Transforming growth factor-β: Multiple effects on cell differentiation and extracellular matrices

DEVELOPMENTAL

BIOLOGY

130,411-422 (1988)

REVIEW Transforming

Growth Factor-P: Multiple Effects on Cell Differentiation and Extracellular Matrices ANGIE RIZZINO

Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, 42nd Street and Dewey Avenue, Omaha, Nebraska 68105 Accepted August 3, 1988 TGF-/3: A FAMILY OF RELATED GROWTH FACTORS

INTRODUCTION

Until recently, growth factors were thought to mainly influence cell proliferation. During the past several years, it has become evident that growth factors also exert a wide range of effects on cell differentiation, at least in vitro. Consequently, two questions have received careful scrutiny. How do growth factors exert their effects on cell differentiation? Do growth factors regulate cell differentiation in viva as they appear to in vitro? Impressive progress has been made in answering both questions. Similar advances have occurred in our understanding of the production of extracellular matrices. For many years, the extracellular matrix surrounding cells was believed to play primarily, if not exclusively, a structural role. However, it is now widely recognized that the extracellular matrix plays an active role in regulating embryological development, tissue repair, and abnormal cell proliferation. As a consequence, there has been a major effort to determine how the extracellular matrix performs its regulatory roles and, more recently, to understand how the production, distribution, and turnover of the extracellular matrix is regulated. Progress has been made on both fronts and there is currently good reason to suspect that growth factors exert many of their effects on differentiation by regulating the metabolism of extracellular matrices. This review will describe some of the recent advances in our understanding of the effects of growth factors on cell differentiation and extracellular matrices. Due to the abundance of recent work on growth factors and differentiation, it is not possible to cover all of the pertinent literature. This is also true for the literature pertaining to the extracellular matrix. Consequently, this review will focus on the effects of transforming growth factor type-p (TGF-P) and how it interacts with other growth factors to affect differentiation. 411

The discovery of transforming growth factors more than 10 years ago generated a great deal of interest because these factors induce anchorage-independent growth of nontransformed cells (DeLarco and Todaro, 1978). Since tumor cells, unlike normal cells, can usually be grown in suspension, it was proposed that transforming growth factors play a major role in the growth of tumors (DeLarco and Todaro, 19’78; Sporn and Todaro, 1980). As work with transforming growth factors progressed, it became apparent that most, if not all, cells produce TGF-8 (Roberts et aZ., 1981) and that TGF-P inhibits the growth of a wide range of cells, in particular the growth of epithelial cells and cells of the immune system (reviewed in Sporn et al, 1986). Although relatively few studies have examined the effects of TGF-@ under serum-free culture conditions where growth factor interactions can be readily determined (Rizzino, 1984, 1987a; Rizzino et al, 1986; Rizzino and Ruff, 1986; van Zoelen et a& 1985), it appears that the most common effect of TGF-@ on cell growth is to potentiate or suppress the growth stimulatory effects of other growth factors (reviewed in Sporn et a& 1986). TGF-/3 refers to a complex and extensively characterized family of growth factors. A detailed review of the structure of TGF-P has been published recently (Massag&, 1987) and it is reviewed only briefly here. Growth factors belonging to this family are biologically active as disulfide-linked dimers (25 kDa). A growth inhibitor produced by an African green monkey kidney cell line was the first member of this growth factor family to be identified (Holley et al, 1978). Several years later, efforts to characterize a novel class of growth factors that induce anchorage-independent growth (DeLarco and Todaro, 1978) led to the identification and purification 001%1606/88 $3.00 Copyright All rights

Q 1988 by Academic Press. Inc. of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

of a different form of TGF-/3, referred to as TGF-@l. TGF-Pl has been isolated from many different sources, including human platelets (Assoian et aZ.,1983). Efforts to purify TGF-fil from porcine platelets led to the purification and characterization of a second form of TGF-/3, referred to as TGF-/32 (Cheifetz et al., 1987). TGF+2 is now known to be identical with the growth inhibitor produced by the African green monkey kidney cell line (Hanks et al, 1988) and is produced by several other cell lines (Ikeda et ah, 1987; de Martin et ah, 1987). TGF-@l and TGF-@2 were also isolated independently from demineralized bone and were originally referred to as cartilage-inducing factors (CIF) A and B, respectively (Seyedin et aZ.,1985, 1987). Sequence analysis indicates that TGF-Pl and TGF-/32 exhibit approximately 70% amino acid sequence similarity (Cheifetz et ah, 1987). Recent studies indicate that other forms of TGF-/3 also exist. Cloning of a third member of the TGF-P family was reported recently. This factor, designated TGF-/33, exhibits 70-75% amino acid sequence similarity with TGF-bl and TGF-P2 (ten Dijke et ah, 1988; Jakowlew et al, 1988; Rik Derynck, personal communication). Even more recently, a fourth TGF-fl has been cloned from a chicken cDNA library (M. Sporn, personal communication). The different forms of TGF-P also share significant amino acid sequence similarity with several other regulatory proteins. Miillerian inhibiting substance (MIS), which is found almost exclusively in mammalian testes, is a homodimer composed of 70- to 72-kDa glycosylated subunits. The C-terminal domain of MIS exhibits approximately 32% amino acid sequence similarity with TGF-@l. MIS causes regression of the Miillerian ducts during the development of the male reproductive tract, but TGF-/3 does not appear to mimic this effect. TGF-@ also exhibits approximately 28-38% amino acid sequence similarity to the inhibins and activins. Inhibins suppress the production of follicle-stimulating hormone, whereas activins activate production of folliclestimulating hormone. Lastly, the TGF-P growth factor family appears to have been highly conserved during evolution. The decapentaplegic gene complex (DPP-C), which is critical for Drosophilia development (Spencer et aL, 1982), and a gene product referred to as Vgl, which is localized to the vegetal hemisphere in frog eggs and is implicated in the induction of mesoderm (discussed below), exhibit 48% amino acid sequence similarity with one another and 35-40% amino acid sequence similarity with TGF-/31 (Padgett et aL, 1987; Weeks and Melton, 1987). DIFFERENTIAL

EFFECTS

OF TGF-01

AND

TGF-P;!

In most in vitro systems, TGF-/31 and TGF-02 exert similar effects and appear to be functionally equivalent, but several findings indicate that they are not func-

VOLUME 130,1988

tionally identical in all systems. Recently it was determined that TGF-Pl and TGF-/32 do not affect colony formation of hematopoietic progenitor cells to the same extent. In this system, colony formation is dependent on growth factors such as interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). TGF-Pl is approximately 100 times more effective than TGF-@2 in the inhibition of colony formation by hematopoietic progenitor cells (Ohta et aL, 1987). Similarly, TGF-@l, but not TGF-/32, can inhibit colony formation by marrow progenitor cells treated with colony-stimulating factor (M-CSF). More recently, it has been determined that TGF-81 (Miiller et aZ.,1987), but not TGF-02 (Andrew Baird, personal communication), can block the effects of fibroblast growth factor (FGF) on endothelial cell proliferation. Furthermore, TGF-/?l has been shown to increase TGF-Pl mRNA levels in a number of different cell lines (van Obberghen-Schilling et aZ., 1988). However, in at least one cell line, AKR-2B cells, TGF-Pl has been found to increase TGF-Pl mRNA, not TGF-/32 or TGF-/33 mRNA levels; whereas TGF-P2 increases the levels of mRNA for TGF-bl, TGF-P2, and TGF-P3 (C. Bascom and H. Moses, personal communication). Lastly, TGF-/32 has been reported to be more effective than TGF-Pl as an inducer of mesoderm formation (discussed below). Together these findings suggest that TGF-@l and TGF-/32 may not be interchangeable in viva and thus their functions in viva are likely to differ both spatially and temporally. EFFECTS

OF GROWTH

FACTORS

ON DIFFERENTIATION

For over 25 years, growth factors have been known to affect differentiation (Cohen and Elliot, 1963). During the past several years, it has become abundantly clear that many growth factors exert a wide range of effects on cell differentiation. For example, growth factors belonging to the FGF family can induce angiogenesis (Esch et aL, 1985; Lobb et al, 1985; Thomas et al, 1985), stimulate neurite extension (Togari et aL, 1983), stimulate release of several proteases (Saksela et ah, 1987; Edwards et al, 1987), block myogenesis (Linkhart et al., 1982), increase the response of primary anterior pituitary cells to thyrotropin-releasing factor and increase their release of prolactin and thyrotropin (Baird et al., 1985). Moreover, growth factors have been implicated recently in the regulation of differentiation during early development, specifically, during mesoderm formation in amphibian embryos. Mesoderm formation during amphibian development is believed to result from inductive interactions between tissues derived from the vegetal and animal poles. Explants of the animal hemisphere from Xenopus embryos are known to form mesodermal tissues when placed in contact with tissues from the vegetal pole or

