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TGF-b and the TGF-b Family: Context-Dependent Roles in Cell and Tissue Physiology

Masato Morikawa,1 Rik Derynck,2 and Kohei Miyazono3

1Ludwig Cancer Research, Science for Life Laboratory, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden 2Department of Cell and Tissue Biology, University of California at San Francisco, San Francisco, California 94143 3Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Correspondence: [email protected]

The transforming growth factor-b (TGF-b) is the prototype of the TGF-b family of growth and differentiation factors, which is encoded by 33 genes in mammals and comprises homo- and heterodimers. This review introduces the reader to the TGF-b family with its complexity of names and biological activities. It also introduces TGF-b as the best-studied factor among the TGF-b family proteins, with its diversity of roles in the control of cell proliferation and differentiation, wound healing and immune system, and its key roles in pathology, for exam- ple, skeletal diseases, fibrosis, and cancer.

lthough initially thought to stimulate cell TGF-b has been well documented in most cell Aproliferation, just like many growth factors, types, and has been best characterized in epithe- it became rapidly accepted that transforming lial cells. The bifunctional and context-depen- growth factor b (TGF-b) is a bifunctional reg- dent nature of TGF-b activities was further con- ulator that either inhibits or stimulates cell pro- firmed in a large variety of cell systems and liferation. TGF-b was originally isolated as a biological responses. For example, TGF-b can cytokine that, together with epidermal growth inhibit EGF-dependent proliferation of cells factor (EGF), induces cellular transformation in monolayer culture, whereas TGF-b and and anchorage-independent growth of selected EGF synergistically enhance anchorage-inde- fibroblast cell lines (Roberts et al. 1981), yet did pendent growth of the same cells in soft agar not require the presence of EGF to induce phe- medium (Roberts et al. 1985). Now, it is widely notypic transformation of other fibroblast cell accepted that TGF-b regulates a variety of lines (Shipley et al. 1984). In contrast, TGF-b key events in normal development and physiol- was also identified as a growth inhibitor se- ogy, and perturbation of TGF-b signaling creted from confluent BSC-1 cells, epithelial has been implicated in the pathogenesis of dis- cells of African green monkey kidney (Tucker eases such as connective tissue disorders, fibro- et al. 1984). The growth inhibitory activity of sis, and cancer.

Editors: Rik Derynck and Kohei Miyazono Additional Perspectives on The Biology of the TGF-b Family available at www.cshperspectives.org Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a021873 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a021873

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The identification of TGF-b family mem- 1995; Sekelsky et al. 1995). In C. elegans, daf-1 bers and their signaling components has en- and daf-4 turned out to also encode serine/thre- abled the characterization of the complex biol- onine transmembrane kinase receptors for ogy of the TGF-b family members. Molecular TGF-b family members. Screening for mutants cloning of TGF-b family members and their with similar phenotypes with daf-4 revealed signaling mediators started in 1985 with the re- three genes, sma-2, sma-3, and sma-4, that ported characterization of complementary DNA were structurally similar to Mad of Drosophila (cDNA) coding for human TGF-b1 (Derynck (Savage et al. 1996). In frog, mouse, and human, et al. 1985). Subsequently, various approaches, genes structurally similar to Mad and sma were based on biochemical purification, developmen- subsequently identified, and the designation tal genetics, and/or targeted cDNA cloning, led “Smad” (Sma and Mad) was adopted. Ligand to the identification of polypeptides structurally binding to specific tetrameric type II/type I re- similar to TGF-b1, which together comprise ceptor complexes stabilizes and activates their the members of the TGF-b family. Now signaling capacities, and the receptors then that the human and mouse genome sequence transduce the signals by phosphorylating car- projects are completed, it is apparent that mam- boxy-terminal serine residues of receptor-regu- malian genomes encode 33 TGF-b-related poly- lated (R-) Smads. In most cell types, TGF-bs and peptides. Table 1 shows the 33 known human activins induce phosphorylation of Smad2 TGF-b family polypeptides, which include three and Smad3 (activin/TGF-b-specific R-Smads), TGF-b isoforms, activins, nodal, bone morpho- and BMPs induce phosphorylation of Smad1, genetic proteins (BMPs), and growth and dif- Smad5, and Smad8 (BMP-specific R-Smads). ferentiation factors (GDFs). Although mostly The activated R-Smads form hetero-oligomeric studied as homodimers, various heterodimeric complexes with a common-partner (co-) Smad, combinations of these have also been identified that is, Smad4 in vertebrate cells (Lagna et al. and characterized as biologically active proteins. 1996; Zhang et al. 1996; Kawabata et al. 1998). In contrast to the large number of TGF-b The complexes translocate into the nucleus ligands, fewer receptors and downstream intra- where they regulate the expression of target cellular effectors, termed Smad proteins, medi- genes, such as those encoding inhibitory (I-) ate transduction of intracellular signaling. In Smads, namely, Smad6 and Smad7 in verte- 1991, a cDNA clone for an activin receptor, cur- brates, which can inhibit R-Smad activation by rently known as ActRII, was isolated (Mathews the receptors. Finally, TGF-b family proteins and Vale 1991). The kinase domain of this re- were also shown to induce PI3K-Akt signaling ceptor closely resembled the Caenorhabditis and to activate the common mitogen-associated elegans daf-1, a transmembrane serine/threo- protein (MAP) kinase pathways that are activat- nine-specific protein kinase, which at that time ed by receptor tyrosine kinases, albeit, generally, was seen as an orphan receptor, and provided the to a lower extent. Now that essential players in first indication that TGF-b family members sig- the signaling pathways have been identified, one nal through transmembrane serine/threonine of the major questions to be addressed in this kinases. In the following years, seven type I re- field is to reveal the precise molecular mecha- ceptors and five type II receptors were identified nisms that define the context-dependent dual in mammals and were shown to form hetero- roles of TGF-b family members. meric type I/type II receptorcomplexes.Genetic In this review, we will introduce the TGF-b analysis in Drosophila and C. elegans led to a family members, which in mammals are encod- breakthrough in how signals are transduced ed by 33 genes. Wewill cluster them into several from the receptors to the nucleus. In Drosophila, subgroups based on the structural or sequence mothers against dpp (Mad) was identified as a similarities of the encoded polypeptides. We gene encoding a component that acts epistati- further focus on the three TGF-b isoforms, cally downstream from Decapentaplegic (Dpp; TGF-b1, -b2, and -b3, as the best-studied fac- Drosophila BMP-2/-4 ligand) (Raftery et al. tors among the TGF-b family proteins. In ad-

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Roles of TGF-b in Cell and Tissue Physiology

