The Discovery and Early Days of TGF-Β: a Historical Perspective

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The Discovery and Early Days of TGF-b: A Historical Perspective

Harold L. Moses,1 Anita B. Roberts,2,4 and Rik Derynck3

1Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232 2Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892 3Department of Cell and Tissue Biology, University of California, San Francisco, California 94143 Correspondence: [email protected]

Transforming growth factors (TGFs) were discovered as activities that were secreted by cancer cells, and later by normal cells, and had the ability to phenotypically and reversibly transform immortalized ﬁbroblasts. TGF-b distinguished itself from TGF-a because it did not bind to the same epidermal growth factor (EGF) receptor as TGF-a and, therefore, acted through different cell-surface receptors and signaling mediators. This review summarizes the discovery of TGF-b, the early developments in its molecular and biological characterization with its many biological activities in different cell and tissue contexts and its roles in disease, the realization that there is a family of secreted TGF-b-related proteins with many differentiation functions in development and activities in normal cell and tissue physiology, and the subsequent identiﬁcation and characterization of the receptors and effectors that mediate TGF-b family signaling responses.

HISTORICAL CONTEXT nant transformation, made the Nobel Prize– winning breakthrough that neoplastic transfor- n the early 1970s, George Todaro and Robert mation of a cell by the avian sarcoma virus was IHuebner, studying the transforming activity mediated by a single viral gene ([src] the “onco- of C-type RNA tumor viruses, proposed the gene”), and most signiﬁcantly that the product viral oncogene hypothesis, that is, that “onco- of the src gene, now known to be a cellular sig- genes” were an important component of the naling mediator of many tyrosine kinase recep- virogene of RNA tumor viruses, whose expres- tors, was a slightly modiﬁed analog of a normal sion was suppressed in “normal” cells, and that cellular protein (Stehelin et al. 1976; Levinson treatment of cells with carcinogens, mutagens, et al. 1978). A few years later, they showed that or radiation could activate expression of this the transforming retroviral oncogene, v-src, viral information by inactivating a repressor arose by transduction of the cellular c-src gene (Todaro and Huebner 1972). A few years later, (Swanstrom et al. 1983). Michael Bishop and Harold Varmus,also study- Around the same time, based on results from ing the effects of RNA tumor viruses on malig- a collaboration with the Nobel laureate Stanley

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Cohen, George Todaro suggested that murine creted by fibroblasts transformed by an RNA and feline tumor viruses transform cells by in- virus, Moloney sarcoma virus (MSV), which ducing cells to secrete a molecule functionally they called sarcoma growth factor (SGF). related to epidermal growth factor (EGF), which Although SGF bound to EGF receptors, it was acted on the same cells to affect their transfor- distinguished from EGF (suggested to be the mation (Todaro et al. 1976) through a process nontransforming product of “normal” cells) later termed autocrine secretion (Sporn and To- by its ability to transform normal fibroblasts daro 1980). Along similar lines, the late Nobel in vitro in the soft agar colony-forming assay. laureate Robert Holley had proposed that one That the secreted factor both bound to EGF re- mechanism of acquisition of malignancy in- ceptors and could induce transformation was volved an increase in the availability of specific based on co-elution of these two activities using hormone (or growth factor) receptor sites on the molecular sieve chromatography (De Larco and cell membrane (Holley 1972) and that “trans- Todaro 1978). In 1981, however, it became ap- formed or malignant cells escape from normal parent through the work in the Harold Moses growth controls by requiring less of such hor- laboratory, then at the Mayo Clinic (Moses et al. mones or growth factors” (Holley 1975). The 1981), and the Michael Sporn and Anita Roberts transforming growth factors (TGFs) described laboratory at the National Cancer Institute by De Larco and Todaro (1978) shared proper- (Roberts et al. 1981), that the EGF receptorcom- ties with the v-src gene, in that they were able to peting activity could be separated from the soft cause normal fibroblasts to form progressively agar colony-forming activity. Both groups pur- growing colonies in soft agar (anchorage-inde- sued purification and characterization of factors pendent growth), a property that was closely with seemingly different transforming proper- associated with the transformed phenotype in ties, dependent on the different cell lines used in vivo (Todaro et al. 1979). In contrast with v-src their assays. These factors turned out to be the and other oncogenes, however, transforma- same TGF-b. tion did not result from cell-intrinsic, genetic At the present time, TGF-b is seen as the changes, but from secreted factors that did not prototype of a large family of secreted, dimeric affect the genotype. The term TGF was chosen growth factors and cytokines, encoded in hu- for this activity because of the induction of a mans and mice by 33 genes. TGF-b family pro- transformed phenotype, in both monolayer teins are now known to be critically involved in and soft agar cultures, of nontransformed cells, embryonic development, cell and tissue differ- and because of the assumption, later proven to entiation, tissue homeostasis in the adult, and be incorrect (see below), that the factor was pro- in many disease states, as detailed in at least duced only by transformed (cancer) cells. This 70,000 publications at this point. This review behavior was reversible on removal of the source will cover the early history of TGF-b in the con- of TGFs, leading to the suggestion that these text of general scientific knowledge at the time. factors acted as proximal effectors of transformation. Considering the highly visible discovery of oncogenes as the genetic basis of malignant PURIFICATION AND CHARACTERIZATION transformation around that time, the notion of OF TGF-b reversible transformation bysecreted factors was Approach of the Sporn/Roberts Laboratory met with skepticism. In the late 1970s, the Sporn laboratory focused on the role of retinoids as chemopreventive DISCOVERY OF TGF-b agents. Based on the identification by the To- The TGF-b field began with three papers, one daro group of peptides thought to be proximal published in 1978 and two in 1981. First, De effectors of transformation, the two laboratories Larco and Todaro (1978) described the partial entered a collaboration to design a large-scale purification of polypeptide growth factors se- isolation of the transforming peptides with the

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Historical Perspective of TGF-b

ultimate goal of obtaining insights into the sensitive than the detection of TGF-b protein mechanisms of chemoprevention attributed to on a silver-stained gel. Based on this advice, the the retinoids. Because previous attempts at pu- Sporn/Roberts laboratory used normal tissues, rification of SGF were based on the use of se- including bovine kidneys, human placenta, and rum-free medium conditioned by MSV-trans- platelets, all available in larger quantities than formed cells whose quantity was rather limited, transformed cells, for purification of TGF-b. the first breakthrough was the identification of a The formation of colonies of NRK cells in soft method that could be used to extract SGF di- agar in the presence of EGF (typically a 7-day rectly from tumor cells. Using an acid–ethanol assay) was used to monitor the purification, extraction procedure, previously described for with image analysis to quantify the number of the isolation of insulin from tissues and blood colonies above a designated size limit. Purifica- (Davoren 1962), and the formation of colonies tion from bovine kidneys (Roberts et al. 1983a) in soft agar of “normal” NRK-2B clone 49F or human placenta (Frolik et al. 1983) required (NRK) fibroblasts as an assay, SGF-like activity very large-scale preparations and several chro- was isolated from a variety of both virally or matography steps (Fig. 1), whereas purification chemically transformed cells and tumors (Ro- from human platelets (Assoian et al. 1983), berts et al. 1980). In attempts to purify SGF to which contain 100-fold greater activity, re- homogeneity by successive chromatographic quired only two chromatography steps to steps, it was noted, as mentioned, that the soft achieve homogeneity (Fig. 1). The colony-form- agar colony-forming response of NRK cells was ing activity in the assay depended on the addi- lost, but could be restored by adding EGF to the tion of EGF,confirming that these TGFs met the assay (Roberts et al. 1981; Anzano et al. 1982). definition of a TGF-b used by this laboratory. This led to the important finding that SGF, as The ED50 (half-maximal effective dose) of puri- well as colony-forming activities with similar fied TGF-b in the NRK colony-forming assay biochemical properties isolated from a variety was 2–3 pM in the presence of EGF.Significantly, of non-neoplastic mouse, bovine, and human the finding that TGF-b could be isolated from tissues, were actually comprised of two synergiz- non-neoplastic tissues and especially from ing activities. One of these provided EGF recep- platelets (Childs et al. 1982; Assoian et al. tor binding and was subsequently assigned to 1983), strongly suggested normal physiological TGF-a, a member of the EGF family of growth roles for this molecule, as subsequentlyshown in factors, whereas the other one, subsequently wound healing (Sporn et al. 1983) and develop- named TGF-b, did not bind to EGF receptors, ment (Heine et al. 1987), and later in diseases but was required to induce phenotypic transfor- such as fibrosis and cancer. mation in combination with EGF.An interesting anecdote is that the assignment of “a” and “b” Approach of the Moses Laboratory to these two TGFs was based on Todaro’s in- sistence that TGF-a, which he thought was In the late 1970s, the Moses group was interest- uniquely expressed by tumor cells, represented ed in the molecular changes associated with the dominant transforming activity and that transformation of cells by chemical carcinogens. TGF-b, then known to be expressed in many As was typical at the time, murine embryonic normal tissues, served merely to modulate the fibroblast cell lines served as model systems; process. methods for culturing nontransformed epithe- Advice from the late Nobel laureate Chris- lial cells were not yet widely available. The Moses tian Anfinsen was to “think big” so that a puri- laboratory had shown that many of the pheno- fication scheme would ultimately yield suffi- typic alterations observed in the chemically cient quantities of material for amino acid transformed AKR-MCA and C3H/MCA-58 sequencing and not simply show an “activity.” cell lines could be accounted for by continuous This was especially relevant because the soft growth factor stimulation (Moses et al. 1978, agar colony-forming assay was 100-fold more 1979). His group further found that the trans-