ANGIE RIZZINO

Growth Factors and LAfferentiation

with extracts from a variety of sources (see Godsave et al., 1988). Although formal proof is lacking, several lines of evidence suggest that FGF- and TGF-P-related growth factors are required for mesoderm induction during Xenopus development. The first indication of this was the finding that various forms of FGF (acidic FGF, basic FGF, and a FGF-related growth factor isolated from mouse embryonal carcinoma cells) could induce the appearance of mesoderm-specific markers (Slack et al., 1987). The physiological importance of this finding was highlighted by two other findings. Heparin, which is known to bind all members of the FGF growth factor family, can inhibit mesoderm formation of Xenopus embryos (Slack et al., 1987). Second, Xenops embryos have been found to contain mRNA that codes for a growth factor with significant amino acid sequence similarity to basic FGF (as high as 89% in some regions) (Kimelman and Kirschner, 1987). In this regard, it is interesting that mammalian embryos (mouse blastocysts cultured in serum-free medium for 3 days) also produce a growth factor that is immunologically related to basic FGF (Rizzino and Kelly, unpublished results). (Detailed information regarding the structure and function of the FGF growth factor family, which is made up of at least five different members, can be found in a recent review-Thomas, 1987; also see Zhan et al., 1988.) Similar studies performed with explants of Xenopus embryos have also implicated TGF-& or a closely related growth factor, during mesoderm formation of amphibians. Two different reports demonstrate that TGF-0, either on its own (Rosa et ah, 1988) or in combination with FGF (Kimelman and Kirschner, 1987), can induce mesoderm-specific markers in explants of Xenopus embryos. Although there are notable differences between the two reports, both groups now agree that TGF-P2 is more effective than TGF-Pl (Rosa et aZ.,1988; Marc Kirschner, personal communication). This conclusion is supported by the finding that the mesoderminducing activity, which is present in conditioned medium prepared from a Xenopus cell line, is substantially reduced by antibodies prepared against TGF-/32, but not by antibodies prepared against TGF-fil (Rosa et al., 1988). In further support of a possible role(s) of TGF-/3 during mesoderm formation is the finding that Xenopus eggs contain mRNA (Vgl) that could code for a member of the TGF-fi growth factor family (Weeks and Melton, 1987). At present, it is unclear how TGF-P and FGF influence mesoderm formation, but part of the answer may lie in the effects of these growth factors on the extracellular matrix (discussed below). For additional details concerning mesoderm inducing factor and assays for monitoring mesoderm induction, the reader is directed to articles by Godsave et al. (1988), Smith (1987), and Cooke et aL (1987).

413

Other studies have established that TGF-/3 is widely distributed during mammalian development in utero (Heine et al., 1987). Using antibodies that recognize TGF-Pl but not TGF-fi2, it has been established that the expression of TGF-/3 in mouse embryos (11-18 days of gestation) is both spatially and temporally regulated. High concentrations of TGF-@ were observed in tissues undergoing morphogenesis and the observed distribution of TGF-fl is consistent with TGF-0 playing important regulatory roles during differentiation. However, these studies do not establish a cause-and-effect relationship between the presence of TGF-P and morphogenesis. Furthermore, this study did not distinguish between active and latent forms of TGF-P (the conditions of fixation used during the preparation of the embryos for immunostaining are likely to have activated latent TGF-0). Thus, it remains to be determined whether the distribution of active and latent TGF-/3 are similar. (Active and latent forms of TGF-P are discussed later.) The above study was not designed to demonstrate that TGF-P regulates differentiation during mammalian development. However, other studies have shown that TGF-P exerts profound effects in viwo. TGF-P has been shown to induce fibrosis and angiogenesis when injected subcutaneously into newborn mice (Roberts et al., 1986). Furthermore, TGF-/3 has been shown to reversibly inhibit growth and morphogenesis of mammary glands in viva (Silberstein and Daniel, 1987). In addition, numerous studies have established that TGF-P can affect the differentiation of many different cell types in vitro (Table 1). The majority of the studies performed thus far have examined only TGF-Pl. However, many of the studies reported during the past year have also examined the effects of TGF-02 and relatively few differences (see earlier section) have been noted between the effects of TGF-Pl and TGF-P2. Overall, this body of in vivo and in vitro work clearly demonstrates that TGF-6 can inhibit or promote differentiation. It is also evident that the effects of TGF-/3 are highly cell type dependent (Table 1). In contrast to the fairly predictable effects of TGF-P on the growth of epithelial cells (reviewed by Sporn et al., 1986), TGF-6 can either inhibit or stimulate the differentiation of epithelial cells. This is also true for mesenchymal cells. Chondrogenesis can be stimulated by TGF-0, whereas myogenesis is inhibited by TGF-/3. Similarly, TGF-@ stimulates steroid production by some cells, but inhibits steroid production by others. Thus far, the most consistent effect of TGF-P is its inhibitory effects on the cells of the immune system and this appears to apply to both their growth and their differentiation (Kehrl et al,, 1986a,b; Rook et al., 1986; Wahl et al, 1987). Although TGF-P has been shown to affect the differentiation of over 20 different cell types in vitro, its effects on cells in culture can be misleading and must be

414

DEVELOPMENTAL BIOLOGY TABLE 1 EFFECZS OF TGF-8 ON CELL DIFFERENTIATION’

Cell/process Bronchial epithelial Pulmonary type II epithelial cells Tracheal epithelial cells Keratinocytes Developing mammary gland, in tivo Intestinal epithelial cells Adipogenesis

Effect on differentiation

Inhibits surfactant apoprotein synthesis Inhibits mucin production Stimulation Inhibits end bud formation and ductal growth Stimulation Inhibition

Myogenesis

Skeletal muscle satellite cells Chondrogenesis

Inhibition Stimulation; inhibition

Bone remolding and formation

Multiple effects

Angiogenesis and fibrosis Monocytes

Stimulation in viva

Natural killer cells B cells Pituitary cells Granulosa cells

Leydig cells Adrenocortical cells oocytes

Stimulates chemotaxis and growth factor production Inhibits cytolytic action Inhibits Ig production Increases release of FSH Increases the response to FSH, increases number of EGF receptors; increases aromatase activity Inhibits steroidogenesis Inhibits steroidogenesis Stimulates meiotic maturation

Reference Masui et al, 1986 Whitaett et al, 1987 Reen Wu, personal communication Reiss and Sartorelli, 1987 Silberstein and Daniel, 1987 Kurokowa et al, 1987 Ignots and Massaguk, 1985 Sparks and Scott, 1986 Massague et d, 1986 Olson et d, 1986 Florini et a& 1986 Allen and Boxhorn, 1987 Seyedin et al, 1985 Rosen et al, 1988 Centrella et al, 1986 Noda and Rodan, 1986,1987 Rosen et al, 1988 Sandberg et al, 1988 Roberts et al, 1986 Wahl et al, 1987 Leibovich et al, 1987 Rook et al, 1986 Kehrl et al, 1986a Ying et al, 1986 Ying et a& 1986 Knecht et &, 1986 Feng et al, 1986 Adashi and Resnick, 1986 Avallet et al, 1987 Lin et a& 1987 Hotta and Baird, 1986 Feige et LX!,1986,1987 Feng et al, 1988

a All examples have not been cited.

interpreted cautiously. A good case in point is the effects of TGF-@ on endothelial cells. TGF-B has been shown to inhibit the growth of endothelial cells in vitro. On its own, this observation could lead one to speculate that TGF-/3 inhibits angiogenesis in vivo. However, TGF-fl has been shown to induce angiogenesis when it is injected subcutaneously into new born mice (Roberts et al., 1986). These contrasting in vitro and in vivo observations can be explained if TGF-8 exerts multiple effects during angiogenesis. Recently, it has been proposed that TGF-@ can induce angiogenesis in vivo by

VOLUME 130,1088

attracting and activating monocytes (Wahl et al., 1987). The activated monocytes in turn release several factors, including tumor necrosis factor-a, which can induce angiogenesis. However, TGF-/? is also likely to directly affect endothelial cells in vivo. During angiogenesis, endothelial cells must perform many functions, including cell proliferation, production of proteases that digest components of the extracellular matrix, migration to the tissue undergoing vascularization, and tube formation. Several of these steps can be induced by growth factors belonging to the FGF family and at least some of the effects of FGF are likely to be regulated by TGF-8. TGF-/3 has been shown to inhibit the ability of FGF to induce proliferation of endothelial cells (Frater-SchrSder et aZ.,1986; Miiller et aL, 1987; Saksela et ab, 1987) and TGF-P can inhibit FGF-induced release of plasminogen activator by endothelial cells (Saksela et ak, 1987). In addition, TGF-P stimulates release of the plasminogen activator inhibitor PAI- by endothelial cells (Saksela et al, 198’7)and also stimulates tube formation of endothelial cells cultured in three-dimensional collagen gels (Madri et al, 1988). Thus, it appears that TGF-P can act either directly on endothelial cells (e.g., by modifying the effects of other growth factors) or it can act indirectly by inducing the production of other factors. The latter effect is reminiscent of the induction of a growth factor related to platelet-derived growth factor (PDGF) by TGF-P (Leof et d, 1986). EFFECTS

