In his original experiments, Temin (1966) proposed that the lowered requirement for serum was related to growth factors within this medium. At that time, only one or two such growth factors had been isolated and characterized, although there had been many experiments in a variety of systems with crude serum or tissue extracts, demonstrating both the enhancement and inhibition of the replication of cells in culture (cf. Keski-Oja et al., 1988; Siegfried, 1992). Furthermore, as pointed out by Siegfried (1992), there is some difficulty in the precise definition of a growth factor, because some molecules may have quite different effects on different tissues within the organism. An example of such overlapping functions may be seen in Figure 16.6, wherein a polypeptide may act as a growth factor in one tissue, as a hormone-controlling secretion in an- other tissue, and as a neurotransmitter in the brain (cf. Siegfried, 1992). Thus, one may consider an operational definition for a growth factor as a peptide that produces a biological signal via a specific high-affinity receptor that results in altered growth or differentiation (Siegfried, 1992). A listing of such growth factors exhibiting either growth enhancement, growth inhibition, or both, is seen in Table 16.9.
The rather extensive listing in Table 16.9 is by no means complete. For example, it omits the trophic hormones, growth hormone, and insulin, and some of the families are incomplete, such as the transforming growth factor-β family (Lawrence, 1996). Most of the factors listed in Table 16.9 are of a molecular weight below 20 kDa and with no known modifications of the polypeptide, such as carbohydrate or lipid. The exceptions are the platelet-derived growth factor family, whose members in their dimeric forms are in excess of 30 kDa, and transforming growth factor-β whose dimers are 25 kDa. Erythropoietin, a circulating hormone produced by kidney and liver and acting as a growth factor for the erythroid progenitor population, is a complex, glycosylated protein of molecular weight 46 kDa. Transforming growth factor-β, the tumor ne- crosis factor, and the interferon families are generally considered to be inhibitors of cell growth (Keski-Oja et al., 1988). Some 50 years ago, investigators proposed that cell replication in vivo may be controlled by a negative feedback mechanism through endogenous inhibitors of replica- tion. Such inhibitors were termed chalones and thought to be cell type–specific (cf. Patt and Houck, 1980). Other cell growth inhibitors have been partially characterized (Miyazaki and Horio, 1989), but the three families noted are perhaps the best characterized of the “chalones” at present.
Growth Factor Receptors
In order for growth factors to exert their trophic effects, it is necessary for such polypeptides to interact with specific receptors on the surface of the target cell. We have already noted this in previous chapters (3, 7, and 15). However, in the case of growth factors, their receptors are pre- dominantly of the type involving a single polypeptide chain or its dimeric or trimeric forms, having both external and cytoplasmic domains with a single transmembrane domain. Examples of a number of diagrammatic structures for the receptors for growth factors may be seen in Fig- ure 16.7. Virtually all of these receptors are glycoproteins having a high affinity for their specific growth factor, with their molecular weights being considerably in excess of those of the ligand growth factors themselves, usually in the range of 50 kDa to 150 kDa. As noted in the figure, many but not all growth factor receptors exhibit tyrosine protein kinase activity in the cytoplas- mic domain, such activity being activated by interaction with the ligand, as was previously dis- cussed in Chapter 7. In virtually all instances, interaction of the receptor with its ligand involves a dimerization or in some instances a trimerization of the receptor molecules in the cell mem- brane (cf. Weiss and Schlessinger, 1998).
Signaling mechanisms that are initiated and result in transduction to the nucleus have al- ready been discussed in relation to receptors containing an intrinsic tyrosine kinase (Chapter 7; Fantl et al., 1993). However, as noted from Figure 16.7, receptors involved in hematopoietic and lymphoid cells do not contain tyrosine kinase in their cytoplasmic domain. In this instance, the signal transduction pathway must involve tyrosine kinases that are separate molecules within the cytoplasm of the cell. A considerable amount of work has elucidated many of the details of this pathway (Silvennoinen et al., 1997; McCubrey et al., 2000). A diagram of the pathway that in- volves a family of tyrosine kinases known as JAK kinases (Janus kinases) and STAT transcrip- tion factors (signal transducers and activators of transcription) is seen in Figure 16.8. As noted in the figure, the JAKs phosphorylate the cytokine receptor and at the same time phosphorylate members of signal transduction pathways, not only the STAT pathway but, as noted in the figure, other pathways involved in signal transduction of receptors having inherent tyrosine kinase within their cytoplasmic domains. This “cross-talk” between the JAK-STAT pathways and other signaling pathways allows such cells to activate a variety of transcription factors and subse- quently genetic expression. Such cross-talk also exists between peptide growth factor transduc- tion pathways and steroid hormone receptor signaling pathways (cf. Ignar-Trowbridge et al.,1995). As discussed in the next chapter, tumor necrosis factors utilize a different signaling path- way, which in many instances directs the cell into apoptosis (Chapter 17).
