intracellular Ca" levels in cells treated with TGF13. Nonetheless, recent studies with GTP.S indicate that mouse fibroblasts treated with TGF131 demonstrate an increase in GTPase activity and GTP.S binding, suggesting the involvement of G proteins in lGF13 signal transduction (10). It should be noted that, at
present, only TGF13I, TGF132, and lGF133 have been shown to bind to the three TGF13 receptors. Lastly, TGF13 receptors have been found to be expressed by differentiated embryonal carcinoma cells, which are thought to mimic primitive embryonic cell types. Hence, this may indicate that one or more ofthe TGF13 may be able to regulate developmental processes in these cells (11).
TGF13 Regulation of Cell Proliferation The most widely studied effect of lGF13 is its ability to govern the proliferation of cells both in culture and in vivo. Although the cellular response to treatment with TGF13 differs according to cell type and culture condition, in general, TGF13 is mitogenic only for cells of mesenchymal origin such as fibroblasts (12) and osteoblasts (13). In contrast to this, lGF13 is a powerful growth inhibitor of cells of epithelial origin and is thought to be the most potent polypeptide growth inhibitor reported (12, 14). Furthermore, the differences in effect of TGF13I, TGF132, and TGF133 on the proliferation of cells in tissue culture appear to be only quantitative and not qualitative (15). Many of the studies of the mitogenic effects of TGF13 have employed embryonic cell types. Examination of TGF~treated embryonic fibroblasts indicates that the effect produced varies significantly depending on the origin and culture conditions of the cells. For example, primary rat fibroblasts grown 1 From the Department of Cell Biology,Vanderbilt University School of Medicine, Nashville, Tennessee. 2 Correspondence and requests for reprints should be addressed to Ron W. Pelton, Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
S31
832
PELTON AND MOSES
II( '"I
II( '"'
'I,J N I
cr II
N·lenninal glycopeptide
29=
219 u
r. ~~ o: o: c.~ tk:W#&7@0wk A'
TGF13 Family Chemical Structure and Sequence Similarity Transforming growth factor 131 (TGF13I) was first recognized and named as a factor that can cause normal rodent fibroblasts to behave in vitro as transformed cells (I, 2). Since the discovery oflGF13I, the prototypic TGF13 family member, cloning experiments and sequence analysis have revealed that this molecule is but one member of a much larger lGF13 family. This family includes the closely related molecules lGF13I, lGF132,lGF133,and lGF134 and the more distantly related TGF~like molecules Miillerian-inhibiting substance, the inhibins and activins, the bone morphogenetic proteins, Xenopus tissue culture mesoderminducing factor, Xenopus Vg-I, murine Vgr-I, and the Drosophila decapentaplegic protein (for a more complete review of the entire TGF13 family see [3]). Biologically active (mature) TGF131 is composed oftwo identicalI2.5-kD monomers that form cysteine-cysteine disulfide bonds to give rise to a 25-kD homodimeric molecule. Each monomer is synthesized in a pre-pro form as a 390 aminoacid protein (figure I); the first 29 amino acids of the monomer form the signal sequence, the next 249 amino acids make up the region known as the N-terminal glycopeptide, and the last 112 amino acids constitute the C-terminal mature region, which forms the 12.5-kD monomer. The 112 amino acid mature region is released from the N-terminal glycopeptide at a dibasic cleavage site and dimerizes to yield TGF13l. TGF131 is generally found to be secreted by the cell in a latent (inactive) form, which can be activated upon exposure to extremes of pH or by chaotropic agents, but it is more likely activated in vivo by molecules such as plasmin (3). The region of highest sequence similarity among the members of the TGF13 family is found in the C-terminal mature region. Within the closely related group, TGF13 I to 4 show amino acid sequence similarity of 70 to 800/0 in this 112 amino acid region, with nine of nine cysteine residues invariably conserved (3). The N-terminal glycopeptide regions of these molecules are much less conserved. Within the distantly related group, the proteins demonstrate amino acid similarity of 30 to 40% in the C-terminal region when compared with TGF131 and have only seven of 9 cysteines invariably conserved. Amazingly, sequence comparison of TGF131 from various species reveals that within the 112 amino acid mature region, human, simian, bovine, and porcine TGF131 demonstrate 1000/0 amino acid
SUMMARY An Increased Interest In the role of growth factors In the regulation of processes eencernlng normal and pathologic lung physiology has spurred a flurry of research In this area. Peptide growth factors are known to control not only cell prollfaratlon but other events such as differentiation, chemotaxis, and matrix deposition 88 well. The transforming growth factor j3 (TGFj3l family of regulatory peptldes serves as a prime exemple to Illustrate the multiplicity of effects elicited by peptide growth factors In varIous lung-derived cell types. At present, the TGFj3 family conslste of at least 17 proteins and, based on sequence analysis, they can be divided Into two groups: a cluster that shows very high sequence similarity to TGFj31, the closely related group, and a cluster that ahows weaker sequence similarity to TGFj31, the distantly related group. The purpose of this brief revl_ls to summarize the ullent faatures of TGFI3 structure and regulatory abilities of the closely related group. In addition, we will outline the evidence suggesting a role for TGFj3 In normal lung devalopment and physiology. Emphasis will be placed on studies with the closely related members TGFj31 and TGFj32 because, until recently, purified proAM REV RE8PIR DI8 1990; 142:831-835 tein was evallable only for these two proteins.