OF TGF-fl

ON EXTRACELLULAR

MATRICES

A major unanswered question is how TGF-@ exerts its effects on differentiation. Several different mechanisms are likely to be involved and, at least in some cases, it is likely that TGF-8 either suppresses or promotes differentiation via its effects on the extracellular matrix. TGF-@ has been shown to influence the extracellular matrices produced by many different cell types (Table 2) and, in virtually all cases, TGF-/3 seems to lead to an increase in the extracellular matrix, either by increasing production or by decreasing degradation of the matrix. Attempts to identify the mechanisms involved are just beginning. Recent studies demonstrate that TGF-@ can increase the transcription of the type I collagen gene, and a TGF-P cis regulatory element has been identified (Rossi et aL, 1988). Similarly, TGF-P has been shown to regulate the transcription of several genes involved in the turnover of the extracellular matrix. Transcription of the genes for the metalloproteases collagenase and stromelysin can be induced by several growth factors, including FGF, but induction of these genes is blocked by TGF-/3 (Edwards et al, 198’7). Moreover, transcription of the specific metalloprotease inhibitor TIMP is stimulated by TGF-@ and this stimulation is potentiated by growth factors such as FGF

ANGIE RIZZINO

Growth Factors and L&ferentiatim

415

chondrocytes. TGF-/3 has been shown to increase their production of fibronectin in vitro (Ignotz et al, 1987; Ignotz and Massague, 1987; Rosen et CAL,1988) and the Reference Effect Cells addition of fibronectin to myoblasts (Podleski et ah, 1979), preadipocytes (Spiegelman and Ginty, 1983), and Fine and Goldstein, Increases production of Human lung 1987 chondrocytes (Rosen et aL, 1988) inhibits their differtype I and III collagen fibroblasts Ignotz et al., 1987 Increases production of Myoblasts entiation. Furthermore, given the important role of fitype I collagen bronectin during early amphibian development (DarRaghow et al, 1987 Increases synthesis of Human dermal rib&e et ah, 1988), it is tempting to speculate that the Varga et al, 1987 fibronectin and type I fibroblasts effects of TGF-/3 on fibronectin production (and other and III collagens Bassols and Increases production of components of the extracellular matrix) are partly reFibroblast cell Massague, 1988 fibronectin lines sponsible for the effects of TGF-/3 during amphibian Blatti et uJ, 1988 mesoderm formation. However, even if this is true, Penttinen et al, other mechanisms, including some rapid effects of me1988 soderm-inducing factor (Symes and Smith, 1987), must Chakrabarty et a& Colon tumor cell Increases production of 1987 laminin and fibronectin line also be considered. Lastly, TGF-P may exert some of its Ignotz and Increases production of Preadipocytes inhibitory effects on growth via its effects on the exMassaguk, 1987 cell adhesion protein tracellular matrix. In the case of endothelial cells, receptors TGF-P stimulates their production of fibronectin and Skantze et aL, 1985 Several cell Increases production of their growth is inhibited by exogenously added TGF-0 Chen et al, 1987 sulfated proteoglycans types Bassols and or fibronectin (Madri et al, 1988). Massague, 1988 If growth factors, such as TGF-/3, influence differenEdwards et al., 1987 Fibroblasts Potentiates growth factortiation indirectly through their effects on the extracelThalacker and dependent release of lular matrix, the larger question is how? Undoubtedly, Nilsen-Hamilton, metalloprotease several mechanisms are involved. One mechanism may 1987 inhibitor and suppresses growth involve the effects of the extracellular matrix on cell factor-dependent shape, which in turn affects RNA and protein syntherelease of collagenase ses. Cell shape, which is affected by various components Several cell lines Increases the release of Laiho et al, 1987 of extracellular matrices (e.g., fibronectin, collagen, and plasminogen activator Lund et al, 1987 laminin), can affect the synthesis and the stability of inhibitor PAIThalacker and Nilsen-Hamilton, mRNA. In a classic study, cells placed in suspension 1987 were shown to change shape and exhibit a dramatic Keski-Oja et al, decline in mRNA synthesis and a compensating in1988 crease in mRNA stability (Benecke et aL, 1978). Even Endothelial Reduces release of Saksela et al, 1987 cells though total mRNA levels did not change significantly plasminogen activator and increases release of under these conditions, protein synthesis declined more plasminogen activator than 80%. Apparently, the reduction in protein syntheinhibitor PAIsis resulted from the withdrawal of mRNA into an unNew born mice Induces fibrosis at site of Roberts et al, 1986 translatable pool, which increased the stability of the injection mRNA. Other data suggest a direct link between a All examples have not been cited. changes in the cytoskeleton and the production of specific gene products. Production of milk proteins (&actalbumin and casein) by primary rat mammary cells is (Edwards et aL, 1987). It is interesting that similar in- highly dependent on the substratum used (Wicha et al, teractions occur in at least two other cases. TGF-j3 has 1982; Medina et aL, 1987) and disruption of the cytoskelbeen shown to block the FGF-induced and the epider- eton by cytochalasin B selectively reduces the producma1 growth factor-induced release of proteases by endo- tion of these milk proteins (Blum and Wicha, 1988), at thelial cells (Saksela et aL, 1987) and 3T3 cells (Chiang least in part, by increasing the turnover of their mRNA and Nilsen-Hamilton, 1986; Thalacker and Nilsen-Ha(Zeigler and Wicha, 1987). For additional details, the milton, 1987), respectively, and TGF-@ increases the re- reader is directed to reviews by Bissell and co-workers lease of the plasminogen activator inhibitor PAI- by (Bissell and Aggeler, 1987; Bissell et al, 1982). Similarly, both cell types. TGF-P has been shown to inhibit phenotypically the The most direct evidence that TGF-8 can affect dif- differentiation of chondrocytes and this effect of TGF-fl ferentiation via its effects on the extracellular matrix is blocked by disruption of the cytoskeleton (Rosen et has been obtained with myoblasts, preadipocytes, and aL, 1988). TABLE 2 EFFECTSOF TGF-/3 ON EXTRACEUULAR MATRICES”

416

DEVELOPMENTAL BIOLOGY

The extracellular matrix may also exert other important regulatory effects. In another classic study, cell shape was shown to affect cell proliferation (Folkman and Moscona, 1978) and several studies have established a link between extracellular matrices and the responses of cells to growth factors. Endothelial cells have been shown to require FGF in order to proliferate on untreated tissue-culture plastic, yet the same cells grown on a cornea1 extracellular matrix do not require FGF (Gospodarowicz et aL, 1979). Although this observation may have been due to FGF contamination of the extracellular matrix preparation used, other studies that illustrate this phenomenon are not subject to this criticism. Recent studies have shown that TGF-@ does not inhibit the growth of endothelial cells cultured in a three-dimensional collagen matrix (Madri et aL, 1988), yet TGF-@ does inhibit the growth of endothelial cells in monolayer cultures. Interestingly, growth of endothelial cells in monolayer culture is dependent on the substratum, as is the extent of growth inhibition observed in the presence of TGF-/3 (Madri et aZ., 1988). Thus, growth factors and extracellular matrices may form a reciprocal regulatory loop in which growth factors influence the production of extracellular matrices and extracellular matrices influence the responses to growth factors.

VOLUME 130,1988

lines leads to large increases in the release of TGF-P (Kryceve-Martinerie et al, 1982; Anzano et ab, 1985). These data clearly demonstrate that TGF-fi production and release can be regulated. However, the molecular regulatory mechanisms involved remain to be elucidated. The action of a growth factor can also be regulated by modulating its activity after it has been produced. For more than a decade, it has been recognized that some growth factors are released in inactive forms that require activation. In the case of TGF-P, this possibility was recognized soon after TGF-@ was characterized (Lawrence et ah, 1984) and several lines of evidence argue that TGF-@ is released from cells primarily as a high molecular weight complex (approximately 210 kDa). Recent work has demonstrated that the inactive or latent form of TGF-fi is composed of three different polypeptides, the mature form of TGF-/3 (derived from the C-terminal portion of the TGF-P precursor), most of the N-terminal portion of the TGF-P precursor, and a third unrelated polypeptide (Miyazono et al., 1988; Wakefield et a& 1988). The latent form of TGF-P can be activated by treatment at low pH, but this is not thought to be the predominant mechanism for its activation in viva. A more likely mechanism involves proteolytic activation; both plasmin and cathepsin D can release biologically active TGF-P from its latent form (Lyons et a& 1988). Once activated, the levels of TGF-/3 REGULATING THE ACTION OF TGF-/3 apparently can be further regulated by binding to proGiven the widespread and diversified effects of teins such as cYz-macroglobulin (O’Connor-McCourt and TGF-/3, its activity must be regulated very carefully. Wakefield, 1987; Huang et al, 1988). The latter mechaThe most obvious means of regulating the effects of a nism is likely to be used to remove or “clear” TGF-8. growth factor is to regulate its production. At present, it appears that virtually all cells produce TGF-P (RoREGULATION OF TGF-/3 RECEPTORS berts et aL, 1981). However, as discussed earlier, the Another means of regulating the action of TGF-/3 is levels of TGF-@l are regulated developmentally and other work has established that the production and/or to regulate its cell surface receptors. Nearly all cells release of TGF-@ by several different cell types can be exhibit receptors for TGF-/31 or TGF-/32, but there are regulated. For example, production of TGF-fi mRNA several notable examples of cells that exhibit few, if and release of TGF-/? by T cells increase when these any, receptors for TGF-/3. Thus far, the only examples cells are activated. Similarly, the release of TGF-/3 in- are tumor cells: two embryonal carcinoma cell lines creases upon activation of macrophages (Assoian et al, (Rizzino, 1987b) and six retinoblastoma cell lines (Kim1987). In addition, it appears that the levels of TGF-/3 chi et al, 1988). In the case of embryonal carcinoma can be regulated hormonally. Parathyroid hormone and cells, differentiation leads to the formation of cells that calcitonin can regulate the release of TGF-@ by cultured exhibit receptors for TGF-P and respond to TGF-/3 by growth inhibition (Rizzino, 1987b). Interestingly, the rat calvaria (Pfeilschifter and Mundy, 1987). Interestingly, parathyroid hormone, which is known to increase differentiated cells, unlike the parental embryonal carbone resorption, increases the release of TGF-P; cinoma cells, are not tumorigenic. In addition to the of emwhereas calcitonin, which is known to decrease bone change in TGF-/3 receptors, differentiation bryonal carcinoma cells also leads to large increases in resorption, decreases the release of TGF-fl. Moreover, the number of receptors for epidermal growth factor antiestrogens increase the release of biologically active TGF-P by a mammary tumor cell line (Knabbe et ah, (EGF) (Rees et aL, 1979), PDGF (Rizzino and Bowen1987). Lastly, the levels of mRNA for TGF-P are ele- Pope, 1985) and FGF (Rizzino et al., 1988b), but binding of insulin-like growth factor II is decreased (Heath and vated in malignant tissues (Niitsu et al, 1988; Akhurst et al, 1988) and viral transformation of established cell Shi, 1986). Myogenesis also has a dramatic effect on the