Table 1. Names and genes for the TGF-b family proteins Official gene Protein name symbol (human) Protein name synonyms TGF-b1 TGFB1 CIF-A (cartilage-inducing factor-A), differentiation-inhibiting factor TGF-b2 TGFB2 G-TsF (glioblastoma-derived, T-cell suppressor factor), BSC-1 GI (BSC-1 cell-growth inhibitor), polyergin, CIF-B (cartilage- inducing factor-B) TGF-b3 TGFB3 Inhibin a INHA Inhibin A and Ba a,b Inhibin bA INHBA Inhibin A and activin A or AB, FRP (follicle-stimulating hormone-releasing protein), EDF (erythroid differentiation factor), XTC-MIF (Xenopus XTC cell mesoderm-inducing factor) a,b Inhibin bB INHBB Inhibin B and activin B or AB, XTC-MIF c Inhibin bC INHBC Activin C c Inhibin bE INHBE Activin E Nodal NODAL BMP-16 (bone morphogenetic protein-16) Myostatin MSTN GDF-8 (growth and differentiation factor-8) BMP-2 BMP2 BMP-3 BMP3 Osteogenin BMP-4 BMP4 BMP-2B BMP-5 BMP5 BMP-6 BMP6 Vgr1 (Vg1-related protein) BMP-7 BMP7 OP-1 (osteogenic protein-1) BMP-8A BMP8A OP-2 BMP-8B BMP8B OP-3 BMP-9 GDF2 GDF-2 BMP-10 BMP10 GDF-1 GDF1 GDF-3 GDF3 Vgr2 GDF-5 GDF5 CDMP-1 (cartilage-derived morphogenetic protein-1), BMP-14 GDF-6 GDF6 CDMP-2, BMP-13 GDF-7 GDF7 CDMP-3, BMP-12 GDF-9 GDF9 GDF-9B BMP15 BMP-15 GDF-10 GDF10 BMP-3b GDF-11 GDF11 BMP-11 GDF-15 GDF15 Placental TGF-b, placental BMP (PLAB), PDF (prostate-derived factor), NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1), MIC-1 (macrophage inhibitory cytokine-1) MIS (Mu¨llerian- AMH AMH (anti-Mu¨llerian hormone) inhibiting substance) Lefty A LEFTY2 EBAF (endometrial bleeding-associated factor), TGF-b4, Stra3 Lefty B LEFTY1 a Inhibin A or B is a heterodimer of inhibin a and inhibin bA or bB, respectively. b Activin A or B is a homodimer of inhibin bA or bB, respectively. Activin AB is a heterodimer of inhibin bA and bB. c Activin C or D is a homodimer of inhibin bC or bD, respectively.

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M. Morikawa et al.

dition to the original “transforming” potential, lowed by a cleavage site for a furin protein con- we introduce their diverse roles in the control of vertase that precedes the carboxy-terminal ma- cell proliferation, differentiation, wound heal- ture TGF-b family polypeptide. In the case of ing, and the immune system, and TGF-b’s key the three TGF-b isoforms, the prosegments roles in the context of pathological processes in are also known as latency-associated peptides vivo, for example, connective tissue disorders, (LAPs). Following secretion, the LAPs remain fibrosis, and cancer. noncovalently associated with the mature TGF- b dimer and keep the TGF-bs latent, that is, unable to bind and activate the receptors, in TGF-b FAMILY LIGANDS IN MAMMALS complexes that are called “small latent complex- The name TGF-b derives from the transforming es” (SLCs). Whereas these prosegments are like- activity of the cytokine, which induces anchor- ly to play important roles in folding and trans- age-independent growth when administered to port of the TGF-b family proteins, they were cells together with EGF (Roberts et al. 1981) or shown to remain associated following secretion without the need to add EGF (Shipley et al. with a number of mature TGF-b family pro- 1984), depending on the cell system. At first, teins, such as with BMP-4, BMP-7, BMP-9 two distinct transforming growth factors, that (also known as GDF-2), BMP-10, GDF-5, is, TGF-a and -b, were identified and isolated. GDF-11, myostatin (also known as GDF-8), TGF-a is related to EGF and binds to the EGF and Mu¨llerian-inhibiting substance (MIS, also receptor, whereas TGF-b is structurally distinct known as anti-Mu¨llerian hormone, AMH) from TGF-a. The most striking characteristic, (Wilson et al. 1993; Wolfman et al. 2003; Brown which distinguished the two cytokines at that et al. 2005; Ge et al. 2005; Sengle et al. 2008). time, was that TGF-b is a 25-kd disulfide-linked However, only some of the TGF-b family mem- dimer that is reduced to a 12.5-kd band on gels bers are produced as latent forms that are unable following treatment with reducing agents, for to bind the receptors. With the exception of the example, b-mercaptoethanol (Roberts et al. TGF- b1 complex, the roles of the prosegments 1983), whereas TGF-a was a monomeric pro- for other TGF-b family proteins have been tein of smaller size (Roberts et al. 1980). poorly characterized, even for the closely related All TGF-b family members are encoded by TGF-b2 and -b3. much larger precursor polypeptides whose se- The latency and activation mechanisms that quences have been deduced through cDNA result in the release of active TGF-b protein have cloning. The precursor polypeptides are com- been primarily studied in the case of TGF-b1, posed of three segments: an amino-terminal with additional knowledge gained on the acti- signal peptide that is removed during translo- vation of myostatin (Wolfman et al. 2003). The cation of the protein into the lumen of the prosegment of TGF-b1 contains a proline-rich rough endoplasmic reticulum, a large precursor loop, or latency lasso, which surrounds a TGF- segment or prosegment, and the carboxy-ter- b1 monomer like a straitjacket (Shi et al. 2011). minal TGF-b family monomer polypeptide Comparison between TGF-b1 LAP and the for the active and fully mature TGF-b family BMP-9 prosegment, which does not confer la- protein (112 amino acid residues in the case of tency, suggests conformational differences (Mi TGF-b). The prosegments vary in length from et al. 2015). Further analysis of the prosegments 150 to 450 residues and are remarkably not con- of other TGF-b family members, especially served in sequence among TGF-b family mem- nonlatent ones, will reveal functional conserva- bers, even among the three TGF-b isoforms tion among them. (Derynck et al. 1988). Indeed, the sequence sim- For TGF-b to bind to its cell-surface recep- ilarities among TGF-b-related proteins pertain tors, the LAP needs to be released from the ma- exclusively to the sequences of the mature poly- ture peptide. Both the TGF-b1 LAPand -b3 LAP peptides that follow the prosegments. In the contain an integrin recognition motif Arg-Gly- precursor, the prosegment is immediately fol- Asp, or RGD, and bind to several different integ-

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Roles of TGF-b in Cell and Tissue Physiology

rins, for example, avb6 and avb8. Biochemical formation of gastrointestinal tumors (Yoshi- and structural studies revealed that contractile naga et al. 2008). LTBPs are efficiently incorpo- force is necessary for the release of mature TGF- rated in the extracellular matrix (ECM) (Taipale b1 after complex formation between integrin et al. 1994; Dallas et al. 1995), where latent TGF- avb6 and TGF-b1 LAP (Annes et al. 2004; Shi b, as a part of LLC, is stored for future mobili- et al. 2011). In agreement with these findings, zation and activation. Thus, the regulation of mice with RGD-to-RGE mutation in TGF-b1 latency of TGF-b has substantial biological sig- LAP (Yanget al. 2007) and integrin b6-deficient nificance, although it has received little atten- mice (Itgb62/2) (Munger et al. 1999) recapitu- tion. The control of TGF-b latency has been late aspects of the phenotype of Tgfb12/2 mice, reviewed elsewhere (Horiguchi et al. 2012). indicating the importance of the interaction be- The mammalian genome encodes three dif- tween LAP and integrins in latent TGF-b1 acti- ferent TGF-b isoforms, TGF-b1, -b2, and -b3. vation. The SLC covalently attaches the large The mature proteins are highly conserved in latent TGF-b-binding protein (LTBP) through sequence and are marked by the presence of LAP, thus, forming the large latent complex nine cysteine residues. The three-dimensional (LLC). The association of LTBP with the SLC is structure of TGF-b2 reveals that four cysteine also important for proper TGF-b1 function. pairs are formed intramolecularly, but the sixth Mice expressing a mutant TGF-b1 precursor cysteine at position 77 forms the single inter- (C33S) with a prosegment that is unable to molecular disulfide bridge that results in dimer bind LTBP, instead of the wild-type TGF-b1 formation (Fig. 1) (Daopin et al. 1992; Schlu- precursor, are hypomorphic for TGF-b1; negger and Gru¨tter 1992). Tgfb1C33S/C33S mice show decreased levels of ac- Another distinct subfamily is the inhibin b tive TGF-b1, decreased TGF-b signaling, and (or activin b) family. Inhibins are heterodimeric

RXXR Intermolecular

TGF-β, inhibin β C CC CC CC CC

Myostatin, GDF-11 C CC CC CC CC

GDF-15 C CC CC CC CC

BMP/GDF, inhibin α C CC CC CC

BMP-8A, BMP-8B C CC C CC CC

GDF-3 C CCCC CC

GDF-9, GDF-9B C CC C CC

Lefty A, lefty B C CC CC C CC

Figure 1. Comparison of the polypeptide organization of TGF-b family proteins. The precursor protein is cleaved by a furin protein convertase, which mainly recognizes the Arg-X-X-Arg (RXXR) motif, but also the Arg-X-Lys/Arg-Arg (RXK/RR) motif. The mature polypeptides (white) contain seven or nine cysteines con- served in most TGF-b family members. These cysteines engage in inter- and intramolecular disulfide bonds. Human BMP-8A, BMP-8B, and GDF-3 have an additional cysteine residue, which is shown in gray, whereas five members lack the cysteine that mediates the disulfide bond in ligand dimerization. Signal peptide, gray; proseg- ment, black; mature polypeptide, white; TGF-b, transforming growth factor b; BMP, bone morphogenetic protein; GDF, growth and differentiation factor.