sequence conservation, and the murine lGF131 differs by only one amino acid (3). This remarkable level of sequence conservation would suggest that lGF131 has critical functions.
TGF13 Receptors Cross-linking studies with lGF131and lGF132 indicate that both of these molecules interact with the same cell surface binding molecules (presumed to be TGF13 receptors). These receptors, three glycosylated proteins numbering from 2 x l()3 to 4 x IQ4 per cell, are found on a wide rangeof normal and transformed cell types of mesenchymal as well as epithelial origin (4,5). They have been classified as type I (60 to 70 kD), type II (85 to 95 kD), and type III (280 to 330 kD) receptors and display dissociation constants on the orderof25 to 140pM. At present it is unclear what the exact role each of these molecules plays in TGF13 signal transduction; however, studies indicate that the type I receptor is most likely the mediator of the TGF13 signal (6). The type III receptor is thought to be a cell surface TGF~binding proteoglycan that, after binding, can be released from the cell (7). Equally unclear is the second messenger system employed by the receptor to transduce the lGF13 signal to the inside of the cell. It appears that the TGF13 receptors possess no intrinsic kinase activities or Na"/H+ activity (8, 9). Likewise, studies have found no fluctuations in inositol phosphate or intracellular Ca" levels in cells treated with TGF13. Nonetheless, recent studies with GTP.S indicate that mouse fibroblasts treated with TGF131 demonstrate an increase in GTPase activity and GTP.S binding, suggesting the involvement of G proteins in lGF13 signal transduction (10). It should be noted that, at
present, only TGF13I, TGF132, and lGF133 have been shown to bind to the three TGF13 receptors. Lastly, TGF13 receptors have been found to be expressed by differentiated embryonal carcinoma cells, which are thought to mimic primitive embryonic cell types. Hence, this may indicate that one or more ofthe TGF13 may be able to regulate developmental processes in these cells (11).
TGF13 Regulation of Cell Proliferation The most widely studied effect of lGF13 is its ability to govern the proliferation of cells both in culture and in vivo. Although the cellular response to treatment with TGF13 differs according to cell type and culture condition, in general, TGF13 is mitogenic only for cells of mesenchymal origin such as fibroblasts (12) and osteoblasts (13). In contrast to this, lGF13 is a powerful growth inhibitor of cells of epithelial origin and is thought to be the most potent polypeptide growth inhibitor reported (12, 14). Furthermore, the differences in effect of TGF13I, TGF132, and TGF133 on the proliferation of cells in tissue culture appear to be only quantitative and not qualitative (15). Many of the studies of the mitogenic effects of TGF13 have employed embryonic cell types. Examination of TGF~treated embryonic fibroblasts indicates that the effect produced varies significantly depending on the origin and culture conditions of the cells. For example, primary rat fibroblasts grown 1 From the Department of Cell Biology,Vanderbilt University School of Medicine, Nashville, Tennessee. 2 Correspondence and requests for reprints should be addressed to Ron W. Pelton, Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
S31
832
PELTON AND MOSES
II( '"I
II( '"'
'I,J N I
cr II
N·lenninal glycopeptide
29=
219 u
r. ~~ o: o: c.~ tk:W#&7@0wk A'