ANGIE RIZZINO

Growth Factors

growth factor receptors exhibited by myoblasts. However, in this case, EGF (Olwin and Hauschka, 1988), FGF (Olwin and Hauschka, 1988) and TGF-/3 (Ewton et ak, 1988) binding decreases as a consequence of myogenesis. Thus, it appears that differentiation can coordinately regulate the appearance or disappearance of receptors for several different growth factors. Growth factor receptors for TGF-/3 and other growth factors can also be regulated by other mechanisms. During the past year, it has been determined that receptors for TGF-/3, EGF, PDGF, and FGF are regulated by cell density. Specifically, the binding of each of these growth factors decreases as cell density increases and, thus far, studies to examine the mechanisms involved have demonstrated that the decreased binding of TGF-/3 and EGF is due to a reduction in receptor number (Rizzino et aZ.,1988a). Although the mechanism involved has not been determined, it is clear that density-induced down-regulation of growth factor receptors is a widespread effect (it has been observed in more than 10 cell lines) and this phenomenon may be partly responsible for the reduction in growth rate of nontransformed cells that occurs at high cell densities. In addition, cell density may regulate the composition of specific growth factor receptors. In the case of endothelial cells, sub-confluent cells seem to express only the high molecular weight form of the TGF-/I receptor, whereas confluent endothelial cells seem to express only the two low molecular weight receptor forms (Mtiller et aZ.,1987) (see discussion of TGF-/3 receptors below). TGF-P receptors are not the only growth factor receptors to exist in multiple forms. Besides the multiple forms of receptors for the insulin-like growth factor family, at least two different receptors for PDGF exist, and PDGF can be crosslinked to at least two different subunits on the same cells (Heldin et aZ.,1988; Gronwald et al, 1988). Thus, the possibility exists that cell density regulates the composition of receptors for several different growth factors. Lastly, it is widely recognized that receptors for specific growth factors can be regulated by other growth factors. For example, PDGF can reduce the number of cell surface EGF receptors (Wrann et aZ., 1980), whereas TGF-fl and insulin have been shown to increase the number of cell surface receptors for EGF (Assoian et aZ., 1984; Takehara et al., 1987) and insulin-like growth factor II (Corvera and Czech, 1985), respectively. Recent studies also indicate that adrenocorticotropic hormone can increase the number of TGF-/3 receptors expressed by adrenocortieal cells (Cachet et ab, 1988). Evidence that TGF-Z31 and TGF$2 can exert different effects raises an important question. What are the mechanisms responsible for the differential effects of TGF-@l and TGF-/32? Part of the answer appears to lie in the complexity of the TGF-fl receptor. Cross-linking

and

417

Dzfferentiatim

studies have established that TGF-fl associates with at least three different glycoproteins (Massague, 1985; Cheifetz et ah, 1986). This has led to the speculation that there are at least three different receptors for TGF-P: a high molecular weight receptor (280-330 kDa) that exhibits high affinity for both TGF-01 and TGF-~32, and two lower molecular weight receptors (65-75 kDa and 85-110 kDa) receptors that exhibit a much higher affinity for TGF-/31 than they do for TGF-P2 (Cheifetz et al., 1987). Furthermore, TGF-P receptors may exhibit additional complexity, since there appears to be a form of the high molecular weight receptor that preferentially binds TGF-P2 (Segarini et ah, 198’7). FUTURE

DIRECTIONS

The findings discussed in this review demonstrate that TGF-@ exerts multiple effects on differentiation. These findings represent a great deal of progress, but it is evident that there is far more to learn. Understanding the different roles of TGF-P will require detailed information regarding its production, secretion, and activation. Moreover, it will be necessary to do more than identify the cellular targets of TGF-& we must also understand how TGF-8 interacts with other growth factors to affect cell function and understand how cell-cell interactions influence responses to TGF-8. In the immediate future, many unanswered questions can be addressed with the technology already available. An important problem that has received too little attention is the regulation of TGF-J3 production. While it is evident that the production of TGF-P can be regulated, this matter has been examined in relatively few systems and the molecular regulatory mechanisms involved are virtually unknown. Progress in this area can be anticipated, since cloning of the genes for the different forms of TGF-P is well underway (Derynck et ab, 1985; Hanks et aZ.,1988; ten Dijke et ah, 1988; Jakowlew et al., 1988), if not already completed, and this will make it possible to determine the mechanisms involved in the regulation of TGF-P gene expression. It will be particularly interesting to identify the regulatory gene sequences responsible for the differential transcription of the genes for each form of TGF-6. Similarly, it will be important to clone the gene or genes for TGF-/3 receptors and to determine how their expression is controlled. Hopefully, this will help explain the differential distribution of TGF-P receptors. It can also be anticipated that additional details on the developmental regulation of TGF-8 will be forthcoming. In particular, it will be important to define the spatial and temporal distribution of TGF-~32, TGF-03 and TGF-P4, especially in comparison to one another and to TGF-Pl. (The latter studies will be limited to TGF-/31 and TGF-P2 until isolation and purification of

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TGF-83 and TGF-@4 is complete and antibodies specific for TGF-/33 and TGF-P4 are prepared.) A more difficult, but no less important, problem is the spatial and temporal distribution of the active versus the latent forms of each TGF-/3. Important progress has been made in defining the structure of the latent form of TGF-fil, but it remains to be determined whether a common structure exists for latent forms of each TGF-P. In addition, detailed mechanisms for the,activation of TGF-P need to be determined. Although there is evidence from in vitro studies that activation can occur by specific proteases, there is currently no evidence that activation occurs by a similar mechanism in viva. Hopefully, the latter issue can be addressed once the molecular mechanisms for activation in vitro have been described in detail. In the same vein, formal proof that TGF-P regulates cell differentiation in viva is lacking. Although it is clear that TGF-/3 from external sources can dramatically affect cell proliferation and differentiation in viva, rigorous proof that endogenous TGF-P regulates cell function in viva has not been obtained. While there is little doubt that TGF-P exerts countless effects under normal physiological conditions, the events involved need to be identified and studied in detail. In this regard, it will be important to formally test the hypothesis that growth factors, such as TGF-P, can indirectly influence differentiation by affecting the extracellular matrix. Moreover, it will be essential to determine whether these effects provide a permissive environment needed for differentiation to occur or whether they act inductively. Finally, the molecular mechanisms by which TGF-/3 exerts its effects have not been discussed in this review since this subject was reviewed recently (Sporn et cd, 1987). However, this area of research warrants continued and expanded effort and two broad questions need to be addressed. First, what are the signal transduction pathways that mediate the effects of TGF-@? There are many facets to this question, including the intracellular signals generated when TGF-/3 binds to its receptor. Second, how does TGF-P potentiate/suppress the responses of cells to other growth factors? Part of the answer appears to lie in the ability of TGF-/3 to regulate receptors for other growth factors and hormones, but other mechanisms are clearly involved (Like and Massag&, 1986, Chambard and Pouyssegur, 1988; Muldoon et al., 1988). Without answers to these questions, we will have only a descriptive understanding of growth factors that surely play major roles in development, tissue repair, and malignant growth. Heather Rizzino is thanked for helpful discussions during the preparation of this review. She is also thanked for excellent editorial assistance. Work in the author’s laboratory was supported by grants from the National Institute of Child Health and Human Development (HD19837, HD21568), the National Cancer Institute (Laboratory