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M. Morikawa et al.

proteins consisting of an inhibin a chain and et al. 1995; Israel et al. 1996). BMP-2/GDF-6 an inhibin b chain, whereas activins are homo- heterodimer is also able to regulate ectodermal dimers of inhibin/activin b chains. The four cell fate determination (Chang and Hemmati- known b chains are closely related to each other Brivanlou 1999), and BMP-7/GDF-7 hetero- and have the corresponding set of nine cys- dimer is more potent than the corresponding teines, which is apparent in the TGF-bs. Inhibin homodimers in neuronal differentiation (Butler a, in contrast, is more divergent and has seven and Dodd 2003). cysteines, corresponding to the seven carboxy- Finally, some TGF-b family proteins, lefty A terminal cysteines seen in all TGF-b proteins. and lefty B, GDF-3, and GDF-9 and GDF-9B All other ligands in the TGF-b family, with (also known as BMP-15), lack the fourth cys- five exceptions discussed further below, have teine in the seven-cysteine pattern and thus have a characteristic seven-cysteine pattern that cor- eight or six cysteines (Fig. 1). As this missing responds to the cysteine pattern observed in cysteine mediates in other ligands the disulfide TGF-b and inhibin b, but without its amino- bond dimerization, one should assume that terminal two cysteines (Fig. 1). In these cases, these TGF-b family proteins do not form disul- the disulfide-linked dimerization is mediated by fide-bonded dimers and have alternative means the fourth cysteine in each monomer. Many of of interacting with other TGF-b family pro- these proteins have been named BMP or GDF teins. Lefty is found to associate with nodal followed by a number, based on their sequence and to inhibit nodal signaling through a dual relationship to other BMPs or GDFs, which mechanism involving its interaction with no- were often identified in the same laboratories. dal and the EGF-CFC coreceptors, Cripto-1, Within this large group of ligands, there is a FRL-1, or Cryptic, which are required for no- “core” BMP/GDF subfamily of proteins with dal signaling (Chen and Shen 2004; Tabibzadeh similar or related activities. These are structur- and Hemmati-Brivanlou 2006). GDF-3, which ally closely related to each other and are shown also lacks the cysteine required for intermolec- as the top 12 ligands in Figure 2. These BMPs ular disulfide formation, may also function as and GDFs signal through heteromeric complex- an inhibitor of TGF-b/BMP signaling, similarly es that comprise the BMP type I receptors, in- to lefty. Indeed, GDF-3 was reported to func- cluding ALK-1 (activin receptor-like kinase 1), tion as a BMPantagonist (Levine and Brivanlou ALK-2, ALK-3/BMPRIA, or ALK-6/BMPRIB. 2006), whereas it was also reported to act as In contrast, some ligands that have the seven- a nodal-like ligand (Chen et al. 2006; Levine cysteine pattern, like the BMPs and GDFs, do et al. 2009) and to signal possibly through not signal through the same BMP/GDFreceptor ALK-7, a type I receptor for activins (Andersson complexes and intracellular mediators as BMPs et al. 2008). GDF-9B has been reported to and GDFs. Most notably, nodal signals through bind to BMP type I receptor ALK-6/BMPRIB ALK-4, the major type I receptor for activins, and phosphorylate Smad1, Smad5, and Smad8, which is also known as ActRIB, whereas myosta- whereas GDF-9 activates Smad2 through tin signals through ALK-4/ActRIB or ALK-5, ALK-5/TbRI (Moore et al. 2003; Mazerbourg the TGF-b type I receptor also known as TbRI. et al. 2004; Peng et al. 2013). In addition, a As with the heterodimeric inhibins, BMP GDF-9/-9B heterodimer is more bioactive for and/or GDF heterodimers have been identified ovarian functions (Peng et al. 2013). Further and shown to be fully active. In mesodermal characterization of the functions of this sub- differentiation, BMP-4/-7 and -2/-7 hetero- family of TGF-b family proteins is expected. dimers potently induce mesoderm in frog (Su- zuki et al. 1997; Eimon and Harland 1999) and COMPLEXITY OF NOMENCLATURE zebrafish (Little and Mullins 2009). In cultured cells, BMP-2/-7, -4/-7, and -2/-6 heterodimers The name “TGF-b family” derives from the are more active than the corresponding homo- identification of TGF-b1 as a founder member dimers in ectopic bone-formation assays (Aono and its activity in the initial phenotypic trans-

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BMP-5 BMP-6/Vgr1 BMP-7/OP-1 BMP-8A/OP-2 BMP-8B BMP-2 BMP-4 BMP-9/GDF-2 BMP-10 GDF-5/CDMP-1 GDF-6/CDMP-2/BMP-13 GDF-7/CDMP-3 GDF-1 GDF-3/Vgr2 BMP-3 GDF-10/BMP-3B Nodal GDF-9 GDF-9B/BMP-15 Lefty A Lefty B Inhibin α MIS/AMH GDF-15/placental TGF-β Inhibin βA Inhibin βB Inhibin βC Inhibin βE Myostatin/GDF-8 GDF-11/BMP-11 TGF-β1 TGF-β2 TGF-β3 0.1

Figure 2. Phylogenetic tree of the 33 TGF-b family polypeptides in human. The amino acid sequences of 33 TGF- b family polypeptides, encoded by their corresponding human genes, were obtained from NCBI’s protein database (www.ncbi.nlm.nih.gov), and the carboxy-terminal mature polypeptides were assigned based on validated or predicted furin cleavage sites. The sequences were aligned by Clustal Omega (www.clustal.org/ omega) (Sievers et al. 2011) and the phylogenetic tree was illustrated using FigTree v1.4.2 (tree.bio.ed.ac.uk/ software/figtree). Ligands that signal through activin- or TGF-b-activated R-Smads or BMP-activated R-Smads are shown in red or blue, respectively. Ligands that may activate these two types of R-Smads, but whose receptors and Smad-signaling pathways have not been fully determined, are shown in orange or light blue, respectively. BMP, Bone morphogenetic protein; OP, osteogenic protein; GDF, growth and differentiation factor; CDMP, cartilage-derived morphogenetic protein; MIS/AMH, Mu¨llerian-inhibiting substance/anti-Mu¨llerian hor- mone; TGF-b, transforming growth factor b.

formation assay. However, this name may be The identification of the BMP/GDF somewhat misleading. The three TGF-b iso- subfamily was based on the isolation and char- forms regulate a wide variety of cellular pro- acterization of BMP-2 and BMP-4 with their cesses, both in development and in the adult ability to elicit ectopic bone formation and af- organisms, and their effects are context-depen- fect skeletal morphogenesis. However, most dent. The three TGF-b isoforms have been es- BMP/GDF proteins were assigned their names tablished as some of the most potent growth because of their sequence similarities to the oth- inhibitors, and inhibit proliferation of most er BMPs/GDFs, whereas their activities remain types of cells. largely uncharacterized. It requires substantial