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Scale of purification of TGF-β in the Sporn laboratory

Bovine kidney Human platelets

Starting material 2 kg 25 units Acid/ethanol extraction 8 L 100 mL Precipitation: ether/ethanol 32 L/16 L 400 mL/200 mL Bio-Gel-P-60 column in Apply 2 L/80 L column Apply 10 mL/2 L column 1 M acetic acid 1 L fractions 5 mL fractions

Bio-Gel-P-60 column in No 0.5 mL/170 mL column 1 M acetic acid/8 M urea 0.5 mL fractions

Cation exchange Ye s No HPLC Bondapak C18 Ye s No HPLC Bondapak CN Ye s No

Final yield 6 mg TGF-β1 10 mg TGF-β1

Amino-terminal sequencing: ALDTNYCFSSTEKNC ...... Amino acid composition (nine cysteines per chain)

Figure 1. Purification to homogeneity of transforming growth factor b (TGF-b) from bovine kidney and human platelets. Human platelets contain 100-fold greater amounts of TGF-b than most other normal tissues. The scale of purification from bovine kidneys (or placenta) was large, requiring volumes of solvents unlikely to be used in laboratories today, and requiring four chromatography steps. In contrast, purification of TGF-b to homogeneity from human platelets was achieved using just two chromatography steps on Bio-Gel P-60. These large-scale purification schemes enabled the first determination of the amino-terminal sequence and amino acid composition of TGF-b1. HPCL, High-performance liquid chromatography.

formed cells had greatly diminished detectable in the soft agar assay was not required to obtain EGF receptors relative to the nontransformed maximum colony–stimulating activity of AKR- precursor cells, similar to the observations of 2B cells. This caused confusion leading to the De Larco and Todaro (1978) with MSV-trans- assumption that the TGF identified by the formed cells. As a result, the Moses laboratory Moses group might be different from that iden- pursued identification of SGF-like transforming tified by the Sporn group, even to the extent that activities by the chemically transformed cells. three types of TGFs were proposed to exist, that They used nontransformed AKR-2B fibroblasts, is, TGF-a, TGF-b, and TGF-g (Roberts et al. the progenitors of the chemically transformed 1983b). However, with exchange of reagents be- AKR-MCA cells, as indicator cells, and this was tween the two laboratories, it was soon apparent different from the assays used by the Todaro and that the requirement for EGF in the transforma- Sporn/Roberts laboratories, which used rat tion assay reflected a difference among the re- NRK fibroblasts. Soft agar–stimulating activity porter cell lines, with EGF needed for stimula- with a molecular sieve chromatography profile tion of colony formation by NRK but not for similar to that reported by De Larco and Todaro AKR-2B cells. was detected in medium conditioned by the The Moses group proceeded to identify chemically transformed cells (Moses et al. TGF-b activity in mouse embryos (Proper 1981). Additional purification using ion ex- et al. 1982) and, more important, in serum change chromatography separated the EGF (Childs et al. 1982). It was further shown that receptor–competing activity from soft agar plasma lacked TGF-b activity and that the TGF growth–stimulating activity. However, in con- activity in serum derived from platelets, which trast to the findings of the Roberts group using are an abundant source for purification of TGF- NRK cells (Roberts et al. 1981), addition of EGF b (Childs et al. 1982). Platelets, as well as re-

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Historical Perspective of TGF-b

combinant protein, are now the source of much tor-related protein (LRP-1) (Huang and Huang of the commercially marketed TGF-b1. 2005). Additionally, the endothelial cell-surface protein endoglin was also shown to act as a TGF-b-binding co-receptor, of particular im- Competitive Binding Assays Identify High- portance in endothelial cells (Cheifetz et al. Affinity Cell-Surface Receptors 1992). The first evidence for a high-affinity TGF-b receptor was provided by Joan Massague´ in col- DISCOVERY OF THE GROWTH-INHIBITORY laboration with Todaro (Massague´ et al. 1982). EFFECTS OF TGF-b Using preparations of SGF that were later shown to contain both TGF-b and TGF-a, they iden- The discovery of growth inhibition by TGF-b tified, by affinity cross-linking, a high-affinity began with a publication in 1978 by Robert Hol- 60-kD band, in addition to the 140- to 170- ley (Holley et al. 1978). Following receipt of the kD EGF receptor. Unlabeled EGF competed Nobel Prize in Physiology or Medicine in 1968 with 125I-labeled SGF binding to the latter for work on transfer RNA and the genetic code, band, but not the 60-kD band. The first com- Holley moved to the Salk Institute and redirect- petitive binding assays with Scatchard analyses ed his efforts to growth control. He showed that for TGF-b receptors were reported in 1984 by BSC-1 cells, derived from African green monkey the Sporn/Roberts and Moses laboratories kidney tissue, secreted a peptide growth inhib- working independently (Frolik et al. 1984; itor, and proceeded to purify this growth inhib- Tucker et al. 1984a). Both groups found very itor from medium conditioned by the BSC-1 high-affinity binding sites, with dissociation cells. By early 1984, the Moses laboratory had constants of 25–40 pM and 10–30 receptors derived data indicating that purified platelet- per cell. Subsequently, Massague´ performed derived TGF-b could inhibit proliferation of competitive binding assays with similar results. AKR-2B cells in monolayer culture. After wit- Using affinity cross-linking, his laboratory nessing at a seminar that the BSC-1 growth in- showed that there were three TGF-b binding hibitor presented itself on a sodium dodecyl species that are now known as the type I and sulfate (SDS)-polyacrylamide gel as a 25-kD type II receptors, and the type III (co)receptor, protein band under nonreducing conditions better known as betaglycan (Massague´ and Like and a 12.5-kD band under reducing conditions, 1985). The development of competitive binding the Moses group hypothesized that the BSC-1 assays for TGF-b was critical in the demonstra- growth inhibitor may well be the same as TGF- tion that TGF-bs are inhibitors of cell prolifer- b. A collaboration was established with Holley ation, particularly in epithelial cells. resulting in the exchange of reagents. It was In addition to the type I and type II recep- shown that platelet-derived TGF-b and BSC-1 tors, and the co-receptor betaglycan, other pu- growth inhibitor had similar biological activi- tative TGF-b receptors were reported, including ties in stimulating colony formation of AKR-2B a 70- to 74-kD band identified by 125I-TGF-b cells in soft agar, inhibiting proliferation of affinity cross-linking in GH3 pituitary cells, AKR-2B cells as well as BSC-1 and Mv1Lu cells which was reported to interact with TGF-b,ac- in monolayer culture, and competing compara- tivin, and inhibin (Cheifetz et al. 1988). A 38- bly for binding of TGF-b in the TGF-b receptor kD binding component was identified by simi- assay (Tucker et al. 1984b; Moses et al. 1985). lar means on choriocarcinoma cells (Mitchell The Sporn/Roberts group at this same time et al. 1992). These and other putative TGF-b- had noted decreased proliferation of a number binding “receptors” received little attention of cancer cell lines in soft agar with the addition subsequent to the initial reports. On the other of TGF-b, assuming that this decrease was the hand, the cell membrane TGF-b-binding com- result of toxicity related to impurities in the ponent called TGF-b type V receptor was later TGF-b preparation. Following the presentation identified as the low-density lipoprotein recep- of some of the growth-inhibition data by Moses