VOLUME 130,1988

search Center Support Grant CA36727), and the American Cancer Society (Core Grant ACS SIG-16). REFERENCES ADASHI, E. Y., and RESNICK,C. E. (1986). Antagonistic interactions of transforming growth factors in the regulation of granulosa cell differentiation. End,mrinology 119,1879-1881. AKHURST,R. J., FEE, F., and BALMAIN, A. (1988). Localized production of TGF-P mRNA in tumour promoter-stimulated mouse epidermis. Nature (London) 331,363-365. ALLEN, R. E., and BOXHORN,L. K. (1987). Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta. J. Cell Physid 133,567-572. ANZANO, M. A., ROBERTS,A. B., DELARCO,J. E., WAKEFIELD, L. M., ASSOIAN, R. K., ROCHE, N. S., SMITH, J. M., LAZARUS, J. E., and SPORN,M. B. (1985). Increased secretion of type fi transforming growth factor accompanies viral transformation of cells. &fo~ Cell Biol 5,242-247. ASSOIAN,R. K., FLEURDELYS,B. E., STEVENSON,H. C., MILLER, P. J., MADTES, D. K., RAINES, E. W., Ross, R., and SPORN,M. B. (1987). Expression and secretion of type /3 transforming growth factor by activated human macrophages. Proc. Natl. Acad. Sci. USA 84, 6020-6024. ASSOIAN, R. K., FROLIK, C. A., ROBERTS,A. B., MILLER, D. M., and SPORN,M. B. (1984). Transforming growth factor-j3 controls receptor levels for epidermal growth factor in NRK fibroblasts. Cell 36, 35-41. ASSOIAN, R. K., KOMORIYA,A., MEYERS, C. A., MILLER, D. M., and SPORN,M. B. (1983). Transforming growth factor-0 in human platelets. J. Biol. C~JWL258,7155-7160. AVALLET, O., VIGIER, M., PERRARD-SAPORI,M. H., and SAEZ, J. M. (1987). Transforming growth factor fi inhibits Leydig cell functions. Biochem. Biophys. Res. Commun. 146,575-581. BAIRD, A., MORMEDE,P., YING, S.-Y., WEHRENBERG,W. B., VENO, N., LING, N., and GUILLEMIN, R. (1985). A nonmitogenic pituitary function of fibroblast growth factor: Regulation of thyrotropin and prolactin secretion. Proc Nat1 Ad Sci USA 82,5545-5549. BASSOLS,A., and MASSAGU~,J. (1988). Transforming growth factor @ regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans. J. Biol Chem 263, 3039-3045. BENECKE,B.-J., BEN-ZE’EV, A., and PENMAN, S. (1978). The control of mRNA production, translation and turnover in suspended and reattached anchorage-dependent fibroblasts. CeU 14,931-939. BISSELL, M. J., and AGGELER,J. (1987). Dynamic reciprocity: How do extracellular matrix and hormones direct gene expression? Prog. Clin Bid Res. 249,251-262. BISSELL, M. J., HALL, H. G., and PARRY, G. (1982). How does the extracellular matrix direct gene expression? J. Theor. Bid 99, 31-68. BLA~TI, S. P., FOSTER,D. N., RANGANATHAN, G., MOSES,H. L., and GETZ, M. J. (1988). Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells. Proc. Nati Acad Sci. USA 85,1119-1123. BLUM, J. L., and WICHA, M. S. (1988). Role of the cytoskeleton in laminin induced mammary gene expression. J. Cell Physid 135, 13-22. CENTRELLA,M., MASSAGU&J., and CANALIS, E. (1986). Human platelet-derived transforming growth factor-b stimulates parameters of bone growth in fetal rat calvariae. Erw!owi?wZo~~119,2306-2312. CHAKRABARTY,S., TOBON,A., BRATTAIN, M. G., and VARANI, J. (1987). Transforming growth factor (TGF@) stimulates fibronectin and laminin synthesis in human colon carcinoma cells. Cancer Rex 28, 58. CHAMBARD,J.-C., and POUYSS~~GUR, J. (1988). TGF-P inhibits growth

ANGIE RIZZINO

Growth Factors and Differentiation

419

FEIGE, J.-J., COCHET,C., RAINEY, W. E., MADANI, C.,and CHAMBAZ, E. M. (1987). Type fl transforming growth factor affects adrenocortical cell-differentiated functions. J. BioL Chem. 262,13,491-13,495. FENG, P., CATT, K. J., and KNECHT, M. (1986). Transforming growth factor p regulates the inhibitory actions of epidermal growth factor BioL Chem 261,9972-9978. during granulosa cell differentiation. J. BioL Chem 261, 14,167CHEIFETZ, S., WEATHERBEE, J. A., TSANG, M. L.-S., ANDERSON, J. K., 14,170. MOLE, J. E., LUCAS, R., and MASSAGUI?., J. (1987). The transforming FENG, P., CAM, K. J., and KNECHT, M. (1988). Transforming growth growth factor-6 system, a complex pattern of cross-reactive ligands factor-p stimulates meiotic maturation of the rat oocyte. Endox+and receptors. CeU48,409-415. nobgy 122,181-186. CHEN, J.-K., HOSHI, H., and MCKEEHAN, W. L. (1987). Transforming FINE, A., and GOLDSTEIN,R. H. (1987). The effect of transforming growth factor type @specifically stimulates synthesis of proteoglygrowth factor-b on cell proliferation and collagen formation by can in human adult arterial smooth muscle cells. Proc. NatL Acad lung fibroblasts. J. BioL Chem. 262.3897-3902. Sci USA 64,5287-5291. CHIANG, C.-P., and NILSEN-HAMILTON, M. (1986). Opposite and selec- FLORINI, J. R., ROBERTS,A. B., EWTON,D. Z., FALEN, S. L., FLANDERS, K. C., and SPORN,M. B. (1986). Transforming growth factor-@ A tive effects of epidermal growth factor and human platelet transvery potent inhibitor of myoblast differentiation, identical to the forming growth factor-8 on the production of secreted proteins by differentiation inhibitor secreted by buffalo rat liver cells. J. BioL murine 3T3 cells and human fibroblasts. J. BioL Chem. 261,10,478Chem. 261, 16,509-16,513. 10,481. FOLKMAN, J., and MOSCONA,A. (1978). Role of cell shape in growth COCHET,C., FEIGE, J.-J., and CHAMBAZ,E. M. (1988). Bovine adrenocortical cells exhibit high affinity transforming growth factor-p control. Nature (London) 273,345-349. FRATER-SCHR~DER,M., MUELLER, G., BIRCHMEIER,W., and B&~HLEN,P. receptors which are regulated by adrenocorticotropin. J. BioL (1986). Transforming growth factor-beta inhibits endothelial cell Chem. 263.5707-5713. proliferation. B&hem. Biophys. Res. Commun 137,295-302. COHEN, S., and ELLIOT, G. A. (1963). The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of GODSAVE,S. F., ISAACS,H. V., and SLACK,J. M. W. (1988). Mesoderminducing factors: A small class of molecules. Development 102, the mouse. J. Invest. DemnatoL 40, l-5. 555-566. COOKE,J., SMITH, J. C., SMITH, E. J., and YAQOOB,M. (1987). The organization of mesodermal pattern in Xenopus luevis: ExperiGOSPODAROWICZ, D., VLODAVSKY,I., GREENBURG,G., and JOHNSON, ments using a Xenopus mesoderm-inducing factor. Development L. K. (1979). Cellular shape is determined by the extracellular ma101,893-908. trix and is responsible for the control of cellular growth and function. “Proceedings, Cold Spring Harbor Conf. Cell Proliferation” CORVERA,S., and CZECH,M. P. (1985). Mechanism of insulin action on membrane protein recycling: A selective decrease in the phosphorVol. 6, p. 561-592. Cold Spring Harbor Laboratory, Cold Spring ylation state of insulin-like growth factor II receptors in the cell Harbor, NY. GRONWALD,R. G. K., GRANT, F. J., HALDEMAN, B. A., HART, C. E., surface membrane. Proc. Natl. Acad Sci USA 82,7314-7318. DARRIB~RE, T., YAMADA, K. M., JOHNSON,K. E., and BOUCAUT,J.-C. O’HARA, P. J., HAGEN, F. S., Ross, R., BOWEN-POPE,D. F., MURRAY, (1988). The 140-kDa fibronectin receptor complex is required for M. J. (1988). Cloning and expression of a cDNA coding for the mesodermal cell adhesion during gastrulation in the amphibian human platelet-derived growth factor receptor: Evidence for more Pleurcdeles waltlii. Dev. BioL 126,182-194. than one receptor class. Proc. NatL AccuL Sci. USA f&5,3435-3439. DE MARTIN, R., HAENDLER, B., HOFER-WARBINEK,R., GAUGITSCH,H., HANKS, S. K., ARMOUR,R., BALDWIN,J. H., MALDONADO,F., SPIES&J., WRANN,M., [email protected], H., SEIFERT,J. M., BODMER,S., FONTANA, and HOLLEY, R. W. (1988). Amino acid sequence of the BSC-1 cell A., and HOFER, E. (1987). Complementary DNA for human gliobgrowth inhibitor (polyergin) deduced from the nucleotide sequence lastoma-derived T cell suppressor factor, a novel member of the of the cDNA. Proc. NatL Acad Sci. USA 85,79-82. transforming growth factor-0 gene family. EMBO J 6,3673-3677. HEATH, J. K., and SHI, W.-K. (1986). Developmentally regulated exDELARCO,J. E., and TODARO,G. J. (1978). Growth factors from mupression of insulin-like growth factors by differentiated murine rine sarcoma virus-transformed cells. Proc. NatL Acad. Sci. USA 75, teratocarcinomas and extraembryonic mesoderm. J. EmbyoL 95, 4001-4005. 193-212. DERYNCK,R., JARRETT,J. A., CHEN, E. Y., EATON, D. H., BELL, J. R., HEINE, U. I., MUNOZ, E. F., FLANDERS,K. C., ELLINGSWORTH,L. R., ASSOIAN,R. K., ROBERTS,A. B., SPORN,M. B., and GOEDDEL,D. V. LAM, H.-Y. P., THOMPSON,N. L., ROBERTS,A. B., and SPORN,M. B. (1985). Human transforming growth factor-8 complementary DNA (1987). Role of transforming growth factor+ in the development of sequence and expression in normal and transformed cells. Nature the mouse embryo. J. Cell BioL 105.2861-2876. (Imdm) 316,701-705. HELDIN, C.-H., B~CKSTR~M,G., &TMAN, A., HAMMACHER,A., RONNEDWARDS,D. R., MURPHY, G., REYNOLDS,J. J., WHITHAM, S. E., DoSTRAND,L., RUBIN, K., NISTER, M., and WESTERMARK,B. (1988). CHERTY,A. J. P., ANGEL, P., and HEATH, J. K. (1987). Transforming Binding of different dimeric forms of PDGF to human fibroblasts: growth factor beta modulates the expression of collagenase and Evidence for two separate receptor types. EMBO J. 7.1387-1393. metalloproteinase inhibitor. EMBO J. 6,1899-1904. HOLLEY, R. W., ARMOUR,R., and BALDWIN, J. H. (1978). Density-deESCH, F., BAIRD, A., LING, N., UENO, N., HILL, F., DENOROY,L., KLEPpendent regulation of growth of BSC-1 cells in cell culture: Growth PER,R., GOSPODAROWICZ, D., BOHLEN,P., and GUILLEMIN, R. (1985). inhibitors formed by the cells. Proc. Natl. Acad. Sci. USA 75, Primary structure of bovine pituitary basic fibroblast growth fac1864-1866. tor (FGF) and comparison with the amino-terminal sequence of HOTTA, M., and BAIRD, A. (1986). Differential effects of transforming bovine brain acidic FGF. Proc. NatL Acad Sci USA 82,6507-6511. growth factor type @on the growth and function of adrenocortical EWTON,D. Z., SPIZZ,G., OLSON,E. N., and FLORINI, J. R. (1988). Decells in vitro. Proc. NatL Acad. Sci. USA 83, 7795-7799. crease in transforming growth factor-beta binding and action durHUANG, S. S., O’GRADY, P., and HUANG, J. S. (1988). Human transing differentiation in muscle cells. J. BioL Chem, 263,4209-4032. forming growth factor 0: ols-macroglobulin complex is a latent form FEIGE, J. J., COCHET,C., and CHAMBAZ, E. M. (1986). Type p transof transforming growth factor p. J BioL Chem 263,1535-1541. forming growth factor is a potent modulator of differentiated adreIGNOTZ,R. A., ENDO, T., and MASSAGU~,J. (1987). Regulation of finocortical cell functions. Biochem. Biophys. Res. Commun. 139, bronectin and type I collagen mRNA levels by transforming growth 693-700. factor+?. J. Biol. Chem. 262, 6443-6446.