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caution to assume their biological functions older mice with GDF-11 improved muscle re- merely because they share a name, especially generation, a contradictory report claims that for BMPs or GDFs. Recently, several BMP/ serum levels of GDF-11 increase with age (Eger- GDF ligands were rediscovered as critical regu- man et al. 2015). Further work on the biological lators of distinct physiological and pathological activity of GDF-11 is required to confirm the processes. For example, BMP-9 and BMP-10, significance of GDF-11 as a potential youth- which were identified as ligands of the ALK-1 promoting factor. In contrast, GDF-5 expres- receptor (David et al. 2007), play essential roles sion was shown to be associated with muscle in the maintenance of homeostasis of vascular hypertrophy through activation of BMP sig- system and heart development (David et al. naling (Sartori et al. 2013). Another TGF-b 2009; Pardali et al. 2010). Additionally, BMP-6 family protein, MIS/AMH, is secreted by Ser- was identified as an endogenous ligand that reg- toli cells in the testis, and plays important roles ulates hepcidin expression and iron metabolism in early gonadal development and sex differen- in vivo (Andriopoulos et al. 2009; Meynard et al. tiation through inhibition of the Mu¨llerian duct 2009). GDF-9 and the closely related GDF-9B development. MIS signals through its type II are secreted by oocytes and regulate female fer- receptor, MISRII, and BMPRIA/ALK-3 in tility in several mammals. vivo, which subsequently activates the Smad1 Myostatin/GDF-8 (encoded by the Mstn pathway (Jamin et al. 2002). Mutations in the gene) is now established as a key regulator of gene encoding MIS (Behringer et al. 1994) or skeletal muscle mass; it inhibits muscle differ- MISRII (Imbeaud et al. 1995) result in persis- entiation and regeneration, and induces muscle tent Mu¨llerian duct syndrome. Intriguingly, the atrophy through receptor complexes of the sex-determining roles of MIS/MISRII signaling ActRII or ActRIIB type II receptors with ALK-4 are also conserved in fish, non-mammalian ver- or -5 type I receptors that activate the Smad2/ tebrates that lack a Mu¨llerian duct (Morinaga 3 pathway (McPherron et al. 1997; Lee and et al. 2007). McPherron 2001). As shown in Mstn-null Figure 2 and Table 1 show the 33 known mice and in loss-of-function mutations in cattle human TGF-b family polypeptides with their with double-muscling phenotype, mutations in evolutionary relationship calculated based on the Mstn gene result in increased muscle mass sequence similarities. Because multiple and of- with a combination of muscle cell hyperplasia ten parallel approaches have led to the identifi- and hypertrophy (Grobet et al. 1997; Kambadur cation of TGF-b family proteins, it is not sur- et al. 1997; McPherron and Lee 1997; McPher- prising that several ligands became known with ron et al. 1997). GDF-11, which is highly related multiple names (Table 1). Now, most investiga- to myostatin, also signals through ActRII and/ tors have settled on a common name for each or ActRIIB (Oh et al. 2002). Similar to myosta- individual TGF-b family protein; this nomen- tin, GDF-11 has been reported to inhibit myo- clature is used in Figure 2 and Table 1. blast differentiation and regulate the same tar- With genome-wide and transcriptome get genes (Egerman et al. 2015). It is worth analyses gaining in popularity, much research noting that soluble ActRIIB-Fc, a ligand trap has involved the control of TGF-b family mem- for myostatin, activin, and GDF-11, prevents bers. However, some discrepancies between the muscle wasting and reverses loss of skeletal mus- “official gene symbols” and the commonly used cle and cardiac atrophy in tumor-bearing mice names for TGF- b family proteins have caused with cachexia (Zhou et al. 2010). Recently, confusion for researchers and students who en- GDF-11 has been identified as a circulating fac- ter the field. For example, BMP-9 is the accepted tor in young mice, which reverses age-related and commonly used name for the protein, but dysfunction in mouse cardiac and skeletal mus- its official gene symbol is GDF2. Lefty A and cle (Loffredo et al. 2013; Sinha et al. 2014). lefty B are widely used names for the proteins, Whereas these reports claimed that serum levels but the corresponding official gene symbols are of GDF-11 decrease with age and that treating Lefty2 and Lefty1, respectively.

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Roles of TGF-b in Cell and Tissue Physiology

TGF-b SIGNALING IN THE CONTROL thermore, chromatin immunoprecipitation OF GENE EXPRESSION (ChIP) with promoter array analysis (ChIP- chip) and ChIP followed by sequencing More than 30 years of research on these multi- (ChIP-seq) revealed chromatin-binding land- functional ligands has revealed different and scapes of Smad proteins (Chen et al. 2008; Koi- sometimes opposite responses to TGF-b, which numa et al. 2009a,b; Qin et al. 2009). Some strongly depend on the cellular context. Most of these are defined through Smad binding information on the activities of TGF-b ligands with lineage-specific transcription factors at derives from studies using cells cultured in me- corresponding enhancer regions (Chen et al. dia that are supplemented with cytokine-con- 2008; Lee et al. 2011; Morikawa et al. 2011; Mul- taining serum, thus allowing for signaling cross len et al. 2011; Trompouki et al. 2011). Forced talk. Although Smads directly target and bind expression of different lineage-specific tran- regulatory gene sequences, theyeitheractivate or scription factors then redirects Smads to novel repress gene expression in cooperationwith high binding regions of different lineages (Mullen affinity DNA-binding transcription factors and et al. 2011; Trompouki et al. 2011). These find- transcription coregulators that are controlled by ings suggest that the binding of Smad complex- and/or dependent on other signaling pathways, es to target DNA sequences is defined through therefore providing scenarios for highlycontext- cooperation with other DNA-binding tran- dependent transcription responses controlled scription cofactors in two different ways: (1) by signaling cross talk. Furthermore, Smad acti- cell-type- or lineage-specific transcription fac- vation and Smad activities are regulated byother tors, or pioneer factors, open up local chroma- kinase and ubiquitylation pathways. Addition- tin structure to make it accessible for Smads, ally, Smad activation in response to TGF-b and (2) DNA-binding cofactors that are in- ligand-induced receptor activation is comple- duced and activated in a context-dependent mented by TGF-b/BMP-induced activation of manner strengthen the interaction between non-Smad-signaling pathways that may be sep- Smads and DNA. The control of gene expres- arately controlled. The combination of this sig- sion by Smads has been reviewed elsewhere naling cross talk at multiple levels, together with (Morikawa et al. 2013; Gaarenstroom and Hill differences in the physiology of different cell 2014). types, provides the basis for the high context In addition to genes that encode proteins, dependence of the responses to TGF-b. TGF-b also regulates expression of noncoding Several genes, such as the SMAD7 gene RNAs, such as microRNAs (miRNAs) and long encoding the inhibitory Smad7, SERPINE1 en- noncoding RNAs (lncRNAs). miRNAs are coding plasminogen activator inhibitor 1, or 19–25 nucleotides long (with an average CDKN1A encoding the cyclin-dependent ki- of 22 nucleotides), and are transcribed as long nase inhibitor p21Cip1/Waf1, have been estab- primary transcripts with hairpin structures lished as direct Smad target genes that are (pri-miRNAs). Most pri-miRNAs derive from generally and rapidly induced after TGF-b introns of coding genes or are independently treatment in many cell types (Lund et al. 1987; transcribed as noncoding transcripts. In the Datto et al. 1995b; Nakao et al. 1997; Koinuma nucleus, a microprocessor complex contain- et al. 2009b). The development of DNA micro- ing the RNase III enzyme Drosha and the dou- arrays, which allows simultaneous monitoring ble-stranded RNA (dsRNA)-binding protein of thousands of transcripts, has revealed “syn- DGCR8 (DiGeorge syndrome critical region 8) expression groups” of genes that are coregulated specifically recognizes and cleaves pri-miRNAs (Niehrs and Pollet 1999). FoxO and C/EBPb to generate precursor miRNAs (pre-miRNAs), proteins were identified as some of the first which are exported to the cytoplasm, where examples of transcription factors that control another RNase III Dicer generates a duplex the expression of TGF-b/Smad-regulated syn- of 19–22 nucleotides long. One strand of the expression groups (Gomis et al. 2006a,b). Fur- duplex is incorporated into the RNA-induced