Transforming Growth Factors
Following the characterization of epidermal growth factor (cf. Carpenter and Cohen, 1990) and nerve cell growth factor (Levi-Montalcini, 1987), the search for other growth factors and their characterization became a popular area of research in the 1970’s. During this time, Todaro and associates reported the striking observation that media from murine sarcoma virus–transformed cells possessed growth factor properties when added to normal or some other neoplastic cells (De Larco and Todaro, 1978). Most striking was the fact that these “growth factors” actually induced the morphological transformation of normal cells and anchorage-independent growth, one property in cell culture that correlates quite well with tumorigenicity in vivo (Table 14.1). This striking effect may be noted microscopically in Figure 16.9. Untreated normal rat cells shown in A exhibit a single monolayer of growth and a quite regular pattern. Treatment with media from murine sarcoma–transformed rat cells induced the normal cells to growth in the pat- tern seen in B, in which cells become quite crowded and pile up on one another as characterized
Figure 16.8 Diagram of interaction between the JAK-STAT and Ras pathways in signaling in hemato- poietic and lymphoid cells. The arrows indicate action of components toward others. (Adapted from Lea- man et al., 1996, with permission of the authors and publisher.)
by the transformed phenotype. Even more striking is the absence of anchorage-dependent growth of the normal rat cells in C but the growth in single colonies of more than 500 cells each in the soft agar, as noted in D (Todaro et al., 1981). Subsequent to these studies, Todaro and associates (Marquardt et al., 1984) isolated and determined the amino acid sequence of the “sar- coma growth factor,” which was subsequently termed transforming growth factor-α (TGF-α). The structural similarity of TGF-α to epidermal growth factor is seen in Figure 16.10. Although not shown in the figure, both growth factors are originally synthesized as a large transmembrane precursor, and the active growth factor as seen in Figure 16.10 is cleaved from the cell surface and released into the surrounding environment (cf. Kumar et al., 1995). Interestingly, the “nor- mal” epidermal growth factor (EGF) may also induce transformed properties in cells in vitro by addition to the medium (Liboi et al., 1986). Other studies have indicated that many cells in cul- ture may respond to a variety of growth factors by exhibiting properties of the transformed phe- notype (Kaplan and Ozanne, 1983; van Zoelen et al., 1988). Furthermore, as might be expected, both TGF-α and EGF interact with the EGF receptor, and both are produced by a variety of normal tissues (Rall et al., 1985; Salomon et al., 1990).
A second transforming growth factor, TGF-β, was independently discovered by two labo- ratories (Moses et al., 1981; Roberts et al., 1981). TGF-β was later shown to be a component of the crude “sarcoma growth factor” present in the media of retrovirus-transformed cells (cf. Lawrence, 1996). Subsequent studies have demonstrated that this growth factor is one of a very
Figure 16.9 A. Untreated normal rat kidney cells. B. Normal rat kidney cells treated with crude “sar- coma growth factor” for 6 days. C. Untreated normal rat kidney cells plated in 0.3% soft agar. D. Colony of normal rat kidney cells plated in 0.3% soft agar, treated with “sarcoma growth factor,” and photographed two weeks after treatment. See text for details. (Adapted from Todaro et al., 1981, with permission of the authors and publisher.)
large family of growth factors occurring normally in a variety of species with quite divergent functionalities. TGF-β itself occurs in three different forms, TGF-β1, -β2, and -β3, which are produced from a large precursor form nearly three times the size of the active growth factor monomer (Figure 16.11). As seen from the figure, there is considerable sequence conservation in the active TGF-β component, but the pro and pre components exhibit much less conservation. The extended polypeptide containing the pro- and active TGF-β are combined within the cell as dimers, which are proteolytically cleaved, and a binding protein [latent TGF-β-binding protein (LTPB)] is linked by a disulfide bond to the pro region. Secretion of these latent complexes of TGF-β are secreted into the cellular environment, as noted in Figure 16.12. The active dimeric form of TGF-β is freed from the complex by one or more of the components seen in the figure, and this dimeric active form may then interact with receptors with subsequent cell signaling
Figure 16.10 Structures of epidermal growth factor (A) and transforming growth factor α (B) in the mouse and rat, respectively. The extreme similarity of the positions of the disulfide (CYS-CYS) bridges and the lengths and sequence similarities of the intervening chains are readily noted. Invariant amino acid residues present in all known epidermal growth factor-like structures are depicted with shaded circles in the transforming growth factor structure.
Figure 16.11 Regions of homology and overall structure of mammalian TGF-β isoforms. The three mammalian TGF-βs are highly homologous to one another in the C-terminal region of 112 amino acids (a.a.) but show much more sequence divergence in the precursor (pre) and pro regions. The black bars indicate regions of sequence identity between TGF-β1, -β2, and -β3. The percentage identity in each do- main is given below the numbers of a.a. Other members of the TGF-β family show virtually no homology in the pro region but still are quite homologous in the C-terminal 112 a.a. (Adapted from Wakefield et al.,1991, with permission of the authors and publisher.)
(Roberts and Sporn, 1996). In contrast to the predominantly growth-stimulatory activity of TGF- α and other growth factors listed in Table 16.9, TGF-β and members of its family have a number of other functions. In fact, TGF-β acts as a growth-stimulatory agent for only a few cell types of mesenchymal origin (cf. Wakefield et al., 1991). TGF-βs are strongly growth-inhibitory for most epithelial cells, as well as cells of the hematopoietic system. TGF-β may also serve to enhance or inhibit the expression of the differentiated phenotype of a number of cells.