factor-induced DNA synthesis in hamster fibroblasts without affecting the early mitogenic events. J. Cell. Physiol. 135,101-107. CHEIFETZ,S., LIKE, B., and MASSAGUI?., J. (1986). Cellular distribution of type I and type II receptors for transforming growth factor-B. J.

420

DEVELOPMENTALBIOLOGY

IGNOTZ,R. A., and MASSAGUB,J. (1985). Type B transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.

VOLUME 130.1988

LIKE, B., and MASSAGU&J. (1986). The antiproliferative effect of type p transforming growth factor occurs at a level distal from receptors Proc. Natl. Ad. Sti USA 82,8530-8534. for growth-activating factors. J. Biol. Chem. 261,13,426-13,429. IGNOTZ,R. A., and MASSAGU~,J. (1987). Cell adhesion protein recepLIN, T., BLAISDELL, J., and HASKELL, J. F. (1987). Transforming tors as targets for transforming growth factor-8 action. Cell 51, growth factor-@ inhibits Leydig cell steroidogenesis in primary 189-197. culture. Biochem Biophys. Res. Commun 146,387-394. IKEDA, T., LIOUBIN, M. N., and MARQUARDT,H. (1987). Human transLINKHART, T. A., LIM, R. W., and HAUSCHKA, S. D. (1982). Regulation forming growth factor type 02: Production by a prostatic adenocarof normal and variant mouse myoblast proliferation and differencinema cell line, purification, and initial characterization. &o&mtiation by specific growth factors. In “Growth of Cells in Hormonistry 26,2406-2410. ally Defined Media, Book B” (G. H. Sato, A. B. Pardee, and D. A. JAKOWLEW,S. B., DILLART, P. J., KONDAIAH, P., SPORN,M. B., and Sirbasku, Eds.), p. 867-876. Cold Spring Harbor Laboratory, Cold ROBERTS,A. B. (1988). Complementary deoxynucleic acid cloning of Spring Harbor, NY. a novel transforming growth factor-p messenger ribonucleic acid LOBB, R. R., ALDERMAN, E. M., and FETT, J. W. (1985). Induction of from chick embryo chondrocytes. Mol. Endocrinol. 2,747-755. angiogenesis by bovine brain derived class 1 heparin-binding KEHRL, J. H., ROBERTS,A. B., WAKEFIELD, L. M., JAKOWLEW, S., growth factor. Biochemistry 24,4969-4973. SPORN,M. B., and FAUCI, A. S. (1986a). Transforming growth factor LUND, L. R., RICCIO,A., ANDREASEN,P. A., NIELSEN, L. S., KRISTEN0 is an important immunomodulatory protein for human B lymSEN, P., LAIHO, M., SAKSELA, O., BLASI, F., and DANNY,K. (1987). phocytes. J. ImmunoL 137,3855-3860. Transforming growth factor-8 is a strong and fast acting positive KEHRL, J. H., WAKEFIELD, L. M., ROBERTS,A. B., JAKOWLEW,S., ALregulator of the level of type-l plasminogen activator inhibitor VAREZ-MON, M., DERYNCK, R., SPORN, M. B., and FAUCI, A. S. mRNA in WI-38 human lung fibroblasts. EMBO J. 6,1281-1286. (1986b). Production of transforming growth factor p by human T LYONS, R. M., KESKI-OJA, J., and MOSES, H. L. (1988). Proteolytic lymphocytes and its potential role in the regulation of T cell activation of latent transforming growth factor-b from fibroblastgrowth. J. Exp. Med 163,1037-1050. conditioned medium. J. Cell Biol. 106, l-7. KESKI-OJA, J., RAGHOW, R., SAWDEY, M., LOSKUTOFF,D. J., POST- MADRI, J. A., PRATT, B. M., and TUCKER, A. M. (1988). Phenotypic LETHWAITE,A. E., KANG, A. H., and MOSES,H. L. (1988). Regulation modulation of endothelial cells by transforming growth factor-b of mRNAs for type-l plasminogen activator inhibitor, fibronectin, depends upon the composition and organization of the extracellular and type I procollagen by transforming growth factor-p: Divergent matrix. J. Cell Biol. 106,1375-1384. responses in lung fibroblasts and carcinoma cells. J. Biol Chem. MASSAGU~,J. (1985). Subunit structure of a high-affinity receptor for 263,3111-3115. type p’ transforming growth factor: Evidence for a disulphideKIMCHI, A., WANG, X.-F., WEINBERG,R. A., CHEIFETZ, S., and MASlinked glycosylated receptor complex. J. BioZ. Chem 260,7059-7066. SAGU~,J. (1988). Absence of TGF$ receptors and growth inhibitory MASSAGU~,J. (1987). The TGF-/3 family of growth and differentiation responses in retinoblastoma cells. Science 240,196-198. factors. Cell 49,437-438. KIMELMAN, D., and KIRSCHNER,M. (1987). Synergistic induction of MASSAGU!&J., CHEIFETZ,S., ENDO, T., and NADAL-GINARD, B. (1986). mesoderm by FGF and TGF-@ and the identification of an mRNA Type /3 transforming growth factor is an inhibitor of myogenic coding for FGF in the early Xenopus embryo. CeU51,869-877. differentiation. Proc. NatL Acad. Sci. USA 83,8206-8210. KNABBE, C., LIPPMAN, M. E., WAKEFIELD, L. M., FLANDERS, K. C., MASUI, T., WAKEFIELD, L. M., LECHNER,J. F., LAVECK, M. A., SPORN, KASID, A., DERYNCK,R., and DICKSON,R. B. (1987). Evidence that M. B., and HARRIS, C. C. (1986). Type @transforming growth factor transforming growth factor-p is a hormonally regulated negative is the primary differentiation-inducing serum factor for normal growth factor in human breast cancer cells. Cell 48,417-428. human bronchial epithelial cells. Proc. Natl Acad Sci. USA 83, KNECHT, M., FENG, P., and CATT, K. J. (1986). Transforming growth 2438-2442. factor-p regulates the expression of luteinizing hormone receptors MEDINA, D., Lr, M. L., OBORN,C. J., and BISSELL,M. J. (1987). Casein in ovarian granulosa cells. Biochem. Biuphys. Res. Commun 139, gene expression in mouse mammary epithelial cell lines: Depen800-807. dence upon extracellular matrix and cell type. Exp. Cell Res. 172, KRY&VE-MARTINERIE, C., LAWRENCE,D. A., CROCHET,J., JULLIEN, 192-203. P., and VIGIER, P. (1982). Cells transformed by Rous sarcoma virus MENKO, A. S., and BOETTIGER,D. (1987). Occupation of the extracelrelease transforming growth factors. J. Cell. PhysioZ. 113,365-372. lular matrix receptor, integrin, is a control point for myogenic difKUROKOWA,M., LYNCH, K., and PODOLSKY,D. K. (1987). Effects of ferentiation. CeU 51,51-57. growth factors on an intestinal epithelial cell line: Transforming MIYAZONO, K., HELLMAN, U., WERNSTEDT, C., and HELDIN, C.-H. growth factor @inhibits proliferation and stimulates differentia(1988). Latent high molecular weight complex of transforming tion. Biochem Biophys. Res. Commun. 142,775-782. growth factor 81. J. BioL Chem 263,6407-6415. LAIHO, M., SAKSELA, O., and KESKI-OJA, J. (1987). Transforming MULDOON, L. L., RODLAND, K. D., and MAGUN, B. E. (1988). Transgrowth factor-p induction of type-l plasminogen activator inhibiforming growth factor fi modulates epidermal growth factor-intor. J. Biol. Chem. 262, 17,467-17,474. duced phosphoinositide metabolism and intracellular calcium LAWRENCE,D. A., PIRCHER, R., KRYC~VE-MARTINERIE,C., and JLJLlevels. J. BioZ. Chem. 263,5030-5033. LIEN, P. (1984). Normal embryo fibroblasts release transforming M~LER, G., BEHRENS,J., NUSSBAUMER,U., B~HLEN, P., and BIRCHgrowth factors in a latent form. J. CeU.Physiol. 121,184188. MEIER,W. (1987). Inhibitory action of transforming growth factor 6 LEIBOVICH, S. J., POLVERINI,P. J., SHEPARD,H. M., WISEMAN, D. M., on endothelial cells. Proc. Natl. Acad Sci. USA 84,5600-5604. SHIVELY, V., and NUSEIR,N. (1987). Macrophage-induced angiogenesis is mediated by tumour necrosis factor-a. Nature (London) 329, NIITSU, Y., URUSHIZAKI, Y., KOSHIDA, Y., TERUI, K., MAHARA, K., KOHGO,Y., and URLJSHIZAKI,I. (1988). Expression of TGF-beta gene 630-632. in adult T cell leukemia. Blood 71,263-266. LEOF, E. B., PROPER,J. A., GOUSTIN,A. S., SHIPLEY,G. D., DICORLETO, NODA, M., and RODAN, G. A. (1986). Type-P transforming growth P. E., and MOSES,H. L. (1986). Induction of c-&s mRNA and activity factor inhibits proliferation and expression of alkaline phoaphataae similar to platelet-derived growth factor by transforming growth in murine osteoblast-like cells. Biochem. Biophys. Res. Commtm factor p: A proposed model for indirect mitogenesis involving auto140,56-65. crine activity. Proc. Natl. Acad. Sci. USA 83,2453-2457.