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silencing complex, which posttranscriptionally epithelial cells and keratinocytes (Siegel and regulates hundreds of target mRNAs (Blahna Massague´ 2003, 2008). The extent of the growth and Hata 2013). inhibitory response to TGF-b varies depending TGF-b regulates directly or indirectly the on the cell type and the particular cell system expression of pri-miRNA transcript and the studied, and reaches a growth arrest in some cell generation of mature miRNAs. Among them, types (Tucker et al. 1984). Confirming obser- miRNA-200 family (miR-200a, -200b, -200c, vations in cell culture, evidence for an antipro- -141, and -429) and miR-205 are markedly liferative effect of TGF-b1 in vivo was first down-regulated in cells that have undergone obtained with polymer beads impregnated with epithelial-mesenchymal transition (EMT) in TGF-b1 and implanted near the epithelial end response to TGF-b. TGF-b indirectly represses buds of immature mammary glands (Silberstein miR-200 family members through induction and Daniel 1987). Also, intravenous injection of of ZEB1 (also known as dEF1) and ZEB2 TGF-b1or-b2 inhibits proliferation of the re- (also known as Smad-interacting protein 1, or generating rat liver (Russell et al. 1988). The SIP1) transcription factors, and miR-200 family cytostatic effects by TGF-b are mediated mainly members target and inhibit the expression of through two sets of mechanisms: induction of ZEB proteins, which stabilize regulatory net- cyclin-dependent kinase inhibitors and elimi- works for TGF-b-induced EMT (Gregory et nation of proliferative drivers. TGF-b induces al. 2008; Park et al. 2008). In addition, TGF-b expression of p15Ink4b, p21Cip1/Waf1, and activates processing of pri-miRNAs. R-Smads p27Kip1 (Hannon and Beach 1994; Polyak et al. interact with RNA helicase p68 (also known 1994; Datto et al. 1995a), and inhibits the ex- as DDX5), a component of the Drosha micro- pression of mediators that contribute to cell processor complex, to promote pri-miRNA proliferation, such as c-Myc (Pietenpol et al. processing by Drosha (Davis et al. 2008). The 1990), Cdc25A (Iavarone and Massague´ 1997), role of TGF-b signaling in the control of and Id proteins (Kang et al. 2003; Anido et al. miRNA generation is more extensively dis- 2010). In addition to direct effects of TGF-b cussed elsewhere (Butz et al. 2012; Hata and signaling on cell-cycle control, TGF-b opposes Lieberman 2015). the action of specific mitogens, such as EGF in Similarly to miRNAs, the emerging physio- keratinocytes (Coffey et al. 1988), or platelet- logical and pathological roles of lncRNAs have derived growth factor (PDGF) in rat fibroblast started to attract the interest of investigators. and embryonic cells (Roberts et al. 1985; An- LncRNAs are noncoding RNAs usually longer zano et al. 1986). How TGF-b signaling inter- than 200 nucleotides. Several lncRNAs have feres with the growth stimulation resulting from been reported to play essential roles in TGF-b- growth factor-induced receptor tyrosine kinase regulated biological processes. Future work will signaling has remained largely unexplored. confirm their biological significance. In contrast to the growth inhibition in many cell types, TGF-b can also promote cell prolif- eration in other cell types under certain condi- CONTROL OF CELL PROLIFERATION tions, as initially reported (Roberts et al. 1981). BY TGF-b TGF-b has been shown to stimulate prolifera- TGF-b ligands convey strong growth inhibitory tion of several cell types, including chondro- activity in most of the cell types, and this inhi- cytes, osteoblasts under some conditions, mes- bition is reversible after ligand removal (Tucker enchymal stem cells (MSCs), some fibroblasts, et al. 1984; Roberts et al. 1985; Ohta et al. 1987; and endothelial cells under some conditions Kimchi et al. 1988). Although the antiprolifer- (Roberts et al. 1981; Goumans et al. 2002; Jian ative response is observed in many cell types, et al. 2006). As mentioned, the effects are often including epithelial, endothelial, hematopoiet- context-dependent because TGF-b can induce ic, and glial cells, most knowledge about the both growth promotion and growth inhibition molecular mechanisms comes from studies in in the same cells depending on the cell culture

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Roles of TGF-b in Cell and Tissue Physiology

conditions (Roberts et al. 1985). Thus, not only phenotype in the immune system (Shull et al. may different cell types respond differently to 1992). In addition, mice deficient in TGF-b2 TGF-b, but also the same cell type may show and -b3 expression reveal defects in the central opposite responses under different experimen- nervous system (Vogel et al. 2010). tal conditions. The molecular mechanisms re- Among a wide variety of effects on many sponsible for inducing proliferation are less lineages, TGF-b ligands play essential roles in defined than those that lead to inhibition of determining the direction and extent of mesen- proliferation. In some contexts, the growth- chymal differentiation, as first shown in ecto- promoting effect seems to be secondary to in- derm explants from frog embryos (Kimelman duction of other cytokines such as PDGF (Leof and Kirschner 1987; Rosa et al. 1988). In cul- et al. 1986; Matsuyama et al. 2003; Bruna et al. tured cells, TGF-b1 inhibits differentiation to- 2007). The control of cell proliferation has been ward adipocytes (Ignotz and Massague´ 1985) reviewed elsewhere (Siegel and Massague´ 2003, and skeletal myocytes (Florini et al. 1986; Mas- 2008). sague´ et al. 1986). In contrast, TGF-b1 and -b2 enhance differentiation toward chondrocytes, consistent with their independent identification CONTROL OF CELL DIFFERENTIATION and purification as cartilage-inducing factors A AND STEMNESS BY TGF-b PROTEINS and B (CIF-A and CIF-B), respectively (Seye- TGF-b family members control the develop- din et al. 1985). Additionally, TGF-b promotes ment, differentiation, and function of diverse or represses the expression of differentiation cell types. For example, activin A, which uses markers by bone matrix-depositing osteoblasts, similar repertoires of receptors and intracellular depending on their extent of differentiation. In- signaling pathways as TGF-b, was identified as a hibition of endogenous TGF-b and activin sig- critical mesoderm-inducing factor in vivo (Asa- naling in human MSCs by pharmacological in- shima et al. 1990) as well as an erythroid differ- hibition of the ALK-4, -5, and -7 type I receptor entiation factor (EDF) (Eto et al. 1987; Yu et al. kinases, induces osteoblast maturation (Maeda 1987) and a nerve cell survival factor (Schubert et al. 2004), and transcriptome analysis further et al. 1990). Additionally, the Spemann’s orga- supports the notion that inhibition of ALK- nizer in early embryonic development secretes 5/TbRI-mediated TGF-b signaling enhances extracellular antagonists, such as follistatin, adipogenic and osteogenic differentiation, and which prevents activin signaling, and the BMP prevents chondrogenic differentiation (Ng et al. inhibitor noggin (Smith and Harland 1992; 2008). Hemmati-Brivanlou et al. 1994), which are crit- Complementing the control of MSC differ- ical for the establishment of morphogen gradi- entiation toward specific lineages, TGF-b helps ents of activins and BMPs. maintain MSCs in an undifferentiated state Similar to the developmental roles of (Jian et al. 2006). In serum-free media with ba- activin, TGF-b ligands are implicated in differ- sic fibroblast growth factor (FGF-2), PDGF-BB, entiation toward a wide variety of lineages, in- and insulin, TGF-b1 enables multiple passages cluding immune cells (Li and Flavell 2008), of MSCs, and inhibition of any of these signal- blood cells (Blank and Karlsson 2015), and neu- ing pathways decreases proliferation of MSCs, ral/neuronal cells (Krieglstein et al. 2011). Con- suggesting a requirement of these tyrosine ki- sistently, TGF-b2-deficient mice show defects nase-activated pathways for MSC proliferation in multiple organs, including heart, lung, cra- (Ng et al. 2008). Thus, the cooperation of TGF- niofacial, limb, spinal cord, eye, inner ear, and b with other cytokines plays important roles urogenital tracts (Sanford et al. 1997), and TGF- in the maintenance and expansion of MSC pop- b3-deficient mice display defects during pul- ulations before differentiation commitment, monary and palate development, suggesting while also restricting their differentiation po- its critical roles in EMT (Kaartinen et al. tential, inhibiting differentiation along the os- 1995), whereas TGF-b1-deficient mice show a teoblast, myoblast, and adipocyte lineages.