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at a national meeting in the spring of 1984, the was by Silberstein and Daniel (1987). They in- Sporn/Roberts group concluded that the de- serted slow-release pellets containing TGF-b creased cell proliferationwasthe result of growth into developing mouse mammary glands and inhibition by TGF-b (Roberts et al. 1985). They observed marked inhibition of mammary duc- also showed that the effects of TGF-b on anchor- tal growth and morphogenesis. They further age-independent growth of cells could be mod- showed that this growth inhibition was revers- ulated by other growth factors. As an example, ible. Subsequently, systemically administered Myc-1 cells formed large colonies in the pres- TGF-b1 was shown to inhibit the proliferative ence of EGF, and formation of these colonies response of liver to partial hepatectomy (Russell was strongly inhibited by addition of TGF-b. et al. 1988). Additionally, transgenic mice over- In contrast, identical concentrations of TGF-b expressing TGF-b1 in epithelial tissues also enhanced colony formation of these same cells, showed inhibition of proliferation in the mam- when in the presence of platelet-derived growth mary gland (Jhappan et al. 1993; Pierce et al. factor (PDGF), which by itself had almost no 1993) or skin (Cui et al. 1996; Fowlis et al. 1996). colony-forming activity. Moreover, in monolay- Numerous later studies involving transgenic ex- erculture, TGF-b inhibited the growth-promot- pression of TGF-b, dominant-negative type II ing effects of PDGF on these same cells, empha- TGF-b receptors and conditional knockout of sizing the highly contextual effects of TGF-b type II receptor expression supported the role of (Roberts et al. 1985). TGF-b as an endogenous negative regulator of It was then also important to evaluate proliferation of several cell types, including ep- whether TGF-b could inhibit the proliferation ithelial, endothelial, and hematopoietic cells. of normal cells in culture. Primary cultures of By the late 1980s, many groups suspected human foreskin keratinocytes were shown to be that, even though TGF-b expression was shown growth inhibited by TGF-b in a reversible man- to be up-regulated in cancers (Derynck et al. ner, and to be arrested in the G1 phase of the cell 1987), the TGF-b pathway may have a tumor- cycle. In contrast, a squamous cell carcinoma suppressive function because of its ability to in- cell line, derived from epithelial cells, was not hibit cell proliferation. Thus, inactivation of the inhibited by TGF-b (Shipley et al. 1986). Addi- growth-inhibitory response to TGF-b would tionally, the Sporn/Roberts and Moses labora- then initiate malignant transformation. Howev- tories showed that several carcinoma cell lines er, solid evidence for tumor suppression by were growth-inhibited by TGF-b, whereas oth- TGF-b signaling was not reported until 1995. ers were not (Moses et al. 1985; Roberts et al. Data supporting TGF-b’s tumor suppressor 1985). Numerous subsequent studies from sev- role were derived from transgenic mouse studies eral laboratories showed that TGF-b could in- (Pierce et al. 1995) and through identification of hibit a wide variety of epithelial cell types in inactivating mutations in the TGF-b type II re- culture, and that, in general, highly malignant ceptor in human colon carcinoma cells with mi- carcinoma cells had lost the growth-inhibitory crosatellite instability (Markowitz et al. 1995). response. The notion that a growth factor could Subsequent studies of genetically modified mice inhibit cell proliferation was at that time sur- and mutations in genes encoding TGF-b signal- prising and counterintuitive. Indeed, numerous ing mediators in human cancers came to sup- growth factors had been identified in the 1970s port a major role for the TGF-b pathway in tu- and early 1980s, based on their abilities to stim- mor suppression (reviewed in Akhurst and ulate cell proliferation, thus equating growth Derynck 2001). Additionally, however, a sub- factors with growth stimulation. Additionally, stantial body of evidence arose that TGF-b sig- transformation was inherently linked to in- naling facilitates tumor progression through ef- creased cell proliferation. The notion of auto- fects on the carcinomacells and on host cells and crine control of growth inhibition was novel. tumor stroma (reviewed in Derynck et al. 2001; The first demonstration that TGF-b could Massague´ 2008; Ikushima and Miyazono 2010; inhibit proliferation of epithelial cells in vivo Pickup et al. 2013).

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COMPLEMENTARY DNA (cDNA) CLONING braries eventually led to a cDNA sequence of AND CHARACTERIZATION OF PRE-PRO- 2439 bp corresponding to the TGF-b precursor TGF-b mRNA (Derynck et al. 1985). The deduced polypeptide sequence established that the puri- cDNA Cloning of TGF-b1 fied biologically active TGF-b, now known as Searching for a new research area of interest, Rik TGF-b1, was the carboxy-terminal 112 amino Derynck, then at Genentech, became interested acid segment of a 390 amino acid precursor in the newly discovered TGF activity that was protein with an amino-terminal signal sequence described to him by George Todaro over a pizza and a long pro-sequence (Fig. 2). It was pro- dinner at Frederick, Maryland, early in 1983. posed, and subsequently verified, that the After all, the induction of reversible transforma- TGF-b monomer was cleaved from its precur- tion by a secreted factor specifically made by sor following a tetrabasic site, Arg-His-Arg-Arg. tumor cells, even though controversial, was a Later studies showed that furin, a member of the remarkable observation, and, if indeed TGF ex- mammalian convertase family of endopro- ists, then one must be able to clone it. Although teases, is the likely protease required for process- Derynck focused his efforts on the cDNA cloning TGF-b1 in vivo (Dubois et al. 1995). Sub- ing of TGF-a (Derynck et al. 1984), Dave Goed- sequent cDNA cloning of TGF-b1 from mouse del, the head of the department, pointed him (Derynck et al. 1986), pig (Derynck and Rhee toward a recent paper from the Sporn/Roberts 1987), and monkey (Sharples et al. 1987) cells laboratory (Roberts et al. 1983a), biochemically showed that the protein was highly conserved, showing that TGF-b was a dimeric protein, un- that mature human and simian TGF-b1 differed like TGF-a, with an amino-terminal sequence by only one amino acid from murine TGF-b1, that was unrelated to TGF-a. Derynck first did and that human and porcine TGF-b1 were not want to deal with it, being too busy with identical. cDNA cloning of TGF-b and subse- TGF-a cDNA cloning, but then contacted the quent northern hybridizations also revealed Sporn group to affirm his interest in cloning that, unlike other growth factors that were typ- TGF-b, which led to a formal collaboration. ically expressed in specific cell types, TGF-b1 The Sporn/Roberts laboratory provided suffi- mRNA was synthesized by almost all normal cient quantities of TGF-b isolated from human and tumor cells examined, yet at higher levels platelets (Assoian et al. 1983) for clostripain by tumor cells (Derynck et al. 1985,1987), al- digestion, eventually resulting in the identifica- luding to the broad spectrum of activities that tion of a 55-residue-long contiguous amino- were to be discovered. The cDNA cloning of terminal amino acid sequence. Because the TGF-a and TGF-b, first presented at a Cold polymerase chain reaction had not yet been de- Spring Harbor meeting in September 1984, scribed (Mullis et al. 1986), a newly developed led to the acceptance that TGFs did exist. approach using long oligonucleotides as hybridization probes was used. As reliable amino Concept of Growth Factor Latency acid sequence runs became available, two 44- residue-long oligonucleotides, designed based Even before TGF-b was cloned, it was seen as on a stretch of TGF-b sequence and the known peculiar that TGF-b was secreted from cells in a bias of human codon usage, and a set of 16 14- biologically inactive form or complex that re- mers complementary to all codons for amino quired activation by acidification (Lawrence acids 13–16 were used as hybridization probes et al. 1984; Pircher et al. 1984). This had initially against a human genomic DNA library. This led not been noted because the isolation procedure to the identification of an exon encoding amino started with the use of acid in the extraction, and acids 10–60 of mature TGF-b that then was subsequent purification steps were also per- used to screen a lgt10-based placenta cDNA formed under acidic conditions. Additional ear- library, and yielded a partial cDNA. Extensive ly studies showed that latent TGF-b could be screening of placenta and tumor cell cDNA li- activated not only by extremes of pH, but also

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A 390 amino acids

NH RHRR COOH 2 * ** ***## **

Signal (Latency-associated peptide, LAP ) Mature TGF-β peptide 249 amino acids 112 amino acids

II Acid pH + II α β v 6 Tsp Active Heat TGF-β Latent complex Proteases, LAP etc. Figure 2. Structure and function of pre-pro-transforming growth factor b (TGF-b). (A) Complementary DNA (cDNA) cloning of TGF-b showed that it derives from a large precursor with a signal peptide, the large pro- segment (latency-associated protein [LAP]), and the carboxy-terminal mature biologically active protein (in dimeric form). Large green arrows show processing sites, black bars and asterisks show the position of cysteine residues, and # shows two adjacent cysteine residues. The pattern of cross-linking of the four intrachain disulﬁdes is shown in brackets. Red arrows (down) show the position of the interchain disulﬁde bridges, whereas a green arrow (up) shows the cysteine that links LAP to latent TGF-b-binding protein (LTBP).A blue underscore shows the position of glycosylation sites and a red underscore shows the position of the Arg-Gly-Asp (RGD)- integrin-binding sequence present in the LAPs of TGF-b1 and TGF-b3 but not the TGF-b2 LAP.(B) TGF-b is secreted from cells in a biologically inactive form (latent TGF-b), with noncovalent association of the dimeric LAP with the dimeric mature carboxy-terminal 112 amino acid TGF-b. Latent TGF-b can be activated by a variety of treatments in vitro resulting in dissociation of the LAP protein from mature TGF-b, thus unmasking its receptor-binding epitopes.

by chaotropic agents, boiling, binding to a2- a member of the fibrillin family of proteins, to macroglobulin, or proteolysis using one of sev- direct extracellular localization of the latent eral proteases (Lawrence et al. 1985; O’Connor- complex (Miyazono et al. 1988, 1991; Wakefield McCourt and Wakefield 1987; Lyons et al. 1988; et al. 1988; Kanzaki et al. 1990; Tsuji et al. 1990). Sato et al. 1990). Moreover, because most cells The extensive sequence divergence among the can respond to TGF-b, physiological activation three mammalian TGF-bs and the existence of of this latent complex was increasingly seen as a four LTBPs suggest differences in extracellular key regulatory step in the control of its biolog- targeting and activation mechanisms. ical activity. The latency of TGF-b was subsequently revealed by Larry Gentry and Tony Expression of Recombinant TGF-b Purchio, and by Kohei Miyazono, then working with Calle Heldin, as well as Lalage Wakefield Recombinant expression of TGF-b was reported and Roberts, to result from noncovalent associ- 2 years after it was cloned (Gentry et al. 1987). ation of the mature TGF-b homodimer with the Latent pre-pro-simian TGF-b was expressed in large dimerized pro-segment that has been re- Chinese hamster ovary (CHO) cells, treated named “latency-associated protein” (LAP), thus with methotrexate to amplify the coexpressed preventing binding of TGF-b to its receptors dihydrofolate reductase gene. One CHO clone (Gentry et al. 1987; Miyazono et al. 1988; Wake- showed TGF-b secretion exceeding by 50-fold field et al. 1988). This “small latent complex” the levels naturally secreted by cultured cells. was also shown to bind to yet another and larger The secreted recombinant TGF-b was latent, protein, latent TGF-b-binding protein (LTBP), requiring acidification for biological activity.

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This observation led to the hypothesis that the showed differential binding efficiencies to the precursor sequences might play a role in the then putative three TGF-b receptor types, iden- activation process. Studies of recombinant tified as the 65-kD type I receptor, the 85-kD TGF-b expression confirmed the processing type II receptor, and the 280-kD type III recep- sites of the signal peptide and the mature tor or betaglycan. As later confirmed, once these TGF-b that were predicted from the cDNA clon- TGF-b receptors had been cloned, TGF-b2 ing of the TGF-b precursor (Gentry et al. 1988). bound only poorly to the type II TGF-b recep- These studies also showed that the precursor tor, but bound to betaglycan with similar was cross-linked by disulfide bonding and likely affinity as TGF-b1 (Cheifetz et al. 1987; Lo- to function as a dimer, and that glycosylation of pez-Casillas et al. 1993). Independent studies TGF-b was restricted to the precursor domain in other laboratories identified TGF-b2asa (Fig. 2). Subsequent complementation assays cartilage-inducing activity from bovine bone showed that the TGF-b prosequence was also (Seyedin et al. 1987), a T-cell suppressor activity required for proper folding, disulfide forma- made by human glioblastoma cells (Wrann et al. tion, dimerization, and secretion of mature 1987), and a growth inhibitor secreted by BSC-1 TGF-b (Gray and Mason 1990), and thus acts monkey kidney cells (Hanks et al. 1988). Sub- as a chaperone. Because TGF-b was shown not sequent cDNA cloning showed that TGF-b2 de- to be glycosylated, attempts were made to ex- rives from a 412 amino acids precursor, and that press the mature form of TGF-b in bacteria its mature 112 amino acid sequence is 71% (Tuan et al. 1996). Bacterially expressed mature identical to TGF-b1 (de Martin et al. 1987; TGF-b was inactive, confirming the need of the Marquardt et al. 1987). precursor domains to assure proper disulfide Shortly after the characterization of TGF- bonding of the mature TGF-b, and required re- b2, cDNA cloning identified yet a third TGF- naturation using the glutathione-redox method b, called TGF-b3, which was 72% identical to to endow some biological activity. TGF-b1 and 76% identical to TGF-b2 (Der- ynck et al. 1988; ten Dijke et al. 1988). All nine cysteines in the 112–amino acid mature TGF-b IDENTIFICATION OF TGF-b HOMOLOGS forms were positionally conserved in the three TGF-bs. TGF-b3 was found to play a unique Three TGF-bs role in fusion of the palatal shelves (Proetzel Following the identification of platelets as the et al. 1995) and, unlike TGF-b2, to bind the most concentrated source of TGF-b, R&D Sys- type II TGF-b receptor with similar affinity as tems (Minneapolis, MN) chose porcine plate- TGF-b1. Although the three mature TGF-bs lets to generate commercially available TGF-b, show 64%–82% sequence identity, their pro- which made sense considering that porcine and domains are only 28%–45% identical, yet human TGF-b had identical sequences (Der- some sequence features are conserved. The three ynck and Rhee 1987). They worked with the prodomains have conserved N-linked glycosyl- Moses laboratory, then at the Mayo Clinic, to ation sites, and, with the exception of the TGF- learn purification techniques based on the b2 pro-domain, an Arg-Gly-Asp (RGD) poten- method published by Assoian et al. (1983). Sur- tial integrin binding site is apparent. In addi- prisingly, they identified three peaks of activity, tion, each pro-region has three cysteines, two of subsequently found to be homodimeric TGF- which are involved in interchain disulfide bonds b1, homodimeric TGF-b2 and a heterodimer, necessary for dimerization of the pro-region in TGF-b1:TGF-b2 (Cheifetz et al. 1987). Al- the small latent TGF-b complex, whereas the though TGF-b2 had the identical size and bio- third cross-links to its binding protein LTBP logical activity of TGF-b1 in growth inhibition (Fig. 2). Mammalian cells encode only these assays, it had a distinct amino-terminal amino three TGF-bs, encoded by three distinct genes acid sequence and was not recognized by anti- (Table 1). Although the term TGF-b isoforms is bodies raised against TGF-b1 sequences. It also often used, these three TGF-bs are not to be

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Table 1. Comparative features of the TGF-b isoforms Number of amino acids Chromosomal locus

Precursor LAP Mature Tetrabasic site Human Mouse mRNA (kb) TGF-b1 390 249 112 RHRR 19q13 7 2.5 TGF-b2 414,442a 281 112 RKKR 1q41 1 4.1,5.1,6.5 TGF-b3 412 279 112 RKKR 14q23-4 12 3.0 aTGF-b2 has a prominent alternative spliced variation encoded by the 5.1-kb mRNA and is expressed only in certain tissues. A 29-amino acid insertion results in a latency-associated protein (LAP) with three additional cysteine residues, suggesting a more complex secondary structure (Webb et al. 1988).

considered as isoforms derived from the same in Drosophila was shown to encode a precursor gene, but are encoded by different genes. with a carboxy-terminal TGF-b-related sequence, demonstrating the evolutionary ancestry of TGF-b proteins, and the first evidence The TGF-b “Superfamily” that TGF-b-related proteins direct develop- Shortly after the cDNA cloning of TGF-b1, and mental patterning (Padgett et al. 1987). Later at the time that TGF-b2 and -b3 were identified, that year, Vg1 mRNA, which localizes to the unrelated cDNA cloning projects in other labo- vegetal hemisphere of Xenopus eggs, was also ratories revealed the existence of other large pre- shown to encode a TGF-b-related protein as cursors with sequence homology with TGF-b in the carboxy-terminal fragment of a larger pre- their carboxy-terminal sequences and conser- cursor, further expanding the TGF-b family, vation of the last seven of TGF-b’s nine cys- and further drawing attention to regulation of teines. Structural analyses later revealed that development, in this case mesoderm formation, the positioning of these seven cysteines, which by TGF-b-related proteins (Weeks and Melton characterizes the class of TGF-b-related pro- 1987). Finally, the characterization, late in teins, defines the “cystine knot” in the mature 1988, of the cartilage- and bone-inducing forms of these molecules (McDonald and Hen- “bone morphogenetic protein (BMP),” isolated drickson 1993). The first two proteins so iden- from bone, resulted in the characterization of tified were the two polypeptides that form the cDNAs encoding the TGF-b-related BMP-2A disulfide-linked inhibin heterodimer, an inhib- (now known as BMP-2) and BMP-3, which in- itor of follicle-stimulating hormone secretion duced cell differentiation, and formed the basis (Mason et al. 1985). The alignment of the in- for the discovery of additional BMPs (Wozney hibin cDNA-encoded polypeptides, isolated by et al. 1988). Tony Mason in the Seeburg laboratory at Gen- The rapid succession of reports identifying entech, with the TGF-b1 cDNA sequence fol- TGF-b-related proteins, the revelation that they lowed a random evening encounter at Genen- function in very diverse contexts and have mul- tech, in which Mason discussed with Derynck tiple developmental roles in patterning, mor- the difficulties in cloning inhibin cDNAs that phogenesis, and cell differentiation, and their seemed to resemble similar frustrations with the evolutionary ancestry and conservation pro- TGF-b1 cDNA cloning. Half a year later, the foundly affected many fields, most notably de- cDNA-derived sequence for the Mu¨llerian-in- velopmental biology and cancer biology. These hibiting substance (MIS), which causes Mu¨l- discoveries also generated enthusiasm for the lerian duct regression in development, revealed notion that TGF-b-related proteins form a again a large precursor sequence with a car- “superfamily” encoded by a “super gene family.” boxy-terminal, TGF-b-related sequence (Cate However, considering the similar organization et al. 1986), and the first role for a TGF-b-relat- of all encoded protein sequences as large pre- ed protein in development. Again, half a year cursors with a signal peptide, a large pro-seg- later, the DPP-C locus, which directs patterning ment and a carboxy-terminal mature TGF-b-

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related sequence, the TGF-b-related proteins Given that TGF-b was expressed by many constitute a family that, by analogy with other cell types found in all tissues examined and classes of proteins, does not meet the definition abundant in platelets, it was evident that the of a superfamily that groups proteins with di- molecule must have physiological functions dis- vergent organization. We now know that the tinct from its transforming activity and roles in mouse and human genomes have 33 genes en- cancer. Early in vivo experiments in the Sporn/ coding precursor monomers of TGF-b-related Roberts laboratory were based on the adage of proteins. With the existence of homodimeric Alexander Haddow (1972) that “the wound is a and heterodimeric TGF-b-related proteins, the tumor that heals itself,” restated by Dvorak number and diversity of homo- and heterodi- (1986) as “tumors are wounds that do not meric combinations with functional roles re- heal,” thus inviting the evaluation of a TGF in mains unclear. TGF-b family proteins play wounds. Release of TGF-b from Hunt–Shilling diverse roles in the control of cell proliferation, wire-mesh chambers implanted into the backs metabolism, and differentiation, and act as of rats profoundly stimulated a stromal re- potent and often essential regulators of devel- sponse, including angiogenesis and ECM de- opmental processes. TGF-bs, BMPs, and acti- position (Sporn et al. 1983). Injection of TGF- vins (inhibin polypeptide homodimers) also b into the nape of the neck of newborn mice emerged as regulators of disease pathogenesis confirmed that TGF-b stimulated formation of and progression. granulation tissue, was chemotactic for inflammatory cells, and stimulated ECM production by fibroblasts (Roberts et al. 1986), thus dem- THE EXPANDING BIOLOGICAL ACTIVITIES onstrating that it was active in multiple aspects OF TGF-b of wound healing. These observations were con- TGF-b research in the latter half of the 1980s firmed and expanded in many follow-up studies was characterized by discoveries of a plethora using models of impaired healing (reviewed in of biological effects on cells in culture. With Roberts 1995). the commercial availability of TGF-b, it had A molecular basis for these effects on wound become apparent that most if not all cell types healing and connective tissue deposition was responded to TGF-b, while they also made and provided by studies in cell culture (Fig. 3). secreted TGF-b, thus allowing for autocrine TGF-b was shown to strongly induce the expres- stimulation. The many effects seen included sion of ECM components, including fibronec- regulation of cell proliferation, cell differentia- tin, certain collagens, chondroitin/dermatan tion, extracellular matrix (ECM) production and remodeling, chemotaxis, lymphocyte function, and growth factor and hormone produc- Major biological responses and diseases β tion. By the mid-1980s, it was widely accepted regulated by TGF-

that TGF-b could either stimulate or inhibit cell Wound healing Inhibits proliferation Regulates apoptosis proliferation depending on the cell type, and Fibrosis Regulates differentiation presence of other growth factors and culture Carcinogenesis conditions (discussed above). TGF-b also in- Regulates immune cell function duced many other cell responses that appeared Stimulates accumulation of ECM to also depend on the cell type and culture con- Promotes chemotaxis ditions. This led to an oversimpliﬁed and cari- catural notion among some that TGF-b acted as Figure 3. Roles of transforming growth factor b a switch: It switched on what was off, and (TGF-b) in pathophysiology. TGF-b plays prominent roles in wound healing, ﬁbrosis, and carcino- switched off what was on. In addition to being genesis, as well as a host of other diseases. Listed biologically confusing, this notion did not set on the right are various TGF-b-dependent cellular an inviting stage to explore therapeutic modal- mechanisms that contribute to its effects. ECM, Ex- ities using TGF-b or TGF-b antagonists. tracellular matrix.

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sulfate, biglycan, decorin, osteopontin, osteo- and Leydig cells, while it was stimulated in gran- nectin, tenascin, and thrombospondin (Mas- ulosa cells. TGF-b was also shown to stimulate sague´ 1990). In addition, TGF-b stimulated expression of follicle-stimulating hormone by the synthesis of inhibitors of matrix-degrading pituitary cells (Ying et al. 1986), whereas other enzymes, including plasminogen activator in- studies revealed that TGF-b induces PDGF ex- hibitor 1 (PAI-1) and a tissue inhibitor of matrix pression by fibroblasts (Leof et al. 1986). Con- metalloproteinases (TIMPs) (Laiho et al. 1986; sequently, TGF-b was now additionally seen as Overall etal. 1989). Conversely,TGF-binhibited a mediator of hormonal control in different expression of matrix-degrading proteases such as contexts, consistent with the discovery that the collagenase, stromelysin, and plasminogen acti- hormone inhibin is a TGF-b-related factor. Fur- vator (Edwards et al. 1987; Kerr et al. 1988). thermore, it was now apparent that some activ- Thus, the increase in inhibitors and decrease in ities of TGF-b occurred secondarily through the matrix proteases caused increased ECM accu- activation of expression of some hormones and mulation. TGF-b treatment was also shown to growth factors. induce marked changes in integrin expression in Studies in the latter part of the 1980s also several cell types resulting in altered cell binding revealed that TGF-b has potent effects on dif- to ECM components (Ignotz and Massague´ ferentiation of a wide variety of cell types. This 1987; Roberts et al. 1988; Heino et al. 1989). stood in remarkable contrast with the “classical” Further, TGF-b revealed itself as potently che- growth factors, whose effects on cells were pri- motactic for fibroblasts (Postlethwaite et al. marily seen to enhance proliferation, and were 1987) and macrophages (Wahl et al. 1987), a thought of primarily in the context of cancers. function that likely contributes to the increased These effects on cell differentiation were espe- abundance of these cells at sites of increased cially remarkable considering TGF-b’s link to TGF-b production such as wounds and cancers. cell transformation. TGF-b was often seen to Around that time, collaborative studies be- inhibit cell differentiation, for example, of pre- tween the laboratories of Sporn/Roberts and adipocytes, skeletal muscle myoblasts, muscle Toni Fauci at the National Institutes of Health satellite cells, and osteoblasts, conceptually revealed that TGF-b could inhibit the differen- resembling inhibitory effects of TGF-b on im- tiation and functions of B and T lymphocytes as mune cell functions. On the other hand, TGF-b well as natural killer cells (Kehrl et al. 1986a,b; stimulated differentiation of prechondrobasts, Rook et al. 1986). These unexpected findings intestinal epithelial cells, keratinocytes, bron- with a factor that was thought to play a major chial epithelial cells, and osteoblasts under role in cancers linked TGF-b to the immune some conditions. These findings led to the no- system and provided the basis for the many tion that TGF-b was a differentiation factor, in subsequent studies that highlight the roles of addition to a growth inhibitor, as summarized TGF-b as a potent immunoregulator, control- around that time by Massague´ (1990). These ling proliferation, differentiation, and function initial observations, together with the realiza- of most lymphocyte classes, as well as macro- tion that defects in developmental patterning phages and dendritic cells, and a potent sup- and tissue differentiation were associated with pressor of immune surveillance (Letterio and dysregulation of TGF-b-related proteins, set the Roberts 1998). These proposed roles of TGF-b stage for a wide range of studies that defined the were supported and in part elucidated by later developmental control of tissue differentiation studies using TGF-b1 knockout mouse (Shull by TGF-b and TGF-b family proteins. et al. 1992; Kulkarni et al. 1993) that further Expanding these observations on cell differ- characterized a role for TGF-b1 in autoimmu- entiation, the Derynck laboratory reported, in nity (Letterio et al. 1994) and in immune toler- 1994, that TGF-b induces epithelial cells in cul- ance (Letterio and Roberts 1998). ture to undergo a reversible transition into cells Additionally, steroidogenesis was reported with mesenchymal appearance (Miettinen et al. to be inhibited by TGF-b in adrenocortical cells 1994). Although this was consistent with obser-

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vations by Ray Runyan that TGF-b is required despite their very similar activities in cell cul- for transition of endocardial cells into mesen- ture, the three TGF-bs have highly divergent chymal cells during heart valve formation (Potts developmental roles. et al. 1991), these results were nevertheless met With TGF-b having been discovered as a with surprise, if not apprehension, primarily factor that induces cell transformation, and because of the overall notion that differentiation the overall notion in the 1980s that growth fac- could not be redirected, and certainly not by tors act predominantly in cancers, thus contrib- one factor. Epithelial–mesenchymal transition uting to cancer progression, many researchers (EMT) had been shown to occur in develop- aimed to elucidate roles of this newly discovered ment (Hay 1995), but the underlying signaling factor in cancer initiation and progression. As mechanisms were not known. The induction of with other growth factors, much emphasis was EMT by TGF-b in cell culture was the basis for on the control of cell proliferation by TGF-b, many subsequent studies characterizing mech- even though, and especially because, it rapidly anisms and roles of TGF-b-induced, Smad-me- became apparent that TGF-b has growth-inhib- diated gene reprogramming during EMT, and itory activities on epithelial, immune, and he- complementary roles of non-Smad pathways. matopoietic cells, which give rise to the bulk of As the roles of EMT in development, starting cancers. As the other activities of TGF-b became with mesoderm induction and gastrulation, be- apparent, researchers incorporated their results came more apparent, many studies gradually and views into a more complex picture of the revealed the developmental control of EMT by roles of the apparently increased TGF-b signal- TGF-b-related proteins (reviewed in Lamouille ing in cancers. Increased ECM protein ex- et al. 2014). pression in response to TGF-b was linked to The increasing realization in the late 1980s increased ECM turnover in cancers. The che- and early 1990s that TGF-b controls cell dif- motactic effects of TGF-b on fibroblasts and ferentiation was complemented by in situ hy- immune cells contributed to tumor stroma for- bridization and immunohistochemical studies mation and the tumor microenvironment. Lo- showing expression of all three TGF-bs during calized immunosuppression by TGF-b was seen embryogenesis, each of these in specific pat- to contribute to cancer growth and progression. terns, both spatially and temporally (Heine et TGF-b was also found to induce angiogenesis in al. 1987; Lehnert and Akhurst 1988; Pelton et al. cancers and to contribute to cancer-associated 1989, 1990a,b, 1991; Millan et al. 1991). These localized inflammation. Finally, EMT in re- observations suggested specific roles for the in- sponse to increased TGF-b signaling was individual TGF-bs in processes such as angiogen- creasingly viewed as a key contributing event esis, cardiogenesis, palate formation, and oste- to carcinoma invasion and dissemination, and ogenesis, and provided the basis for subsequent recently to the generation and properties of can- studies on developmental defects resulting from cer stem cells (Derynck et al. 2001; Massague´ targeted inactivation of the individual TGF-b 2008; Ikushima and Miyazono 2010; Katsuno genes in mice (Shull et al. 1992; Kulkarni et al. et al. 2013). 1993; Proetzel et al. 1995; Sanford et al. 1997). Besides cancer, other pathological connec- Although Tgfb12/2 mice highlighted the roles tions with dysregulated TGF-b production and of TGF-b1 in the control of the immune system responsiveness became gradually apparent. and inflammation (Shull et al. 1992; Kulkarni Most notably, the suppression of experimental et al. 1993), as well as angiogenesis, vasculogen- glomerulonephritis by TGF-b antiserum (Bor- esis, and hematopoiesis (Dickson et al. 1995), der et al. 1990) provided the impetus for many inactivation of TGF-b2 and TGF-b3 expression subsequent studies highlighting the contribu- primarily resulted in defects in organs that de- tions of increased TGF-b signaling to different velop through epithelial–mesenchymal tissue types of fibrosis. Again, increased TGF-b activ- interactions (Proetzel et al. 1995; Sanford et al. ity, TGF-b’s abilities to activate ECM protein 1997). These observations also revealed that, expression, its potent chemotactic activities

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for fibroblasts and immune cells, and the induc- cell surface (Frolik et al. 1984; Tucker et al. tion of EMT by TGF-b became incorporated in 1984a), when compared with average numbers the etiology and progression of fibrosis (Chap- for RTKs. Around 1987–1990, several laborato- man 2011; Nieto 2011). In addition to fibrosis, ries started projects aimed at cDNA cloning of several connective tissue and skeletal diseases the receptors, using receptor purification or ex- were also seen to result from increased TGF-b pression cDNA cloning approaches, in combi- signaling, which, in a number of cases, was as- nation with radiolabeled ligand-binding assays. sociated with genetic mutations that lead to The cDNA cloning of the first known recep- dysregulated TGF-b activation (Shore and Kap- tor for a TGF-b family protein was achieved by lan 2010; Doyle et al. 2012). Lawrence Mathews working in the laboratory of The sheer number and overwhelming vari- Wylie Vale(Mathews and Vale1991). An expres- ety of biological activities described for TGF-b sion cDNA cloning strategy in combination during the 1980s was unprecedented in the with 125I-activin binding to transfected cells al- growth-factor field, and attracted many investi- lowed him to isolate cDNAs for the activin type gators from different disciplines to the field. II receptor, now known as ActRII, noting that it Although this generated rapid progress with was likely a transmembrane serine/threonine new insights into the biology of TGF-b, one kinase. The cytoplasmic domain of this receptor negative aspect of so many biological effects showed extensive sequence similarity to the was that development of pharmacologic agents transmembrane protein daf-1 in Caenorhabditis to target and modulate TGF-b actions by indus- elegans, which had been cloned the year before, try was discouraged for a number of years be- and was proposed as the first transmembrane cause of the potential plethora of side effects. serine/threonine kinase receptor with a ligand still to be identified (Georgi et al. 1990). The identification of ActRII was rapidly followed cDNA CLONING AND IDENTIFICATION by the cDNA cloning of a related activin recep- OF TGF-b RECEPTORS AND SMADs tor, ActRIIB (Attisano et al. 1992; Mathews et al. 1992), and of alternatively spliced variants (At- cDNA Cloning of the TGF-b Receptors tisano et al. 1992). A similar expression cloning Once specific high-affinity receptors for TGF-b approach as for ActRII, but using 125I-TGF-b as had been identified in 1984 (Massague´ et al. ligand, resulted in the cDNA cloning of the 1982), the next goal was to characterize these TGF-b type II receptor, now known as TbRII, with the ultimate aim to define the TGF-b sig- by the laboratories of Harvey Lodish and Bob naling pathways that lead to the diversity of Weinberg (Lin et al. 1992). Also, this receptor emerging biological activities. Using growth- was shown to phosphorylate on serine and thre- factor-activated tyrosine kinase receptors as a onine. A few years later, the MIS binding type II reference point, it was rapidly concluded that receptor was cloned, based on its homology with the TGF-b receptors would act through a dif- previously isolated TGF-b family receptors ferent class of receptors, because they induced (Baarends et al. 1994; di Clemente et al. 1994), growth inhibition, rather than growth stimula- and the type II BMP receptor BMPRII was tion, and many other responses hitherto not cloned based on either its association with seen with receptor tyrosine kinases (RTKs). the type II TGF-b receptor in a yeast two-hybrid Furthermore, three types of receptors were con- approach (Kawabata et al. 1995; Liu et al. 1995) sistently identified, with both the type I and or sequence homology using a polymerase chain type II receptors required for TGF-b responses, reaction (PCR)-based strategy (Nohno et al. based on analyses of mutant cell lines (Laiho 1995; Rosenzweig et al. 1995). Five TGF-b fam- et al. 1991). This was again very different from ily type II receptors are now known to be en- RTKs. The task to purify or clone these recep- coded by human cells. tors would, however, be challenging considering cDNA cloning of the type I receptors was the very low levels of the TGF-b receptors at the accomplished in large part using PCR-based

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strategies with oligonucleotide primers for con- for signaling activation complemented the ex- served sequences in the ActRII, TbRII, and/or tracellular interdependence of the type II and Daf1 receptors (Ebner et al. 1993a; He et al. type I receptors for productive ligand binding. 1993; ten Dijke et al. 1993). This strategy result- Wecurrently know many of the functional com- ed in the identification of type I receptors with binatorial type II/type I receptor interactions overall similarity to the type II receptors, easily and the corresponding ligands that activate recognizable by their distinct Gly-Ser (GS)-rich these complexes, but, considering the complex- sequence immediately upstream of the kinase ity of homomeric and heteromeric ligands, ad- domains (Wrana et al. 1994a). As it was initially ditional assignments will be forthcoming. not clear what ligands they would bind, Peter Finally, the type III TGF-b receptors, beta- ten Dijke coined the term ALK for “activin re- glycan and endoglin, differ from the other TGF- ceptor-like kinase” for these receptors (ten b family receptors in the sense that they do not Dijke et al. 1993), although various other names directly initiate TGF-b signaling, but rather were chosen by other laboratories. Seven type I control ligand presentation to the type I and receptors, designated ALK1-7, are now known type II receptors (Bernabeu et al. 2009). Al- to be encoded by human cells (ten Dijke et al. though betaglycan is in vivo primarily expressed 2000; ten Dijke and Hill, 2004), whereas ALK-8 in mesenchymal cells, endoglin expression is re- was found in zebrafish (Bauer et al. 2001; stricted to endothelial cells. The proteoglycan Mintzer et al. 2001). With five type II and seven betaglycan was cloned by Fernando Lopez-Ca- type I receptors, and a much larger number of sillas, working with Joan Massague´, following ligands, the assignment of the type I receptors to protein purification and based on peptide se- individual ligands was, and continues to be, quences that allowed the generation of oligonu- complex and tedious. It was proposed that the cleotides as hybridization probes (Lopez-Casil- type II and type I receptors formed a hetero- las et al. 1991), and by Xiao-Fan Wang then meric complex (Wrana et al. 1992), thus requir- working with Bob Weinbergusing an expression ing the ligand binding and function of a type I cDNA cloning strategy (Wang et al. 1991). receptor to be assessed in combination with the cDNA cloning of the glycoprotein endoglin was type II receptor. Furthermore, it became appar- reported at a time that endoglin was only known ent that, at least in the case of TGF-b and acti- to be a cell-surface protein specifically expressed vin, the type I receptors only bound ligand in vascular endothelium (Gougos and Letarte when combined with the appropriate ligand- 1990) and, thus, preceded the cDNA cloning binding type II receptor, which provides ligand of the first TGF-b family receptors and the re- specificity (Attisano et al. 1993; Ebner et al. alization that it participates in TGF-b signaling. 1993b). In contrast, the type II or type I BMP Endoglin was subsequently found to bind TGF- receptors, when expressed individually, gener- b, allowing it to play a role in ligand presenta- ally showed only a weak affinity for BMPs, and tion to type II/type I TGF-b receptor complexes only bind BMPs efficiently when coexpressed in (Cheifetz et al. 1992), and to link genetically heteromeric receptor combinations (Miyazono with hereditary hemorrhagic telangiectasia et al. 2001, 2010). Using a type II/type I recep- type I (McAllister et al. 1994). tor combinatorial strategy, TGF-b was shown to signal primarily through the type I receptor that Discovery and Characterization of the is now known as TbRI or ALK-5 (Franzeń et al. Smad Pathway 1993, Bassing et al. 1994). The functional interdependence of the type II and type I receptors At the time that the TGF-b receptors were was subsequently shown to result from ligand- cloned, little was known about any downstream induced, type II receptor-mediated phosphory- mechanisms, and of course the next big step was lation of the GS domain in the type I receptor to elucidate how TGF-b induces the many di- that leads to activation of the type I receptor verse responses. TGF-b had been shown to in- kinase (Wrana et al. 1994b). This dependence duce Ras and Erk mitogen-activated protein

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(MAP) kinase activation (Mulder and Morris yielded “mothers against dpp” (mad) and medea 1992; Yan et al. 1994; Mucsi et al. 1996), but as genes that encode downstream components activation of this pathway was at odds with the required for dpp signaling (Raftery et al. 1995), substantial differences in responses when com- and revealed sequence similarity between Mad pared with RTKs that activate the Ras-Erk MAP and three C. elegans genes (Sekelsky et al. 1995). kinase pathway. Additionally, TGF-b was shown Parallel studies in C. elegans had identified daf-4 to activate protein phosphatase 1 (Gruppuso as a BMP-2 and -4 homolog receptor (Estevez et al. 1991) and to induce phosphorylation of et al. 1993). Three genes, sma-2, -3, and -4, were the transcription factor cAMP response ele- shown to function in the same cells as daf-4, and ment-binding (CREB) protein (Kramer et al. to be required for daf-4 function (Savage et al. 1991), but these findings again did not explain 1996). The identification of Mad in Drosophila the major TGF-b responses. With the receptors and Sma proteins in C. elegans defined a new in hand, several laboratories embarked on iden- family of signaling mediators downstream tifying associated proteins and kinase substrates from BMP-related proteins, and prompted an for the TGF-b receptors. This led, within the intense search by several groups for homologs next few years, to the identification of FKBP12 in mammals. Within a year, five Mad homologs (Wang et al. 1996a; Chen et al. 1997) and the a were identified in different laboratories resulting subunit of farnesyltransferase (Ventura et al. in a diverse set of names (Derynck and Zhang 1996; Wang et al. 1996b) as associated proteins, 1996). One of these, initially named human and the translation initiation factor eIF2a/ Mad4, was identical to DPC4 (“deleted in pan- TRIP-1 (Chen et al. 1995) and Ba subunit of creatic carcinoma, locus 4”), whose gene had protein phosphase 2A (Griswold-Prenner et al. just been identified as a putative tumor suppres- 1998) as kinase substrates, but these again were sor genewith inactivating mutations in pancreas not seen as signaling effectors that mediate the cancer (Hahn et al. 1996). The nomenclature predominant TGF-b responses. The identifica- was soon resolved by agreement of the involved tion by Matsumoto and colleagues of the “TGF- investigators to use the term Smad as a combi- b-activated kinase 1” (TAK1) as a signaling nation of the Mad and Sma names (Derynck mediator that is activated by TGF-b and BMP et al. 1996). (Yamaguchi et al. 1995) generated considerable The assignment of Smads as effectors of excitement. However, its identity as a MAPKK TGF-b/activin or BMP ligands and key aspects kinase that could activate MAP kinase pathways of the overall signaling mechanism progressed and the inability of TAK1 to activate TGF-b at a frantically competitive pace in 1995–1996. target gene responses raised the general ques- Individual Smads, when overexpressed, were tion to what extent TAK1 contributed to TGF-b shown to mimic effects of corresponding li- and BMP responses. Furthermore, TAK1 was gands (Baker and Harland 1996; Graff et al. subsequently also shown to be an effector of 1996; Liu et al. 1996; Zhang et al. 1996), and signaling in response to ligands that are not re- dominant-negative mutants were found to in- lated to TGF-b and act through different recep- hibit ligand-induced responses (Zhang et al. tor classes. 1996). Ligand-induced receptor activation in- A major breakthrough toward the identifi- duced Smad phosphorylation (Eppert et al. cation of TGF-b-activated signaling effectors 1996, Hoodless et al. 1996; Zhang et al. 1996), came from genetic analyses of Drosophila and Smad3 was found to interact with the type I, but C. elegans. The Drosophila decapentaplegic (dpp) not the type II receptor (Zhang et al. 1996), and gene, which encodes the homolog of vertebrate Smad4 cooperated with Smad3 (Zhang et al. BMP-2 and -4, was by then knownto act through 1996). Additionally, a carboxy-terminal frag- the type II receptor, Punt, and two type I recep- ment of Smad1 or Smad4 was shown to have tors, Thickveins and Saxophone (Ruberte et al. transcription activation potential (Liu et al. 1995). A genetic screen for enhancers of weak 1996), and activin or BMP-2/-4-induced nuclear dpp alleles in Bill Gelbart’s laboratoryat Harvard accumulation of Smad2 or Smad1, respectively

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Historical Perspective of TGF-b

(Baker and Harland 1996; Hoodless et al. 1996; through association with the DNA-binding Liu et al. 1996). These observations provided transcription factor FAST1 (Chen et al. 1996), the basis for the model of Smad activation that now known as FoxH1. However, Mad was sub- conceptually paralleled the well-established sequently shown to bind DNA directly, to a GC- model of signal transducers and activators of rich sequence in the vestigial promoter (Kim et transcription (STAT) activation in response to al. 1997), and a PCR-based selection procedure cytokine receptors (Fig. 4). Thus, Smads were defined the palindromic sequence GTCTAGAC thought to interact with type I receptors and be as the consensus “Smad-binding element” for activated through phosphorylation by the Smad3 and Smad4 (Zawel et al. 1998). There- receptor complexes, form a complex with fore, it was concluded that receptor-activated Smad4, translocate into the nucleus, and target Smads were DNA-binding transcription fac- regulatory gene sequences for direct transcrip- tors, similarly to STATs. On the other hand, the tional activation (Derynck and Zhang 1996). association and cooperation of Smad2 with This model was rapidly refined (e.g., by the FoxH1 at target promoters (Chen et al. 1996; demonstration that the type I TGF-b receptor Labbe´ et al. 1998), the association and cooper- directly phosphorylates Smad2, and not ation of Smad3 with c-Jun/c-Fos at overlapping Smad4) at two carboxy-terminal serines, thus promoter sequences (Zhang et al. 1998), and leading to its activation (Hoodless et al. 1996; the cooperation of Smad3 with the transcrip- Macıás-Silva et al. 1996), and that Smad4 co- tion factor TFE3 in the control of PAI-1 expres- operates with all receptor-activated Smads test- sion (Hua et al. 1998), which were revealed ed (Lagna et al. 1996; Zhang et al. 1997). around the same time, all emphasized the How Smad complexes could activate tran- need for cooperation of Smad complexes with scription was again pursued at a high pace by high-affinity DNA-binding transcription fac- several laboratories. An early report found tors. These and other observations led to the Smad2 to interact with the Mix2 promoter now generally accepted model that Smads acti-

AB Ligand Type II receptor Ligand Type I receptor Receptor

JAK JAK

Cytosol P P P P Smad3 P P

P P P Smad4 STATs P Translocation Translocation

Nucleus P P Transcription P Transcription

Figure 4. Initial working model of Smad signaling proposed in 1996. Following the identiﬁcation of Smads as transforming growth factor b (TGF-b) family signaling effectors and several reports on how they might do so, this ﬁrst model was proposed, based on these results and the perceived analogy with the established mode of signal transducers and activators of transcription (STAT) signaling. (From Derynck and Zhang 1996; reprinted, with permission, from the authors.)

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H.L. Moses et al.

vate target gene transcription, through physical by Smad3 with the formation of functional association and functional cooperation with a myogenic transcription factor complexes at E- variety of high-affinity DNA-binding transcrip- box DNA sequences in target genes (Liu et al. tion factors, thus enabling DNA binding to ad- 2001, 2004). In contrast, in TGF-b-induced jacent DNA sequences, and that Smad complex- repression of c-myc expression, Smad3/4 com- es use CBP or p300 as coactivators (Derynck plexes were seen to associate with the DNA- et al. 1998). This model explained why, despite binding E2F4/5 and DP1 transcription factors, extensive efforts, no consensus TGF-b respon- and the co-repressor p107, thus enabling these sive DNA sequences could ever be found in complexes to bind at a composite Smad-E2F- TGF-b target genes, and illustrates the high ver- binding site in the c-myc promoter, and to exert satility of Smad signaling dependent on the na- transcription repression through p107 (Chen ture of the interacting transcription factor. This et al. 2002). At the osteocalcin and Runx2 pro- mode of cooperation also predicted direct moter, TGF-b-activated Smad3/4 complexes cross-talk of Smads with other signaling path- were seen to associate with Runx2 at a Runx2- ways that target the Smad-associated transcrip- binding DNA sequence and to directly recruit tion factors, thus providing a mechanistic basis histone deacetylases (Alliston et al. 2001; Kang for the context-dependence of the TGF-b re- et al. 2005). sponses. When this model was proposed in As these mechanistic insights were unfold- the context of an invited minireview for Cell, ing, two novel Smads were discovered that had a the editor, Ben Lewin, called Derynck to argue conserved transactivation domain that charac- that this model was totally wrong, indicating terizes the Smads, but lacked the preceding ami- that Smads were plain DNA-binding transcrip- no-terminal Mad homology 1 (MH1) domain tion factors with a DNA-binding consensus se- that is seen in all receptor-activated Smads and quence ( just like STATs), as just published in Smad4. These Smads, Smad6 and Smad7, with Molecular Cell (Zawel et al. 1998). After vigor- Dad as the Drosophila counterpart, were shown ous arguing, he very reluctantly agreed to have to associate with the type I receptors or other this model published. With an extensive body of Smads, thus competitively preventing activation ongoing research on many target genes in the of the effector Smads, which is why they were next few years, Smads were shown to associate named inhibitory Smads (Hayashi et al. 1997; and cooperate with more than 100 DNA-bind- Imamura et al. 1997; Nakao et al. 1997; Tsunei- ing transcription factors, depending on the na- zumi et al. 1997; Hata et al. 1998). With Smad6 ture of the target promoter, and with many and Smad7 expression activated in response to co-repressors and coactivators that help define several signaling pathways, it became apparent the amplitude of the TGF-b-induced transcrip- that the inhibitory Smads provide endogenous tion responses (Feng and Derynck 2005; Mas- control of the level of Smad activation by TGF-b sague´ et al. 2005). family proteins (ten Dijke and Hill 2004; Although we started understanding the Briones-Orta et al. 2011). Additional observa- mechanisms of TGF-b-induced, Smad-mediat- tions suggested that they may also directly re- ed activation of gene expression, these observa- press transcription of target genes in the nucleus tions did not address how TGF-b family pro- (Bai and Cao 2002; Gro¨nroos et al. 2002). teins induce repression of gene expression. With With the overall mechanisms of Smad-me- research on different target genes as model sys- diated transcription control defined, at least in tems, it became subsequently apparent that also general terms, several laboratories have been repression of gene expression was mediated by focusing on the activation of non-Smad signal- Smad complexes, thus fully attributing the con- ing pathways by TGF-b and TGF-b-related trol of gene expression by TGF-b family pro- proteins. Indeed, TGF-b family proteins were teins to Smads. Smad3-mediated repression of shown to activate Erk, p38, and JNK MAP ki- myogenic transcription factor target genes was nase pathways, as well as the PI3K-Akt-TOR seen to result from direct physical interference pathway, and to act through TAK1, an upstream

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Historical Perspective of TGF-b

Figure 5. Early contributors to the development of the transforming growth factor b (TGF-b) ﬁeld. The picture taken in February 2006 shows (seated from left) Rik Derynck, Anita Roberts, Harold Moses, and (standing from left) Michael Sporn and Joan Massague´.

mediator of MAP kinase pathways (Derynck greatly contributed to our current mechanistic, and Zhang 2003; Moustakas and Heldin 2005; structural, developmental, and physiological Zhang 2009). The mechanisms through which understanding of the biology of TGF-b and TGF-b proteins induce activation of these path- have made this into a highly exciting area of ways, or cross-talk with the Smad pathway, are biology with substantial implications for our being characterized, whereas other studies ad- understanding of various diseases. The effects dress how cross-talk between these pathways of TGF-b family proteins on many individual and the Smad pathway controls the cellular re- cell types and the very nature of Smad signaling, sponses in cell differentiation and growth con- complemented by non-Smad pathways, empha- trol and during development and in cancer. size and have made us appreciate the contextual nature of TGF-b effects at the cell, tissue, and organismal level. Certainly, 35 years after its dis- SUMMARY covery, the future of TGF-b research is bright The pioneering efforts of the Todaro, Holley, and sure to yield exciting new insights and phar- Moses, Roberts/Sporn, Derynck, and Massague´ macological approaches to manipulate this laboratories (Fig. 5), resulting in the discovery pathway for clinical beneﬁt. and characterization of TGF-b1 in the early 1980s, have now led to the description of an almost unfathomable range of activities of REFERENCES TGF-b, and the identiﬁcation of a large family Akhurst RJ, Derynck R. 2001. TGF-b signaling in cancer—A of related growth and differentiation factors double-edged sword. Trends Cell Biol 11: S44–S51. with important effects in development and Alliston T, Choy L, Ducy P, Karsenty G, Derynck R. 2001. TGF-b-induced repression of CBFA1 by Smad3 decreases adult physiology and disease pathogenesis. cbfa1 and osteocalcin expression and inhibits osteoblast Many highly committed research groups have differentiation. EMBO J 20: 2254–2272.

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26 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a021865 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press

The Discovery and Early Days of TGF-β: A Historical Perspective

Harold L. Moses, Anita B. Roberts and Rik Derynck

Cold Spring Harb Perspect Biol 2016; doi: 10.1101/cshperspect.a021865 originally published online June 21, 2016

Subject Collection The Biology of the TGF-β Family

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TGF-β Family Signaling in Neural and Neuronal Signaling Cross Talk between TGF-β/Smad and Differentiation, Development, and Function Other Signaling Pathways Emily A. Meyers and John A. Kessler Kunxin Luo

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