ANGIE RIZZINO

Growth

NODA, M., and RODAN, G. A. (1987). Type P transforming growth factor (TGFP) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells. J. Cell. PhysioL 133,426-43’7. O’CONNOR-MCCOURT, M. D., and WAKEFIELD, L. M. (1987). Latent transforming growth factor+ in serum. J. BioL Chem 262,14,09014,099. OHTA, M., GREENBERGER,J. S., ANKLESARIA, P., BASSOLS,A., and MASSAGU~,J. (1987). Two forms of transforming growth factor-0 distinguished by multipotential haematopoietic progenitor cells. Nature

(London) 329.539-541.

OLSON,E. N., STERNBERG,E., Hu, J. S., SPIZZ, G., and WILCOX, C. (1986). Regulation of myogenic differentiation by type /3 transforming growth factor. J. Cell BioL 103,1799-1805. OLWIN, B. B., and HAUSCHKA, S. D. (1988). Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture. J. Cell BioL, in press. PADGET~,R. W., ST. JOHNSTON,R. D., and GELBART,W. M. (1987). A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-p family. Nature (London) 325,81-84. PENTTINEN, R. P., KOBAYASHI, S., and BORNSTEIN,P. (1988). Transforming growth factor p increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc. Nat1 Acad Sci USA 85,1105-1108. PFEILSCHIFTER,J., and MUNDY, G. R. (1987). Modulation of type p transforming growth factor activity in bone cultures by osteotropic hormones. Proc. Natl. Acad. Sci. USA 84,2024-2028. PODLESKI, T. R., GREENBERG,I., SCHLESSINGER,J., and YAMADA, K. M. (1979). Fibronectin delays the fusion of L6 myoblasts. Exp. Cell Res. 122, 317-326. RAGHOW,R., POSTLETHWAITE,A. E., KESKI-OJA, J., MOSES,H. L., and KANG, A. H. (1987). Transforming growth factor-p increases steady state levels of type I procollagen and fibronectin messenger RNAs posttranscriptionally in cultured human dermal fibroblasts. J. Clin. Invest. 79,1285-1288. REES, A. R., ADAMSON,E. D., and GRAHAM, C. F. (1979). Epidermal growth factor receptors increase during the differentiation of embryonal carcinoma cells. Nature (Lon&nj 281,309-311. REISS, M., and SARTORELLI,A. C. (1987). Regulation of growth and differentiation of human keratinocytes by type p transforming growth factor and epidermal growth factor. Cancer Res. 47, 6705-6709.

RIZZINO, A. (1984). Behavior of transforming growth factors in serum-free media: An improved assay for transforming growth factors.Zn vitro,20,815-822. RIZZINO,A. (1987a). Soft agar growth assays for transforming growth factors and mitogenic peptides. In “Methods in Enzymology” (S. P. Colowick et al., Eds.), Vol. 146, pp. 341-352. Academic Press, Orlando, FL. RIZZINO,A. (198713).Appearance of high affinity receptors for type p transforming growth factor during differentiation of murine embryonal carcinoma cells. Cancer Res. 47, 43864390. RIZZINO,A., and BOWEN-POPE,D. F. (1985). Production of PDGF-like growth factors by embryonal carcinoma cells and binding of PDGF to their endoderm-like differentiated cells. Dev. BioL 110, 15-22. RIZZINO,A., and RUFF, E. (1986). Fibroblast growth factor induces the soft agar growth of two non-transformed cell lines. In lritro Cell. Dev. BioL 22,749-755. RrzzINo, A., RUFF, E., and RIZZINO,H. (1986). Induction and modulation of anchorage-independent growth by platelet-derived growth factor, 6broblast growth factor, and transforming growth factor-p.

Factors

and Differentiation

421

J. (I988a). Regulatory effects of cell density on the binding of transforming growth factor-p, epidermal growth factor, plateletderived growth factor and fibroblast growth factor. Cancer Res. 48, 4266-4271. RIZZINO,A., KUSZYNSKI,C., RUFF, E., and TIESMAN,J. (1988b). Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells. Dev. Biol. 129,61-71. ROBERTS,A. B., ANZANO,M. A., LAMB, L. C., SMITH, J. M., and SPORN, M. B. (1981). New class of transforming growth factors potentiated by epidermal growth factor: Isolation from non-neoplastic tissues. Proc. NatL Acad. Sci. USA 78,5339-5343.

ROBERTS,A. B., SPORN,M. B., ASSOIAN, R. K., SMITH, J. M., ROCHE, N. S., WAKEFIELD, L. M., HEINE, U. I., LIOTTA, L. A., FALANGA, V., KEHRL, J. H., and FAUCI, A. S. (1986). Transforming growth factor type 6: Rapid induction of fibrosis and angiogenesis in viva and stimulation of collagen formation in vitro. Proc. NatL Acad. Sci. USA 83,4167-4171.