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TGF-b signaling also controls the mainte- the cancer-initiating cell populations in certain nance of the undifferentiated state and pluri- cancers, including breast cancer, pancreatic can- potency of embryonic stem cells (ESCs). In cer, and diffuse-type gastric cancer (Tang et al. mouse ESCs, leukemia inhibitory factor (LIF), 2007; Ehata et al. 2011; Hoshino et al. 2015). an interleukin 6 (IL-6) class cytokine, and These observations suggest that targeting the BMP-4 play essential roles in the maintenance TGF-b/activin-signaling pathways could be an of pluripotency. However, in this model of attractive therapeutic basis for some advanced “naı¨ve pluripotency,” the role of TGF-b and ac- cancers, although inhibition of these pathways tivin signaling remains to be better defined. in normal tissues may increase the risk for de- Whereas kinase inhibitors for TGF-b/activin velopment of other tumors. The roles of TGF-b type I receptors were reported to help maintain in cancer stem cells and cancer progression are the naı¨ve pluripotent state (Hassani et al. 2012), reviewed elsewhere (Caja et al. 2012; Katsuno nodal/activin signaling was also shown to en- et al. 2013). hance self-renewal of mouse ESCs (Ogawa et al. 2007). TGF-b CONTROLS WOUND HEALING Human ESCs, which share characteristics with mouse epiblast stem cells derived from Normal wound healing is a complex process postimplantation embryos, represent a more that involves (1) cell migration and inflamma- mature stage in pluripotency, and are “primed” tion, (2) proliferation of fibroblasts with forma- for differentiation. These cells are usually main- tion of granulation tissue and ECM deposition, tained with FGF-2 and activin A (Beattie et al. and (3) remodeling of scar tissue for an extend- 2005; James et al. 2005; Vallier et al. 2005), ed time period. TGF-bs have been shown to which stabilize the primed pluripotent state. regulate these different steps through effects They no longer require LIF, which has essential on multiple cell types, and to promote the roles in the naı¨ve state of mouse ESCs. Defined wound healing process in vivo. Indeed, early culture conditions were reported to achieve a studies, initiated shortly after the isolation and naı¨ve pluripotent state for human ESCs and, purification of TGF-b1, revealed that TGF-b1 in this context, two groups used TGF-b or acti- strongly accelerates wound healing in vivo vin ligands (Gafni et al. 2013; Theunissen et al. (Sporn et al. 1983). 2014) rather than their inhibitor(s). The roles of TGF-b1 expression and activation are rap- TGF-b family proteins in stem cells are reviewed idly induced in response to injury, progress- elsewhere (Oshimori and Fuchs 2012; Sakaki- ing outward from the site of injury (Kane Yumoto et al. 2013). et al. 1991). Platelets, which immediately after Accumulating evidence also indicates essen- wounding facilitate the formation of the hemo- tial roles of TGF-b/activin signaling in the static plug, store large amounts of TGF-b1 (As- maintenance of stem-cell-like properties of soian et al. 1983) that is then released at the site some cancer-initiating cell populations, such of injury. Because TGF-b1 acts as a potent che- as glioma-initiating cells (Ikushima et al. moattractant for monocytes (Wahl et al. 1987) 2009; Penuelas et al. 2009), mammary cancer and fibroblasts (Postlethwaite et al. 1987), the stem cells (Mani et al. 2008), pancreatic can- platelet-derived TGF-b1 is able to attract both cer–initiating cells (Lonardo et al. 2011), and cell populations to sites of inflammation and leukemia-initiating cells in chronic myeloid leu- repair. Accordingly, administration of TGF-b1 kemia (Naka et al. 2010). Consistent with these into wound chambers, or to incisional wounds, observations, pharmacological inhibition of stimulates the accumulation of granulation TGF-b/activin type I receptor kinases reduces tissue and the cellularization of the wound cancer progression in some animal models bed, and accelerates the wound healing re- (Ikushima et al. 2009; Penuelas et al. 2009; sponse in experimental models (Sporn et al. Naka et al. 2010; Lonardo et al. 2011). On the 1983; Roberts et al. 1986; Mustoe et al. 1987). other hand, TGF-b was also shown to reduce TGF-b1 is also a potent inducer of the expres-

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Roles of TGF-b in Cell and Tissue Physiology

sion of major ECM proteins, such as fibronectin phylactic use of recombinant TGF-b3 ligand in and collagens (Ignotz and Massague´ 1986; Rob- phase I/II trial (Ferguson et al. 2009). erts et al. 1986), thus promoting ECM deposi- tion at the site of wound healing. Furthermore, TGF-b CONTROLS THE IMMUNE SYSTEM TGF-b also induces inhibition of ECM degra- dation as a result of repressed expression of TGF-b acts as a potent immunosuppressive cy- metalloproteinases and induction of tissue in- tokine through effects on both cell differentia- hibitor of metalloproteinase synthesis (Edwards tion and cell proliferation. For example, TGF-b et al. 1987). inhibits proliferation of T-lymphocytes (Kehrl The repression of the epithelial phenotype et al. 1986) and thymocytes (Ristow 1986), and with its apical-basal polarity and epithelial junc- TGF-b2 was isolated as glioblastoma-derived tion architecture in response to TGF-b, and T-cell suppressor factor, based on the observa- concomitant induction of mesenchymal traits tion that glioblastoma is frequently accompa- with increased cell motility also contribute to nied by immunosuppression (de Martin et al. epithelial wound healing. At the site of injury, 1987). The key roles of TGF-b1 in suppressing epithelial cells undergo partial or complete immune responses also explain the rapid neo- EMT as a result of increased TGF-b signaling, natal development of overt inflammation in therefore promoting epithelial resurfacing, and TGF-b1-deficient mice (Shull et al. 1992). then reacquire their epithelial phenotype (Kal- In contrast, TGF-b2- and -b3-deficient mice luri and Weinberg 2009). TGF-b has also been display developmental defects (Kaartinen et al. shown to induce the expression of a-smooth 1995; Sanford et al. 1997), emphasizing their muscle actin (Desmouliere et al. 1993), a marker roles in development rather than in immune of myofibroblasts. In addition, TGF-b treat- regulation. Consistent with the phenotype of ment enhances acquisition of the myofibroblas- Tgfb12/2 mice, T-cell-specific expression of a tic phenotype, and costimulation with FGF-2 is dominant-negative TGF-b type II receptor shown to inhibit the process (Shirakihara et al. (TbRII) mutant, and T-cell-specific ablation 2011). This and other observations raise the of TbRII or TbRI/ALK-5 show that inhibition possibility that increased TGF-b stimulation of TGF-b signaling in T cells causes neonatal during wound healing may result in or contrib- lethal inflammatory disease. ute to the generation of myofibroblasts. Conversely, TGF-b1 promotes T-cell differ- Although TGF-b1, -b2, and -b3 act through entiation. Similarities in the autoimmune phe- the same receptors and Smad2/3 pathway, they notype of mice lacking expression of TGF-b1 show differences in the control of wound heal- (Tgfb12/2) or Foxp3, the transcription factor ing that remain to be fully explained. Adminis- required for CD4þCD25þ regulatory T cells tration of TGF-b1or-b2 promotes ECM de- (Treg) (Brunkow et al. 2001), led to the discov- position in the early stages of wound healing, ery that TGF-b, in combination with IL-2, but wounds treated with TGF-b1or-b2donot induces Treg cell differentiation through in- differ from control wounds in the final quality of duction of Foxp3 expression in CD4þ T cells scarring (Shah et al. 1995). However, inhibition (Chen et al. 2003; Liu et al. 2008). TGF-b is of TGF-b/Smad3 signaling, for example, in the also essential for the generation of IL-17- absence of Smad3 expression or in the presence expressing proinflammatory T helper cells of an antibody to TGF-b1and-b2, enhances the (TH17), in combination with IL-6 or -21 (Veld- quality of wound healing and reduces scarring hoen et al. 2006; Korn et al. 2007). Thus, TGF-b (Ashcroft et al. 1999; Amendt et al. 2002). Ad- can induce both regulatory/inhibitory and ditionally, administration of TGF-b3, but not proinflammatory T cells, depending on whether TGF-b1nor-b2, also reduces cutaneous scarring proinflammatory cytokines such as IL-6 are (Shah et al. 1995). Thus, recombinant TGF-b3 present. The roles of TGF-b in the immune sys- could be considered to promote scarless wound tem are reviewed elsewhere (Li and Flavell 2008; healing, which is supported by results from pro- Flavell et al. 2010).

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ANOMALOUS TGF-b ACTIVATION rysms and dissections, and aneurysms-osteoar- IN CONNECTIVE TISSUE AND SKELETAL thritis syndrome, are also caused by enhanced DISEASES TGF-b signaling. Mutations that affect TGF-b- signaling pathway components have been re- Gene mutations that result in dysregulation of ported; these include mutations in loci encoding TGF-b latency and confer increased release of TGFB2 (Boileau et al. 2012; Lindsay et al. 2012) activated TGF-b have been implicated in the or TGFB3 (Bertoli-Avella et al. 2015), as well as pathogenesis of connective tissue diseases and other signaling components or regulators, that skeletal diseases. Camurati–Engelmann disease is, TGFBR1 (Loeys et al. 2005, 2006), TGFBR2 (CED) is characterized by a progressive diaphy- (Mizuguchi et al. 2004; Loeys et al. 2005, 2006), seal dysplasia with hyperostosis and sclerosis of SMAD3 (van de Laaret al. 2011), and SKI (Doyle the diaphyses of long bones. Most individuals in et al. 2012). These syndromes share some clinical CED families have a mutation in the region of features in the aorta, including dilation of the the TGFB1 gene locus that encodes LAP (Jans- aortic root or ascending aorta and degeneration sens et al. 2000; Kinoshita et al. 2000), which of the medial layer of the aorta. Another com- affects LAP dimerization and results in hyper- mon feature is paradoxical activation of TGF-b activation of TGF-b signaling (Saito et al. 2001; signaling in aortic lesions, although they harbor Tang et al. 2009), whereas no mutations are loss-of-function mutations in TGF-b-signaling found in the domain encoding the mature components. The molecular mechanisms that TGF-b1 peptide. As an underlying mechanism, explain the paradoxical activation have not increased TGF-b may promote migration of been elucidated. The dysregulation of TGF-b bone marrow stromal cells to sites of bone re- signaling in connective tissue and skeletal dis- sorption (Tanget al. 2009). Accordingly, a type I eases has been discussed elswhere by Gallo receptor kinase inhibitor partially rescues the (Lindsay and Dietz 2011; Akhurst 2012). uncoupled bone remodeling in a mouse model for CED with the mutant TGF-b1 precursor b gene (Tang et al. 2009). INCREASED TGF- SIGNALING DRIVES FIBROSIS Mutations in the gene encoding fibrillin-1 have been causally linked to Marfan syndrome A pivotal role of TGF-b in fibrogenesis was first (Neptune et al. 2003), and their etiology high- revealed when subcutaneous injection of puri- lights important roles of TGF-b in Marfan fied TGF-b1 was seen to induce fibrotic lesions syndrome or Marfan-like connective tissue dis- at the injection site (Roberts et al. 1986). This orders. The fibrillins, which form extracellular potent effect was further confirmed by the fi- microfibrils, associate with LTBP-1, which in- brotic response in lungs of rodents following teracts with the LAP in the LLC, consequently intratracheal administration of adenovirus that linking this complex to elastic microfibrils (Iso- expresses active TGF-b1 (Sime et al. 1997), gai et al. 2003). The absence of fibrillin-1 in whereas neutralization of TGF-b with antise- Fbn1-deficient mice results in excessive TGF-b rum ameliorated experimental fibrosis in the activity in vivo, which could be at the basis of kidney, heart, and liver (Border et al. 1990; De- the pathogenesis of emphysema and other man- nis 1994; Kuwahara et al. 2002). In addition, ifestations of Marfan syndrome (Neptune et al. inhibitors or antagonists of the TGF-b/Smad 2003). Additionally, mutant fibrillin-1 disturbs pathway, such as soluble TbRII (George et al. the microfibril structure (Judge et al. 2004; 1999) or adenovirally expressed Smad7 (Nakao Charbonneau et al. 2010) and activates TGF-b et al. 1999), were shown to prevent liver and signaling, which may then over time generate lung fibrosis in mice, respectively. Most fibro- the pathological features of Marfan syndrome. genic effects resulting from increased TGF-b Clinically overlapping conditions, which re- signaling are thought to be mediated by the sult in aortic aneurysms, for example, in Loeys– Smad pathway because Smad3-null mice are Dietz syndrome, familial thoracic aortic aneu- resistant to bleomycin-induced lung fibrosis

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Roles of TGF-b in Cell and Tissue Physiology

(Zhao et al. 2002), radiation-induced skin fi- vitamin D receptor, PPAR-g (peroxisome pro- brosis (Flanders et al. 2002), and tubulo- liferator-activated receptor-g), and NR4A1, di- interstitial fibrosis following unilateral ureteral rectly repress fibroblast activation induced by obstruction (Sato et al. 2003). In addition, phe- TGF-b (Wu et al. 2009; Ding et al. 2013; Pa- notypes of integrin avb6-deficient mice support lumbo-Zerr et al. 2015). a role of integrin-mediated TGF-b activation in progressive fibrosis because bleomycin-induced TGF-b SIGNALING IN CANCER inflammation can occur without progression to PROGRESSION fibrosis in these mice (Munger et al. 1999). These and other observations suggest that dys- TGF-b induces an antiproliferative response in regulation and sustained activation of TGF-b/ many cell types, including both normal epithe- Smad3 signaling play essential roles in the ini- lial cells and transformed cells. Initial reports tiation and maintenance of the fibrotic tissue showed that antisense inhibition of TGF-b en- phenotype. hances tumorigenicity in vivo (Wu et al. 1992), The profibrotic effects of TGF-b involve a and that certain tumor cells can become un- combination of mechanisms and cell types, in- responsive to TGF-b, which was primarily cluding enhanced infiltration and/or prolifera- assessed by its effects on proliferation (Shipley tion of preexisting fibroblasts, generation of et al. 1986; Kimchi et al. 1988). It was, therefore, myofibroblasts, increased ECM synthesis, and assumed that disruption of TGF-b signaling is inhibition of collagenolysis, all of which are implicated in the pathogenesis of tumor devel- also apparent in wound healing. In addition, opment. In line with this hypothesis, inacti- lineage tracing strongly suggests important con- vating mutations and deletions in the TGFBR2 tributions of mesenchymal cells arising through and SMAD4 gene loci were found in cancers TGF-b-induced EMT. For example, in a model (Markowitz et al. 1995; Hahn et al. 1996) and of tubulointerstitial renal fibrosis, the use of the thought to drive tumorigenesis (reviewed in promoter region of the gene encoding g-gluta- Garraway and Lander 2013; Vogelstein et al. myltranspeptidase, a proximal tubule marker, 2013). for cell lineage tracing suggests that 36% of renal As advocated by Vogelstein and colleagues, fibroblasts derive through EMT from renal tu- the multistep tumorigenesis model (Fearon and bular epithelium (Iwano et al. 2002). Addition- Vogelstein 1990; Vogelstein et al. 2013) involves ally, using the promoter of surfactant protein the requirement of sequential genetic alter- C, a type II alveolar epithelial cell marker, for ations over time, which enables the develop- lineage tracing highlights a substantial con- ment of tumors and promotes cancer progres- tribution of EMT of alveolar epithelial cells sion. In colon cancers, for example, gatekeeping in TGF-b1-induced pulmonary fibrosis (Kim mutations most often occur in the adeno- et al. 2006). Endothelial cells have also been matous polyposis coli (APC) gene, and second identified as a source of fibroblasts in fibrosis. mutations in another gene, such as KRAS, then In heart fibrosis, for example, 27%–35% of all stimulate clonal growth and expansion. Muta- fibroblasts are estimated to originate from en- tions in genes such as PIK3CA, SMAD4, and dothelial cells through endothelial-mesenchy- TP53 follow this mutation process, resulting mal transition (Zeisberg et al. 2007). in more invasive and metastatic tumors. Con- Although TGF-b has been well established ceptually similarly, TGF-b’s function as a sup- as a key profibrotic cytokine, its activity is at- pressor of epithelial cell tumorigenesis may tenuated or aggravated by several other cyto- need to be alleviated at an early stage in tumor kines. For example, IL-1 and tumor necrosis development, which does not preclude roles of factor-a, which cross talk with TGF-b signaling TGF-b signaling at later stages. at multiple levels, are well-characterized profi- On the other hand, tumor cells show in- brotic cytokines. Conversely, some members of creased expression of TGF-b, most commonly the nuclear receptor superfamily, such as the TGF-b1, when compared with normal sur-