ROOK,A. H., KEHRL, J. H., WAKEFIELD, L. M., ROBERTS,A. B., SPORN, M. B., BURLINGTON,D. B., LANE, H. C., and FAUCI, A. S. (1986). Effects of transforming growth factor 0 on the functions of natural killer cells: Depressed cytolytic activity and blunting of interferon responsiveness. J. ZmmunoL 136,3916-3920. ROSA,F., ROBERTS,A. B., DANIELPOUR,D., DART, L. L., SPORN,M. B., and DAWID, I. B. (1988). Mesoderm induction in amphibians: The role of TGF-fi2-like factors. Science 239,783-785. ROSEN,D. M., STEMPIEN,S. A., THOMPSON,A. Y., and SEYEDIN,S. M. (1988). Transforming growth factor-beta modulates the expression of osteoblast and chondroblast phenotypes in vitro. J Cell. Ph,ysiol. 134,337-346. ROSSI,P., KARSENTY,G., ROBERTS,A. B., ROCHE,N. S., SPORN,M. B., and DE CROMBRUGGHE, B. (1988). A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-p. Cell 52,405-414. SANDBERG,M., VUORIO,T., HIRVONEN, H., ALITALO, K., and VUORIO, E. (1988). Enhanced expression of TGF-8 and c-fos mRNAs in the growth plates of developing human long bones. Development 192, 461-470.

SAKSELA,O., MOSCATELLI,D., and RIFKIN, D. B. (1987). The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells. J. Cell BioL 105, 957-963. SEGARINI, P. R., ROBERTS,A. B., ROSEN,D. M., and SEYEDIN, S. M. (1987). Membrane binding characteristics of two forms of transforming growth factor-p. J. BioL Chem. 262, 14,655-14,662. SEYEDIN, S. M., SEGARINI, P. R., ROSEN, D. M., THOMPSON,A. Y., BENTZ,H., and GRAYCAR,J. (1987). Cartilage-inducing factor-B is a unique protein structurally and functionally related to transforming growth factor+‘. J. BioL Chem. 262,1946-1949. SEYEDIN, S. M., THOMAS,T. C., THOMPSON,A. Y., ROSEN,D. M., and PIEZ, K. A. (1985). Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc. Notl. Acad. Sci. USA 82,2267-2271.

SILBERSTEIN,G. B., and DANIEL, C. W. (1987). Reversible inhibition of mammary gland growth by transforming growth factor-p. Science 237,291-293. SKANTZE,K. A., BRINKERHOFF,C. E., and COLLIER,J. P. (1985). Use of agarose culture to measure the effect of transforming growth factor /3and epidermal growth factor on rabbit articular chondrocytes. CancerRes.

45,4416-4421.

SLACK,J. M. W., DARLINGTON,B. G., HEATH, J. K., and GODSAVE,S. F. (1987). Mesoderm induction in early Xenopus embryos by heparinbinding growth factors. Nature (Londm) 326,197200. CancerRes. 46,2816-2820. SMITH, J. C. (1987). A mesoderm-inducing factor is produced by a RrzzrNo, A., KAzAKOFF, P., RUFF, E., KUSZYNSKI,C., and NEBELSICK, Xenopus cell line. Development 993-14.

422

DEVELOPMENTALBIOLOGY

VOLUME 130,1988

SPARKS,R. L., and Scorr, R. E. (1986). Transforming growth factor SPORN,M. B., and ROBERTS,A. B. (1988). Transforming growth type /3is a specific inhibitor of 3T3 T mesenchymal stem cell differfactor @l positively regulates its own expression in normal and entiation. Exp. Cell Res. 165,345-352. transformed cells. J. Biol Chem. 263.7741-7746. SPENCER,F. A., HOFFMANN,F. M., and GELBART,W. M. (1982). Deca- VAN ZOELEN,E. J. J., VAN OOSTWAARD,T. M. J., VAN DER SAAG, P. T., pentaplegic: A gene complex affecting morphogenesis in Drosophila and DE LAAT, S. W. (1985). Phenotypic transformation of normal melanoga&r. Cell 28,451-461. rat kidney cells in a growth-factor defined medium: Induction by a neuroblastoma-derived transforming growth factor independently SPIEGELMAN,B. M., and GINTY, C. A. (1983). Fibronectin modulation of cell shape and lipogenic gene expression in 3T3-adipocytes. Cell of the EGF receptor. J. Cell. Physiol 123,151-160. 35,657-666. VARGA, J., ROSENBLOOM, J., and JIMENEZ, S. A. (1987). Transforming growth factor /3(TGFB) causes a persistent increase in steady-state SPORN,M. B., ROBERTS,A. B., WAKEFIELD, L. M., and ASSOIAN,R. K. (1986). Transforming growth factor-& Biological function and amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem. J. 247,597-604. chemical structure. S&m% 233,532~534. SPORN, M. B., ROBERTS,A. B., WAKEFIELD, L. M., and DE CROM- WAHL, S. M., HUNT, D. A., WAKEFIELD, L. M., MCCARTNEY-FRANCIS, N., WAHL, L. M., ROBERTS,A. B., and SPORN,M. B. (1987). TransBRUGGHE,B. (1987). Some recent advances in the chemistry and forming growth factor type /3 induces monocyte chemotaxis and biology of transforming growth factor-beta. J. Cell Biol. 105, growth factor production. Prop NatL Acad Sci. USA 34,5788-5792. 1039-1045. SPORN,M. B., and TODARO,G. J. (1980). Autocrine secretion and maWAKEFIELD, L. M., SMITH, D. M., FLANDERS,K. C., and SPORN,M. B. lignant transformation of cells. New En& J Med 303,878-880. (1988). Latent transforming growth factor-beta from human plateSYMES,K., and SMITH, J. C. (1987). Gastrulation movements provide lets: A high molecular weight complex containing precursor sean early marker of mesoderm induction in Xenopus lo&s. Devebpquences. J. Bid Chem 263,7646-7654. Tm?nt101,339-349. WEEKS, D. L., and MELTON,D. A. (1987). A maternal mRNA localized TAKEHARA, K., LEROY, E. C., and GROTEND~RST,G. R. (1987). TGF-/3 to the vegetal hemisphere in Xenopus eggs codes for a growth factor inhibition of endothelial cell proliferation: Alteration of EGF bindrelated to TGF-@. Cell 51,861-867. ing and EGF-induced growth-regulatory (competence) gene ex- WHITSE?T,J. A., WEAVER, T. E., LIEBERMAN,M. A., CLARK,J. C., and pression. Cell 49,415-422. DAUGHERTY, C. (1987). Differential effects of epidermal growth TEN DIJKE, P., HANSEN, P., IWATA, K. K., PIELER, C., and FOULKES, factor and transforming growth factor-8 on synthesis of Af, J. G. (1988). Identification of another member of the transforming = 35,000 surfactant-associated protein in fetal lung. J. Biol. Chem, growth factor type j3 gene family. Proc Natl. Acud Sci USA 85, 262,7908-7913. 4715-4719. WICHA, M. S., LOWRIE,G., KOHN, E., BAGAVANDOSS,P., and MAHN, T. THALACKER, F. W., and NILSEN-HAMILTON, M. (1987). Specific indue(1982). Extracellular matrix promotes mammary epithelial growth tion of secreted proteins by transforming growth factor-8 and and differentiation in vitro. Proc. Natl. Acad. Sci. USA 79, 12-0-tetradecanoyl-phorbol-13-acetate. J. Biol. Chem. 262, 3213-3217. 2283-2290. WRANN, M., Fox, C. F., and Ross, R. (1980). Modulation of epidermal THOMAS, K. A. (1987). Fibroblast growth factors. FASEB J. 1, growth factor receptors on 3T3 cells by platelet-derived growth 434-440. factor. Science 210.1363-1364. YING, S.-Y., BECKER,A., LING, N., UENO,N., and GUILLEMIN, R. (1986). THOMAS, K. A., RIOS-CANDELORE,M., GIMENEZ-GALLEGO, G., DIInhibin and beta transforming growth factor (TGFB) have opposite SALVO, J., BENNETT, C., RODKEY, J., and FITZPATRICK, S. (1985). modulating effects on the follicle stimulating hormone (FSH)-inPure brain-derived acidic fibroblast growth factor is a potent anduced aromatase activity of cultured rat granulosa cells. Biochem. giogenic vascular endothelial cell mitogen with sequence homology Biophys. Res. Commun. 136,969-975. to interleukin 1. Proc Natl Acad Sci. USA 82,6409-6413. TOGARI, A., BAKER, D. G., DICKENS, G., and GUROFF,G. (1983). The ZHAN, X., BATES, B., Hu. X., and GOLDFARB,M. (1988). Mol. Cell Biol. 8,3487-3495. neurite-promoting effect of fibroblast growth factor on PC12 cells. ZEIGLER,M. E., and WICHA, M. S. (1987). Regulation of casein RNA B&hem Biophys. Res. Commun. 114,1189-1193. stability by basement membrane. J. Cell Biol 105,48a. VAN OBBERGHEN-SCHILLING, E., ROCHE, N. S., FLANDERS, K. C.,