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rounding tissue, and secrete TGF-b ligands. Ad- multiple roles in developmental patterning, tis- ditionally, elevated TGF-b expression correlates sue differentiation, and maintenance of ho- with tumor progression and poorer prognosis, meostasis. As discussed, the biological effects indicating pro-oncogenic roles for TGF-b at of TGF-b family proteins are contextual, and late stages. Substantial evidence now supports even the same cell type may show different or the notion that the increased TGF-b expression opposite responses to the ligand under different by tumor cells promotes tumor progression by biological contexts. Some TGF-b family mem- enhancing migration, invasion, and survival of bers are made available to cells with concentra- tumor cells during later stages of tumorigenesis, tion gradients in vivo, resulting in graded effects through stimulating ECM deposition and tissue of cell differentiation and function by the re- fibrosis, perturbing immune and inflammatory sponsive cells. The activation of distinctive sets function, stimulating angiogenesis, promoting of genes by the ligand in a dosage-dependent EMT that enables increased migration and in- manner plays essential roles in the specification vasion, and maintaining cancer stem cells. of cell fates during normal development. Cells Platelets attached to tumor cells in the circula- also respond to TGF-b family proteins in com- tion provide an additional source of TGF-b that bination with other external stimuli. Costimu- promotes an invasive mesenchymal-like pheno- lation by other cytokines modifies the respon- type and enhances metastasis in vivo through siveness to the ligand, as observed in the stimulating the TGF-b/Smad and NF-kB path- immune system. In addition, the complexity of ways in the tumor cells (Labelle et al. 2011). the TGF-b family may reflect some functional With the sequential acquisition of genomic redundancy. It is, therefore, possible that some mutations, tumor cells were shown to display a family members could play redundant roles in range of mutations in different signaling path- the regulation of some biological processes. ways, which lead to changes in TGF-b respon- Therefore, detailed profiling of expression and siveness. This is illustrated by a model system activation of all TGF-b family ligands in each consisting of a set of human breast epithelial context could lead to a better understanding of MCF10A–derived cell lines, in which TGF-b the complex nature of TGF-b family members. functions as a tumor suppressor early in the car- Recent advances in single-cell assays have cinogenic process, but switches to a pro-onco- revealed intertumor heterogeneity between tu- genic agent in the later stage. Intriguingly, in the mors of the same histopathological subtype, most aggressive cell line, M-IV,which resembles and intratumor genetic and epigenetic hetero- a high-grade breast carcinoma, loss of TGF-b geneity within individual cancer masses. In ad- response does not affect primary tumorigenesis, dition, characterization of the transcriptome but suppresses metastasis (Tang et al. 2003). and histone modification markers in normal As illustrated with these observations, TGF- cells, such as mouse ESCs, similarly suggests b signaling controls both the initial develop- substantial cell-to-cell variability even in cells ment of tumors as well as the cancer progression with identical genetic backgrounds. It is, there- of a large variety of tumor types. Its roles in fore, possible that the heterogeneity could con- tumor suppression and its extensive roles in tribute to different responsiveness to TGF-b cancer progression through direct effects on family members. Thus, spatial and temporal the cancer cells themselves, as well as the micro- profiling of signaling at the single-cell level environment, are the subject of many reviews will help us to understand the complex mecha- (Bierie and Moses 2006; Ikushima and Miya- nisms of TGF-b family members. zono 2010; Pickup et al. 2013). REFERENCES CONCLUSIONS Akhurst RJ. 2012. The paradoxical TGF-b vasculopathies. Nat Genet 44: 838–839. The TGF-b family comprises a large number of Amendt C, Mann A, Schirmacher P, Blessing M. 2002. secreted and structurally related proteins with Resistance of keratinocytes to TGFb-mediated growth

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TGF-β and the TGF-β Family: Context-Dependent Roles in Cell and Tissue Physiology

Masato Morikawa, Rik Derynck and Kohei Miyazono

Cold Spring Harb Perspect Biol 2016; doi: 10.1101/cshperspect.a021873

Subject Collection The Biology of the TGF-β Family

TGF-β Family Signaling in Early Vertebrate TGF-β Family Signaling in Mesenchymal Development Differentiation Joseph Zinski, Benjamin Tajer and Mary C. Mullins Ingo Grafe, Stefanie Alexander, Jonathan R. Peterson, et al. Bone Morphogenetic Protein−Based Therapeutic TGF-β1 Signaling and Tissue Fibrosis Approaches Kevin K. Kim, Dean Sheppard and Harold A. Jonathan W. Lowery and Vicki Rosen Chapman TGF-β Family Signaling in Ductal Differentiation Bone Morphogenetic Proteins in Vascular and Branching Morphogenesis Homeostasis and Disease Kaoru Kahata, Varun Maturi and Aristidis Marie-José Goumans, An Zwijsen, Peter ten Dijke, Moustakas et al. TGF-β Signaling in Control of Cardiovascular TGF-β Family Signaling in Epithelial Function Differentiation and Epithelial−Mesenchymal Marie-José Goumans and Peter ten Dijke Transition Kaoru Kahata, Mahsa Shahidi Dadras and Aristidis Moustakas TGF-β Family Signaling in Tumor Suppression TGF-β Family Signaling in Connective Tissue and and Cancer Progression Skeletal Diseases Joan Seoane and Roger R. Gomis Elena Gallo MacFarlane, Julia Haupt, Harry C. Dietz, et al. Targeting TGF-β Signaling for Therapeutic Gain The TGF-β Family in the Reproductive Tract Rosemary J. Akhurst Diana Monsivais, Martin M. Matzuk and Stephanie A. Pangas Regulation of Hematopoiesis and Hematological TGF-β Family Signaling in Drosophila Disease by TGF- β Family Signaling Molecules Ambuj Upadhyay, Lindsay Moss-Taylor, Myung-Jun Kazuhito Naka and Atsushi Hirao Kim, et al.

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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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Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved