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Regulation of the Bioavailability of TGF-b and TGF-b-Related Proteins

Ian B. Robertson1 and Daniel B. Rifkin1,2

1Departments of Cell Biology, New York University School of Medicine, New York, New York 10016 2Departments of Medicine, New York University School of Medicine, New York, New York 10016 Correspondence: [email protected]; [email protected]

The bioavailability of members of the transforming growth factor b (TGF-b) family is con- trolled bya number of mechanisms. Bona fide TGF-b is sequestered into the matrix in a latent state and must be activated before it can bind to its receptors. Here, we review the molecules and mechanisms that regulate the bioavailability of TGF-b and compare these mechanisms with those used to regulate other TGF-b family members. We also assess the physiological significance of various latent TGF-b activators, as well as other extracellular modulators of TGF-b family signaling, by examining the available in vivo data from knockout mouse models and other biological systems.

he bioavailability of transforming growth tent TGF-b to its active form, a process known Tfactor b (TGF-b) family ligands is not on- as activation. ly regulated by the release of the growth factor In the last decade, it has become clear that from cells, but it is also controlled by extracel- the properties of the TGF-b LLC are critical in lular mechanisms that modulate, inhibit, acti- modulating the action of the cytokine and that vate, or enhance the binding of these signaling controlled release of TGF-b from the ECM by molecules to their receptors. activation is central to understanding TGF-b This is especially true of the bona fide TGF- signaling in a larger biological context. Here, b dimer; as when TGF-b is secreted from cells it we review the extracellular regulation of TGF-b is tightly bound in a latent complex consisting and examine the available in vivo data to place of its dimeric pro-peptide (referred to as la- these regulatory mechanisms in a physiological tency-associated peptide [LAP]), and a latent context. TGF-b-binding protein (LTBP). This tripartite complex of TGF-b, LAP, and LTBP is called the BIOSYNTHESIS AND LATENCY OF TGF-b large latent complex (LLC). Within this com- b plex, LAP confers latency to the cytokine and TGF- Processing, Latency, and Interaction with LTBPs the LTBP functions to direct and sequester the growth factor into the extracellular matrix TGF-b is initially translated as a 50-kDa pro- (ECM) and assists in the conversion of the la- protein containing both growth factor and

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.a021907 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a021907

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I.B. Robertson and D.B. Rifkin

LAP. This pro-protein is vectorially discharged (Dubois et al. 1995; Hyytiainen et al. 2004). The into the endoplasmic reticulum where it dimer- complex of TGF-b and LAP is referred to as the izes and folds (Fig. 1, lower right). Within the small latent complex (SLC). endoplasmic reticulum, the dimeric pro-TGF-b One of the remarkable properties of the is linked to a single LTBP by a pair of disulfide TGF-b latent complex is the fact that the inter- bonds between LTBPand LAP (Miyazono et al. action of LAP with the growth factor is of such 1991; Gleizes et al. 1996; Saharinen et al. 1996). high affinity that all TGF-b isoforms are secret- The pro-peptide (LAP) is subsequently cleaved ed as part of a latent complex, either LLC or from the mature cytokine in the trans-Golgi by SLC. The one known exception is Camurati– furin or furin-type enzymes (Fig. 1, lower left) Engelmann disease, in which patients have spe-

4. Matrix association 5. Activation

Numerous proposed factors

Fibronectin fibers

Fibrillin microfibril

Active TGF-β released from Modulating latent complex factors

Type II Type I 3. Secretion receptor receptor

P LAP Type III SLC receptor TGF-β LLC 6. Receptor binding

LTBP 1. Folding and dimerization

2. Processing pro-TGF-β pro-LTBP

trans-Golgi Furin cleavage ER LLC folded and assembled

Figure 1. Scheme for the secretion and extracellular regulation of transforming growth factor b (TGF-b). Starting at the bottom right (1), TGF-b and latent TGF-b-binding protein (LTBP) are translated into the endoplasmic reticulum (ER) where pro-TGF-b dimerizes and is then disulfide bonded to LTBP to form a ternary complex. The TGF-b dimer is cleaved from its pro-peptide (latency-associated peptide [LAP]) in the trans-Golgi network, but TGF-b and LAP remain strongly associated via noncovalent interactions forming the large latent complex (LLC) (lower left) (2). Once secreted (middle left) (3), the LTBP may bind various matrix fibers that sequester latent TGF-b until it is released by an activator (upper left) (4). The latent complex is then activated (upper right) (5), by one of several potential mechanisms, releasing the mature TGF-b. Active TGF-b may bind to cell-surface receptors (lower right) (6), although other factors may also bind the active growth factor at this stage and either inhibit or promote receptor binding.

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Regulation of the Bioavailability of TGF-b

cific mutations in TGF-b1 LAP or in the TGF- Significant amounts of mature TGF-b are also b1 signal peptide that yield increased levels of secreted when HEK-293T cells are transfected secreted, constitutively active TGF-b that may with TGF-b expression constructs alone (Wal- be the cause of the bone thickening observed in ton et al. 2010). Thus, the pro-TGF-b dimer can this rare condition (Saito et al. 2001; Janssens properly fold and be processed on its own, but et al. 2003). covalent interaction with an LTBP may still en- LTBPs may serve a chaperone-like function hance the rate of folding and secretion. for pro-TGF-b by enhancing its folding and se- Unlike the TGF-b LAP interactions, which cretion (Miyazono et al. 1991). Without LTBP, are noncovalent, the binding of LAP with LTBP the cysteines in LAP that bind LTBP may form is mediated by a pair of disulfide bonds between incorrect disulfide bonds, and this complex the most amino-terminal cysteines in LAP and would be degraded within the cell (Brunner a unique cysteine pair in LTBP. The LTBPs are et al. 1989). This may account for the enhanced large multidomain glycoproteins that consist of secretion of TGF-b in the presence of LTBP tandem arrays of calcium-binding epidermal (Miyazono et al. 1991; Rifkin 2005) and is con- growth factor–like domains (cbEGFs), 8-Cys sistent with the heightened secretion of TGF-b1 type domains (which can be subcategorized C33S compared with wild-type protein (Annes into TGF-b-binding [TB] domains and hybrid et al. 2004). However, some cell types, such asthe domains), and regions with no clear domain osteosarcoma line UMR-106, produce only the homology that introduce flexibility into these SLC, and the ROS 17/2.8 line produces a mix- proteins (Fig. 2) (Robertson et al. 2014). LTBPs ture of both SLC and LLC (Dallas et al. 1994). bind LAP through a pair of cysteines in the third

Transglutaminase LAP binding Fibrillin binding cross-linking

LTBP1 (L) ?

LTBP2

LTBP3 ?

LTBP4 (L)

Fibrillin1

Signal EGF cbEGF Hybrid domain TB domain Fibrillin unique peptide (8-cys domain) (8-cys domain) carboxy-terminal domains Fibrillin unique Novel domain with 4-cys domain Regions containing no amino-terminal eight cysteines (LTBPs only) recognized domains (FUN) domain (LTBP4L only)

Figure 2. The multidomain structure of latent transforming growth factor b (TGF-b)-binding proteins (LTBPs). Domain structures of the four LTBPs found in the human genome, along with human fibrillin-1 for comparison. Some regions associated with specific functions are highlighted in LTBP-1. The key at the bottom of the figure describes the different domain types. L in LTBP-1 and -4 denotes the long form. The question marks on domains in LTBP-1L and LTBP-3 denote that it is unclear from their sequences whether these EGF domains bind calcium. EGF, Epidermal growth factor domain; cbEGF, calcium-binding epidermal growth factor domain.

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I.B. Robertson and D.B. Rifkin

8-Cys domain (or second TB domain) (Gleizes both long and short forms of LTBP-1 resulted in et al. 1996; Saharinen et al. 1996), but within the only minor craniofacial abnormalities (Drews LTBP family (LTBP-1, -2, -3, and -4), there is et al. 2008). The lack of a more severe phenotype variability with respect to the ability to bond to appears to be a result of compensatory splicing LAP (Saharinen and Keski-Oja 2000). LTBP-1 around the deleted exon (Todorovic and Rifkin and -3 bind well to all three TGF-b isoforms, as 2012), and a study of mice with conditional de- shown by cotransfection assays, whereas LTBP-2 letion of exon 8, which prevents expression of does not bind at all, and LTBP-4 binds only to both isoforms of LTBP-1, showed similar lethal TGF-b1-LAP and does so inefficiently. LTBP-4- cardiovascular defects to those seen with LTBP- binding to LAP may be enhanced in an amino- 1L-null mice (Horiguchi et al. 2015). Gene tar- terminally extended LTBP-4 splice form, but the geting of LTBP-2 on the other hand results in nature and significance of this binding are un- only minor defects, such as disorganization and clear (Kantola et al. 2010). detachment of the ciliary zonules (Inoue et al. The binding of LTBPto the TGF-b pro-pep- 2014). These results are consistent with LTBP- tide is primarily determined by the availabilityat 2’s inability to bind LAP and suggest LTBP-2 the protein surface of a cysteine pair in the third may be important for microfibril assembly. 8-Cys (second TB) domain. LTBP-1, -3, and -4 LTBP-3-null mice are viable but display each contain a dipeptide insertion found only in bone defects, including osteopetrosis and pre- TB domains that bind to LAP (Saharinen and mature ossification of the skull synchondroses Keski-Oja 2000). The dipeptide is missing in the (Dabovic et al. 2002; Colarossi et al. 2005), phe- third 8-Cys domain of LTBP-2 and in all other notypes that indicate reduced TGF-b signaling. 8-Cys domains of the LTBPs and the structural- Interestingly, morpholino gene silencing of ly related fibrillins. Addition of the dipeptide LTBP-3 in zebrafish causes cardiovascular de- into the LTBP-2 third 8-Cys domain converts a fects not seen in the mouse model (Zhou et al. nonbinding domain into a LAP-binding do- 2011). These defects were phenotypically simi- main. The initial docking interaction between lar to those caused by inhibition of TGF-b LAP and LTBP involves a number of acidic and signaling and were rescued by expression of a basic residues, the importance of which has been constitutively active TGF-b receptor, demon- highlighted by mutagenesis studies (Chen et al. strating the importance of LTBP-3 in facilitating 2005; Walton et al. 2010). The higher number of TGF-b action. negatively charged residues arranged around the Studies of mice with a disrupted Ltbp4 gene reactive cysteines may explain the more effective revealed multiple developmental defects includ- binding of the third 8-Cys domains of LTBP-1 ing impaired alveolar septation, cardiomyopa- and -3 to SLC compared with LTBP-4. Secretion thy, and rectal prolapse (Sterner-Kock et al. of LTBP-3 is dependent on association with 2002), phenotypes reflected in human patients TGF-b (Penttinen et al. 2002), but LTBP-1, -2, with LTBP-4 deficiency (Urban et al. 2009). Re- and -4 are secreted efficiently without bound duced deposition of extracellular TGF-b and re- TGF-b (Saharinen et al. 1999). ductions in phospho-Smad2 in various tissues The potential functions of all four LTBPs in were also seen in this study. However, detailed vivo have been investigated using LTBP-null examination of the lungs of Ltbp4-null animals mice (Robertson et al. 2015). LTBP-1 and -4 revealed a surprising increase in phospho- exist as both long (L) and short (S) forms gen- Smad2 (Dabovic et al. 2009). Furthermore, erated by the use of separate promoters and ini- breeding these mice onto a Tgfb22/2 back- tiation codons. Deleting exons specific to the ground rescued the alveolar septation defect generation of the long form of LTBP-1 in mice (Dabovic et al. 2009), suggesting that this defect results in cardiac outflow tract defects that cause was actually caused by increased TGF-b signal- embryonic lethality, potentially a result of de- ing. This rescue effect was specific to TGF-b2as creased levels of TGF-b (Todorovic et al. 2007, a Tgfb12/2 or Tgfb32/2 background did not 2011). Interestingly, deleting an exon shared by rescue the lung defect (Dabovic et al. 2014). To

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Regulation of the Bioavailability of TGF-b

investigate the importance of the LTBP-4–LAP Interactions between the LLC and the ECM interaction, a mouse was generated in which the cysteines (Cys 1235 and 1260) in LTBP-4 respon- Besides promoting effective secretion of latent sibleforputativebindingtoLAPweremutatedto TGF-b from cells, another major function of serinetoinhibitdisulfidebondformation.These LTBPs is localization of latent TGF-b to the mice appeared phenotypically normal (Dabovic ECM (Fig. 1, upper left) (Koli et al. 2005). et al. 2014), which suggests that sequestering This feature has many important implications TGF-b via LAP is not a significant function of for latent TGF-b activation and TGF-b bio- LTBP-4. Additional evidence shows that the pri- availability. mary role of LTBP-4 appears to be the regulation The best-studied interactions of an LTBP of elastic fiber assembly through its interactions with the ECM are those of LTBP-1. Initial stud- with fibulin-5 and fibrillin (Noda et al. 2013; ies using immunofluorescence to monitor Dabovic et al. 2014). This makes LTBP-1 and binding of recombinant fragments to matrix LTBP-3 the LTBPs of greatest interest when con- showed that LTBP-1 possessed two ECM-bind- sidering the extracellular pool of latent TGF-b. ing regions at the amino terminus and one at Latency is dependent solely on the presence the carboxyl terminus (Unsold et al. 2001). Fur- of LAP as mutant TGF-b1 LAP (C33S) that ther experiments showed specific interactions cannot bind LTBP still maintains TGF-b in with fibronectin (Dallas et al. 2005a; Fontana the latent state (Gentry and Nash 1990; Annes et al. 2005; Kantola et al. 2008) and fibrillin et al. 2004). This SLC is efficiently secreted with- fibers (Isogai et al. 2003; Ono et al. 2009; Mas- out LTBP (Yoshinaga et al. 2008). However, the sam-Wu et al. 2010), as well as the formation Cys33 residue in LAP plays an important role in of transglutaminase cross-links with the ECM TGF-b1 function in vivo, as mice with the C33S (Nunes et al. 1997). The specific ECM compo- TGF-b1 mutation develop inflammation that is nent to which LTBP-1 is cross-linked was not similar to that observed in TGF-b1-null mice determined. (although not as severe), implying defective ac- The last three carboxy-terminal domains of tivation of SLC (Yoshinaga et al. 2008). These LTBP-1 interact with the amino terminus of fi- mutant mice also develop gastrointestinal tu- brillin (Isogai et al. 2003; Ono et al. 2009), and mors (Yoshinaga et al. 2008; Shibahara et al. this region of LTBP-1 contains flexible linkers 2012). If the primary function of this cysteine that may facilitate this interaction (Robertson is binding to LTBP, these results reflect the cru- et al. 2014). The carboxyl termini of LTBP-2 and cial importance of SLC–LTBP interactions for LTBP-4 are closely homologous with the car- proper extracellular TGF-b function. boxyl terminus of LTBP-1 and interact with fi- Substitution of Cys223 and Cys225 in TGF- brillin in a similar manner (Isogai et al. 2003; b1 LAP with serines, on the other hand, pre- Ono et al. 2009). LTBP-3, on the other hand, has vents the dimerization of LAP and results in the strikingly different sequence features at its car- secretion of constitutively active TGF-b (Brun- boxyl terminus (Robertson et al. 2011, 2014), ner et al. 1989). This mutant form of TGF-b has and a recombinant fragment of this region of been used in many transgenic studies that show LTBP-3 does not interact with the fibrillin ami- the destructive potential of the release of con- no terminus in solid phase assays, suggesting stitutively active TGF-b and highlight the im- that the LTBP-3 carboxyl terminus may have portance of latency in proper TGF-b function distinct properties (Isogai et al. 2003). The in- (Sellheyer et al. 1993; Sanderson et al. 1995). teraction of LTBP-1 with fibronectin has not However, although these transgenic studies pro- been well characterized, and, in some assays, vide useful information on the effects of a gen- this interaction is mediated by heparan sulfate eralized abundance of active TGF-b, they are proteoglycans (Chen et al. 2007). Fibronectin is less informative for examining the more target- essential for the incorporation of LTBP-1 into ed mechanisms of latent TGF-b localization extracellular fibers in cell culture. Studies con- and activation that occur in normal tissues. ducted by Ono et al. showed LTBP-1 ECM in-

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I.B. Robertson and D.B. Rifkin

corporation by murine neonatal fibroblasts that cellular responses to a defective matrix ac- (cultured for 13 days) was dependent on fibril- tually cause the increase in TGF-b activity. Ad- lin-1 (Ono et al. 2009). However, more recently, ditionally, direct inhibition of TGF-b in young Zilberberg et al. (2012) showed that fibrillin-1 is mice can actually worsen the aortic phenotype not essential for incorporation of LTBP-1 into (Cook et al. 2015), whereas loss of LTBP3 in the ECM of aortic smooth muscle cells or pri- fibrillin-deficient mice protects them from de- mary lung fibroblasts. But fibrillin-1 is still re- veloping aortic aneurysms (Zilberberg et al. quired for matrix incorporation of LTBP-3 and 2015). Both studies show that the relationship LTBP-4 in these cultures (Zilberberg et al. between fibrillin and TGF-b may be more com- 2012), suggesting a link between LTBP-3 and plex and nuanced than first thought. the fibrillin matrix, although no direct inter- Disruptions of the ECM associated with action between these molecules has yet been enhanced levels of active TGF-b have been re- shown. The assembly of fibrillin fibers is also ported in a variety of genetic diseases of con- dependent on fibronectin in most cell cultures nective tissue, including multiple myopathic (Kinsey et al. 2008; Sabatier et al. 2009), making states (Cohn et al. 2007), fibromuscular dyspla- it difficult to tease apart the relative importance sia (Ganesh et al. 2014), and geleophysic and of the fibrillin and fibronectin networks in LTBP acromicric dysplasias (Le Goff et al. 2011; Le incorporation and TGF-b regulation. Goff and Cormier-Daire 2012). This may indi- The functional significance of TGF-b’s cate that a primary response to abnormal ma- ECM localization is still unclear. Some clues trix is the activation of latent TGF-b. One report may derive from studies of Marfan syndrome of particular interest shows dysregulated TGF-b (MFS), a genetic condition caused by mutations signaling in mouse models of osteogenesis im- in the fibrillin-1 gene resulting in a number of perfecta (OI), a brittle bone disease usually symptoms, including tall stature, arachnodac- caused by mutations in type I collagen genes tyly, ectopia lentis, and aortic dilatation. Fibril- (Grafe et al. 2014). Mouse models of both dom- lin is an important structural component of the inant and recessive forms of OI showed increas- ECM (Jensen et al. 2012), but interestingly the es in TGF-b signaling, and bone density was reduction of fibrillin-1 levels in MFS also leads significantly improved by treatment with the to increased levels of active TGF-b (Neptune TGF-b neutralizing antibody 1D11 (Grafe et et al. 2003). Defects in MFS mice, such as im- al. 2014). These observations reinforce the idea paired alveolar septation, mitral valve prolapse, of latent TGF-b activation as a process respon- and aortic dissection, can be prevented by inhi- sive to generalized perturbations in matrix bition of TGF-b signaling (Judge et al. 2004; Ng structure and integrity. et al. 2004; Habashi et al. 2006, 2011; Cohn et al. Recently, LTBP-1 has been shown to bind 2007). One hypothesis is that an insufficiency of and colocalize with extracellular deposits of fibrillin-1 fibers leads to a shortage of available Notch3 present in the cerebral arterioles of ce- sites to anchor latent TGF-b in the matrix, re- rebral autosomal dominant arteriopathy with sulting in excess soluble LLC available for acti- subcortical infarcts and leukoencephalopathy vation. An alternative hypothesis is based on the (CADASIL) patients, and although the precise observation that fibronectin contributes to la- implications for TGF-b signaling are unclear, tent TGF-b activation by integrins by providing this further highlights the potential sensitivity cell-ECM traction (see below). Therefore, per- of the LLC to extracellular perturbations (Kast turbing the strength or structure of the ECM by et al. 2014). reducing fibrillin-1 levels in MFS could directly affect latent TGF-b activation via traction, rath- Other Sources of Latent TGF-b er than passively releasing more latent cytokine from the matrix. It is also conceivable that per- As well as LTBPs, latent TGF-b binds covalently turbations of LTBP–fibrillin interactions are to the trans-membrane leucine-rich repeat pro- not directly involved in MFS pathology and tein glycoprotein-A repetitions predominant

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Regulation of the Bioavailability of TGF-b

protein (GARP) (Wang et al. 2012). GARP ex- with ECM components. Fibrillin, in particular, pression appears restricted to regulatory T cells interacts directly with the pro-peptides of a and platelets (Wang et al. 2008; Stockis et al. number of TGF-b family members, including 2009; Tran et al. 2009), whereas LTBPs are ex- bone morphogenetic protein 2 (BMP-2), BMP- pressed ubiquitously (Rifkin 2005). GARP-like 4, BMP-7, BMP-10, and growth and differen- leucine-rich repeat proteins do not appear until tiation factor 5 (GDF-5) (Sengle et al. 2008a). the evolution of bony fish (Robertson and Rif- Surface plasmon resonance studies showed kin 2013), although TGF-b and LTBPs evolved that the pro-peptides of these growth factors earlier, as they are found in sea urchins, acorn interact with the amino-terminal regions of fi- worms, and lancelets (Robertson et al. 2011; brillin-1 and -2 with similar affinities (Gregory Robertson and Rifkin 2013), suggesting that et al. 2005; Sengle et al. 2008a), and some pro- LTBPs may have more general functions in peptides associate with other regions of the fi- TGF-b biology, with GARP activities restricted brillin molecule. The binding sites for BMP-7 to T-cell and platelet-based functions. In fact, and BMP-5 have been localized more precisely use of anti-GARP antibodies has been reported in the fibrillin amino terminus and have been to suppress the activity of regulatory T-cells and shown to be dependent on the presence of the aid with anticancer immunotherapy (Cuende fibrillin unique amino-terminal (FUN) domain et al. 2015). (Sengle et al. 2008a, 2011; Yadin et al. 2013). E-selectin-ligand-1 binds intracellular pro- However, not all pro-domains of the TGF-b TGF-b (Olofsson et al. 1997); however, its role family members interact with the amino termi- appears to be that of a negative regulator of nus of fibrillin. For example, the myostatin TGF-b by inhibiting pro-TGF-b intracellular (GDF-8) pro-domain does not bind this region, processing and secretion (Yang et al. 2010). but it does associate with the ECM via perle- The existence of other extracellular latent TGF- can domain V glycosaminoglycan (GAG) side b complexes, independent of LTBPs, cannot be chains (Sengle et al. 2011). ruled out. Mice lacking expression of TGF-b1 Although the interactions between latent and TGF-b3 (Mu et al. 2008), or TGF-b2 and growth factors and fibrillin have been clearly TGF-b3 (Dunker and Krieglstein 2002) display shown in vitro, their in vivo significance is significant deleterious effects in embryonic de- unclear. BMP-7 and BMP-4 colocalize with fi- velopment, but double-null mutations of LTBP- brillin-1 in tissue sections (Gregory et al. 2005; 3 and LTBP-4 are viable after birth and do not Sengle et al. 2008a), suggesting that these in- show clear phenotypic similarities with TGF- teractions occur in vivo, but their importance b-null mutations (Dabovic et al. 2011). As pre- is unknown. Functional hypotheses might be viously mentioned LTBP-1-null mice display proposed similar to those suggested for LLC lethal defects in embryonic heart development binding to fibrillin; for example, BMP binding (Todorovic et al. 2007, 2011; Horiguchi et al. to fibrillin may localize and direct growth fac- 2015), but direct phenotypic similarities with tor signals to certain regions of the ECM, and TGF-b-null mice are not clearly apparent. Inac- so determine specific features of tissue develop- tivation of both LTBP-1 and LTBP-3 in the same ment (Ramirez and Rifkin 2009). TGF-b, BMP- mice has not yet been reported, but these may be 10, and myostatin are latent when complexed the most informative for testing the overall sig- with their pro-domains and unable to induce nificance of LTBPs in TGF-b regulation in vivo. signaling without the help of an activator, for example, BMP-1 can activate BMP-10 by cleav- ing its pro-domain (Lee and McPherron Latency and ECM Interactions of Other TGF-b 2001; Sengle et al. 2011). However, BMP-4, -5, Family Members -7, and -9 pro-domain complexes are not la- TGF-b is not alone in its localization to ECM tent, and their pro-domains can be displaced fibers; many other members of the TGF-b fam- from the growth factor allowing these molecules ily and their pro-peptides form associations to bind to their receptors without activation

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I.B. Robertson and D.B. Rifkin

(Brown et al. 2005; Sengle et al. 2008b, 2011). ranging from extremes of pH, to proteases, to The fibrillin interaction might confer “latency” cell-surface integrins. to growth factors not rendered latent by their pro-peptides alone, as localization to the ECM ACTIVATION BY PROTEINS may shield the growth factors from cell-surface Integrins receptors. Signaling then could be regulated by displacement of the growth factor from fibrillin Cell-surface integrins are well-established acti- or contact of fibrillin microfibrils with the cell. vators of latent TGF-b (Munger et al. 1999; Alternatively, fibrillin interaction could pro- Annes et al. 2002; Mu et al. 2002). Integrins mote growth factor activity by preventing in- avb6 and avb8 are the best described activators, hibitor binding. but other av integrins, such as avb1, avb3, and Increased BMP activity in fibrillin-1 defi- avb5, have also been implicated as interacting cient osteoblasts is consistent with the hypoth- with LAP and activating latent TGF-b (Munger esis that fibrillin may inhibit BMPs activity et al. 1998; Lu et al. 2002; Ludbrook et al. 2003; (Nistala et al. 2010), but whether this involves Wipff et al. 2007; Wipff and Hinz 2008; Munger a direct mechanism of fibrillin-binding BMP and Sheppard 2011; Tatler et al. 2011; Hinz pro-domains was not tested. 2013; Sarrazy et al. 2014). Clues to the in vivo mechanisms by which The best-studied mechanism for integrin- fibrillins regulate BMP activity are also sparse mediated activation of latent TGF-b is direct but there are some suggestive data. For example, activation by traction between cells and matrix fibrillin-2-null mice display syndactyly, whereas (Fig. 3A). Early studies showed that overexpres- BMP-7-deficient mice display polydactyly (Ar- sion of b6 integrin-activated latent TGF-b1, teaga-Solis et al. 2001). Heterozygous Bmp7þ/2 even in the presence of protease inhibitors or Fbn2þ/2 mice are normal, but a combined (Munger et al. 1999). Further studies showed heterozygous deficiency of BMP-7 and fibrillin- that latent TGF-b1 activation by avb6 was de- 2 yields mice with both polydactyly and syn- pendent on covalent attachment of LAP to dactyly. These enhanced digit abnormalities in LTBP-1 (Annes et al. 2004), the presence of spe- double heterozygotes may indicate a form of cific ECM-binding regions in the hinge domain epistasis. In fibrillin-2 deficient mice transcrip- of LTBP-1 (Annes et al. 2004), and the presence tion of several genes, including Msx, was ren- of a fibronectin matrix (Fontana et al. 2005). dered insensitive to implanted BMP-4 beads. The integrin-binding RGD (Arg-Gly-Asp) se- These results suggest that fibrillin-2 and BMPs quence in LAP and the cytoplasmic sequences play interrelated roles in specific developmental of b6 integrin that anchor it to the actin cyto- pathways, but the full significance of their inter- skeleton were also required for TGF-b activa- action is unclear. tion (Munger et al. 1999; Annes et al. 2004). In summary, there are several pieces of These results are consistent with an activation in vivo and in vitro evidence connecting the mechanism whereby the conformation of LAP ECM and signaling by other molecules of the anchored to the ECM by LTBPand bound to the TGF-b family, but the precise mechanisms at cell surface by integrins is distorted by traction work are not yet understood. between matrix and cells, liberating active TGF- b (Annes et al. 2004; Fontana et al. 2005; Wipff et al. 2007). This mechanism has been further ACTIVATION OF LATENT TGF-b elaborated by experiments describing the integ- The latent TGF-b complex can be considered as rin-dependent release of active TGF-b by con- a sensor that remains in the ECM until triggered tracting myofibroblast cytoskeletons (Wipff by a specific signal to release TGF-b (Annes et al. 2007), as well as from cell-free matrix in et al. 2003); a process referred to as activation a force-dependent manner by ferromagnetic (Fig. 1, upper right). A number of different la- beads coated with either integrins or anti-LAP tent TGF-b activators have been described, antibodies (Buscemi et al. 2011). Indeed, the

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Regulation of the Bioavailability of TGF-b

Integrin-mediated TGF-β activation mechanisms A Traction-mediated activation

Cell surface

α β Integrin (e.g., v 6)

LAP RGD RGD RGD RGD Actin cytoskeleton anchors s s ss s s s s integrins and can exert traction TGF-β

s s s s s s s s

LTBP1

Strain between matrix TGF-β is released and cell surface deforms LAP Insoluble ECM fibers

B Protease-mediated activation

α β Integrins v 8 Cell surface LAP RGD RGD ? RGD LAP degraded and s s MT1-MMP s s s s TGF-β s s TGF-β is released

s s s s

s s s s

Figure 3. Proposed mechanisms of latent transforming growth factor b (TGF-b) activation by integrins. Two mechanisms have been proposed by which integrins activate latent TGF-b.(A) Traction between cells and extracellular matrix (ECM) is transmitted to latency-associated peptide (LAP) via latent TGF-b-binding pro- teins (LTBPs) bound to the matrix and cell-surface integrins bound to the cell’s actin cytoskeleton. This deforms LAPand releases active TGF-b.(B) Integrins bind LAPat the cell surface and this may make LAP more accessible to proteases, such as membrane-type matrix metalloproteinase 1 (MT1-MMP), that cleave LAP and release the active growth factor, by localizing it and recruiting proteases into its vicinity, or by changing the structure of LAP so that it is rendered more protease sensitive.

state of the latent complex may be sensitized for by the presence of matrix metalloproteinase activation by previous modifications of the ma- (MMP) inhibitors and did not require the trix (Klingberg et al. 2014). The traction mech- cytoplasmic domain of avb8, indicating a trac- anism is supported by the crystal structure of tion-independent mechanism (Mu et al. 2002). TGF-b bound to LAP, which shows that Cys33, This suggests an alternative protease-dependent required for LTBP attachment, and the RGD mechanism, perhaps involving recruitment of motif are on opposite sides of the molecule, MMPs by integrins to facilitate LAP cleavage with TGF-b sandwiched between two arms of and release of TGF-b (Fig. 3B). Alternatively, a LAP “straightjacket” (Shi et al. 2011). This binding by the integrin may induce changes in straightjacket can be deformed by traction ap- LAP structure that promote protease cleavage. plied to the molecule, as shown by atomic force MT1-MMP (membrane-type matrix metallo- microscopy experiments (Buscemi et al. 2011). proteinase 1) was suggested as a potential can- The mechanism of latent TGF-b activation didate protease, as it both conferred the ability by avb8 is more obscure. Latent TGF-b activa- to activate latent TGF-b, when transfected into tion by this integrin was reported to be blocked the avb8-expressing cancer line H1264 and co-

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I.B. Robertson and D.B. Rifkin

localizes with integrin avb8 at substrate contacts mouse models (Henderson et al. 2013), and (Mu et al. 2002). However, as described below, deletion of integrin b6 helps protect against ra- inactivation of MMPs does not produce a TGF- diation-induced lung fibrosis (Puthawala et al. b, null-like phenotype. 2008). TGF-b is also an important mediator of As well as activating latent TGF-b within the immune tolerance, and conditional deletion of LLC, both integrins avb6 and avb8 can activate b8 integrin in dendritic cells causes severe in- latent TGF-b bound to GARP on the surface of flammatory bowel disease and autoimmunity cells expressing this complex (Wang et al. 2012; (Travis et al. 2007). Integrin b8 expressed by Edwards et al. 2014). This form of latent TGF-b effector regulatory T cells is also important for may play an important role in regulating im- suppressing aberrant T-cell-mediated inflam- mune responses, as GARP is primarilyexpressed mation (Worthington et al. 2015), consistent by T-cells. with the role of this integrin in activating The critical nature of integrin-mediated la- TGF-b and promoting immune tolerance. In- tent TGF-b activation in vivo was illustrated by tegrin b8 expressed by lung fibroblasts also plays a mouse model in which the integrin-binding a critical role in activating TGF-b to regulate RGD motif in TGF-b1-LAP was replaced with dendritic cell trafficking in lung tissue, which an RGE sequence (Yang et al. 2007; Aluwihare can in turn contribute to fibrosis and inflam- et al. 2009). These mice phenocopy TGF-b1- mation (Kitamura et al. 2011). In addition, in- null mice and display severe multiorgan inflam- tegrin b8 expressed on dendritic cells regulates mation and absence of epidermal Langerhans maturation of TH17 cells, which contribute to cells. As the mutant TGF-b is secreted normally, autoimmunity (Melton et al. 2010), and inte- this phenocopy shows that the integrin-binding grin b8-mediated TGF-b activation also inhibits RGD motif in TGF-b1 LAP is essential for la- epithelial proliferation in bronchial tissue (Fjell- tent TGF-b1 activation. TGF-b3 also contains birkeland et al. 2003). Therapeutic strategies an RGD motif, the importance of which has not using monoclonal antibodies to target TGF-b been conclusively tested in vivo. However, mice activation by integrins have shown promise in with null mutations in both avb6 and avb8 treating fibroinflammatory airway disease in integrins display cleft palate, also observed in animal models (Minagawa et al. 2014). TGF-b3 knockout mice. Pharmacological in- As well as playing roles in fibrosis and in- hibition of avb6 in mice lacking avb8 causes flammation, TGF-b activation by integrin b8 inflammation similar to that seen in TGF-b1- plays a role in wound closure in vitro (Neurohr deficient mice (Yanget al. 2007; Aluwihare et al. et al. 2006) and may act as an angiogenic control 2009). These observations suggest that integrins switch regulated by perivascular astrocytes play a key role in the in vivo activation of both (Cambier et al. 2005). Specific expression of TGF-b1 and 3. Replacement of the TGF-b3 integrin b8 by nonmyelinating Schwann cells coding sequence with a TGF-b1 sequence into in bone marrow may assist in maintaining dor- the Tgfb3 locus partially rescues the palate clo- mancy in hematopoietic stem cells by generat- sure defect seen in TGF-b3-null mice (Yangand ing a highly localized niche of active TGF-b Kaartinen 2007), indicating that TGF-b1 LAP near blood vessels (Yamazaki et al. 2011). shares some critical properties with TGF-b3 Unlike TGF-b1 and 3, TGF-b2 lacks an RGD LAP, as latent TGF-b1 can be appropriately ac- motif and was not activated in experiments test- tivated in place of TGF-b3 in this context. ing latent TGF-b activation by specific integrins In addition to being important for latent (Annes et al. 2002). TGF-b2 contains other con- TGF-b activation during development, inte- served motifs in the RGD region that are not grins also play a significant role in diseases like found in TGF-b1 and -b3 (Robertson and Rif- fibrosis, in which TGF-b is a major mediator kin 2013), which suggests the possibility that (Leask and Abraham 2004). Conditional dele- TGF-b2 is the target of specific activators that tion of the av integrin gene significantly atten- bind TGF-b2 LAP in a similar fashion to inte- uated hepatic, pulmonary, and renal fibrosis in grin binding to TGF-b1 and TGF-b3 LAP RGD.

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Regulation of the Bioavailability of TGF-b

Proteases phenotypes are reminiscent of defective TGF-b signaling. A number of proteases of various classes can The BMP-1/tolloid MMPs also play a role activate latent TGF-b in vitro, including cys- in latent TGF-b activation. However, rather teine proteases like calpain (Abe et al. 1998), than directly degrading LAP, they catalyze the aspartyl proteases like cathepsin D (Lyons cleavage of LTBP-1 from the ECM, releasing la- et al. 1988), and numerous serine and metal- tent TGF-b bound to LTBP-1, which requires loproteases (Maeda et al. 2001; Jenkins 2008). activation by other factors, such as MMPs However, the relevance of these assorted prote- (Ge and Greenspan 2006). Mice with null mu- ases for latent TGF-b activation in vivo is un- tations in BMP-1 and the closely related tolloid1 clear, as mice deficient in individual proteases (Tll1) protease display excessive accumulation fail to display TGF-b, null-like phenotypes. of LTBP-1 in embryonic tissues and reduced Although this may seem to suggest a minor bi- phospho-Smad2/3 staining, suggesting defec- ological role for protease-mediated activation, tive activation of latent TGF-b (Ge and Green- it may also reflect the general ability of multiple span 2006). Mice deficient in BMP-1 or Tll1 proteases to activate latent TGF-b, creating sig- expression die before birth and these proteins nificant redundancy in vivo. process a variety of other matrix and signal- Many of the MMPs, including MMP2, ing molecules; therefore, it is difficult to attain MMP9, MMP3, MMP13, and MMP14 (MT1- a clear assessment of their importance for latent MMP) (Jenkins 2008), activate latent TGF-b in TGF-b activation in vivo (Pappano et al. 2003). vitro. MMP2 and MMP9 activate latent TGF-b Various serine proteases, including kalli- b when localized by CD44 to the surface of tumor kreins and plasmin, activate TGF- (Lyons et al. 1990). Plasmin mediates TGF-b activation cells during angiogenesis (Yu and Stamenkovic in cocultures of endothelial cells and pericytes 2000), and MMP3 from chondrocyte-derived (Antonelli-Orlidge et al. 1989; Sato and Rifkin matrix vesicles activates latent TGF-b1 (Maeda 1989), in cultures of endothelial cells treated et al. 2001). Interestingly, although MMP2 is with retinoids (Kojima and Rifkin 1993), and present in these chondrocyte vesicles, it did on the surface of activated macrophages (Nunes not activate TGF-b in this context. et al. 1995; Yehualaeshet et al. 1999; Jenkins Mice deficient in many of these proteases 2008). Activation in endothelial-pericyte cocul- have been generated, but none clearly show tures was inhibited by antibodies to LTBP-1 TGF-b-related phenotypes. Inactivation of (Flaumenhaft et al. 1993) and inhibitors of the MMP3 yields micewith novisible abnormalities mannose-6-phosphate receptor (Dennis and (Mudgett et al. 1998; Johnson et al. 2011), and Rifkin 1991). Activation by macrophages was loss of MMP13 yields only mild skeletal defects dependent on plasmin and latent TGF-b bound in mice (Stickens et al. 2004). Mice lacking ex- at the cell surface by thrombospondin1 (TSP1) pression of both MMP2 and MMP9 are viable and CD36 (Yehualaeshet et al. 1999; Jenkins (Garg et al. 2009), as are mice lacking MMP9 2008). Despite the importance of plasmin in and MMP13 expression (Stickens et al. 2004), several TGF-b-activating cell systems, mice although these mice are runted because of with targeted deletion of the plasminogen defects in their growth plates. MT1-MMP gene show no clear developmental abnormali- (MMP14)-deficient mice suffer from osteope- ties and are viable (Bugge et al. 1995), which nia, dwarfism, and fibrosis, but this is likely a rules out a nonredundant role for this protease result of deficiencies in collagen turnover and in latent TGF-b activation. not because of insufficient TGF-b activation Kallikreins are a large family of serine pro- (Holmbeck et al. 1999). MMP2- and MT1- teases, several of which activate latent TGF-b. MMP-deficient mice display respiratory failure, Kallikreins appear to play a role in TGF-b-me- abnormal blood vessel development, imma- diated immunosuppression in seminal plasma ture muscle fibers, and die immediately after (Emami and Diamandis 2010) and lipopoly- birth (Oh et al. 2004). However, none of these saccharide impairment of liver regeneration

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I.B. Robertson and D.B. Rifkin

(Akita et al. 2002). Plasma kallikrein (PLK) TSP-1 has been the subject of particularly cleaves TGF-b1 LAP between Arg58 and intense study with respect to latent TGF-b ac- Leu59. Antibodies have been raised against the tivation. Recombinant TSP-1 activates latent resulting LAP peptides and used to show that TGF-b when added to cell cultures or purified the specific PLK-LAP cleavage products are sig- latent TGF-b in vitro (Murphy-Ullrich and Poc- nificantly more abundant in the liver tissue of zatek 2000). TSP-1 interacts with both LAP and patients with hepatic fibrosis (Hara et al. 2014). active TGF-b in copurification and pull-down Prostate-specific antigen (PSA), a member of assays (Murphy-Ullrich et al. 1992; Yang et al. the kallikrein family, activates TGF-b2 but not 1997; Ribeiro et al. 1999). A KRFK peptide that TGF-b1 (Dallas et al. 2005b). The large number mimics the RFK sequence between the first and of kallikrein proteases again provides significant second TSP-1 domains activates latent TGF-b potential redundancy and, at present, only lim- (Schultz-Cherry et al. 1994, 1995) and inhibits ited data on kallikrein-null mice are available TSP-1 binding. The ability of an LSKL peptide (Bergaya et al. 2001). to inhibit this activation further suggested that Although many proteases activate latent interactions between the RFK motif of TSP-1 TGF-b in a variety of contexts, most of these and the LSKL motif in the amino terminus of studies have been conducted in vitro, and ob- LAP were responsible for TGF-b release (Ri- taining clear in vivo data on the role of proteases beiro et al. 1999). Consistent with this hypoth- in latent TGF-b activation is difficult for the esis is the observation that TSP-2 does not acti- reasons discussed above. One way to gain future vate latent TGF-b, as the RFK motif is absent insight into the significance of protease-medi- from TSP-2 (Schultz-Cherry et al. 1995). ated latent TGF-b activation in vivo might be to Although these are interesting results, some identify specific cleavage sites in LAP or LTBP groups have failed to observe latent TGF-b ac- responsible for activation, and generate mice tivation in response to TSP-1 (Grainger and with mutations replacing these sites with prote- Frow 2000), and surface plasmon resonance ase-insensitive sequences. studies failed to show TSP-1 binding to either LAP, TGF-b, or SLC (Bailly et al. 1997). More- Deglycosylation over, platelet a-granules activate latent TGF-b and are a rich source of TSP-1, but a-granules Deglycosidases, including endoglycosidase F derived from TSP-1-null platelets activate latent (Miyazono and Heldin 1989) and influenza TGF-b just as effectively as those from wild- neuramidase, activate latent TGF-b (Schultz- type platelets (Abdelouahed et al. 2000). Cherry and Hinshaw 1996; Carlson et al. TSP-1-null mice suffer from inflammation 2010). The LAPs of human TGF-b1 and -b2 of the lung and pancreas as well as various other possess three potential N-glycosylation sites, defects (Lawler et al. 1998). The sites of inflam- whereas TGF-b3 LAP has only two sites. It is mation overlap, in part, with those observed in not clear which sites are important foractivation the multiorgan inflammation phenotype of by deglycosidases or what the activation mech- TGF-b1 knockout mice (Kulkarni et al. 1993; anism may be. One possibility is that removing Crawford et al. 1998). Reduced inflammation sugar groups exposes protease-sensitive sites, as was observed in Tsp12/2 animals treated with activation by neuramidase is at least partially the latent TGF-b-activating KRFK peptide, protease dependent (Carlson et al. 2010). The whereas treating wild-type mice with the LSKL in vivo role of deglycosylation-driven latent peptide that inhibits activation resulted in in- TGF-b activation also remains undefined. creased inflammation (Crawford et al. 1998). However, Tsp12/2 mice fail to mimic all of Other Protein Factors the features of TGF-b1-null mice, such as As well as the molecules discussed above, a di- cardiac inflammation. Tsp12/2 animals also verse range of other proteins have been impli- display phenotypes, like kyphosis, not seen cated as latent TGF-b activators. in TGF-b1-null animals. Moreover, although

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Regulation of the Bioavailability of TGF-b

TSP-1 and the KRFK peptide-activated latent cluding detergents, ionizing, and ultraviolet TGF-b2 in vitro (Ribeiro et al. 1999), TSP-1- (UV) radiation (Barcellos-Hoff et al. 1994; Bar- null animals do not display any of the serious cellos-Hoff 1996; Ehrhart et al. 1997; Wang and defects seen in TGF-b2 knockout mice. This Kochevar 2005; Anscher et al. 2006; Biswas et al. leaves the debate over the exact role of TSP-1 2007), reactive oxygen species (ROS) (Barcel- in vivo unresolved, but rules out TSP-1 as a los-Hoff and Dix 1996), heat (Brown et al. central activator of all three TGF-b isoforms. 1990), physical shear (Ahamed et al. 2008), F-spondin (or spondin-1) increases TGF-b and extremes of pH (Lyons et al. 1988). activity in cartilage explants (Attur et al. 2009). Not all of these conditions are encountered An antibody targeted to the F-spondin throm- in vivo, but some may be biologically relevant. bospondin type 1 repeats blocks this activation, For example, ROS activate latent TGF-b both in suggesting parallels with activation by TSP-1. cell cultures and in cell-free systems (Barcellos- However, more information is needed on Hoff and Dix 1996), and ROS are proposed to whether F-spondin activates latent TGF-b by mediate latent TGF-b activation by asbestos, binding directly to LAP or if some other mech- which can lead to mesothelioma in exposed anism is at work in these cultures. lungs (Pociask et al. 2004). ROS produced by Neuropilin (Nrp) is a cell-surface protein HIV-infected regulatory T cells are also pro- reported to act as an activator of latent TGF-b posed to activate latent TGF-b and lead to im- on the surface of T cells (Glinka and Prud’- munosuppression (Amarnath et al. 2007). ROS homme 2008; Glinka et al. 2011) and in some may mediate latent TGF-b activation caused by tumor lines (Glinka et al. 2011). The mecha- ionizing (Barcellos-Hoff 1996) and UV-B radi- nism by which Nrp activates latent TGF-b re- ation (Wang and Kochevar 2005). One study mains undefined, but in solid-phase assays Nrp suggested that ROS activation was specific to binds mature TGF-b1, LAP,and both type I and TGF-b1 and relied on the presence of Met253 II TGF-b receptors (Glinka et al. 2011). Nrp (Jobling et al. 2006), but this residue is not well may initiate intracellular signaling events and conserved (Robertson and Rifkin 2013) and the could act as a co-receptor for TGF-b (Glinka exact mechanism by which ROS activate latent and Prud’homme 2008; Glinka et al. 2011; TGF-b is unclear. One possibility is that ROS Grandclement et al. 2011). catalyze direct breaks in the backbone of LAP to Pregnancy specific glycoprotein 1 (PSG1) is release the active growth factor (Barcellos-Hoff another protein with no known enzymatic ac- and Dix 1996), but it is not known if LAP is tivity recently shown to activate both latent particularly sensitive to ROS-driven scission. TGF-b1 and 2. PSG1 also inhibits dextran so- Latent TGF-b activation by low pH could dium sulfate–induced colitis in a TGF-b-de- play a physiological role in some circumstances. pendent manner (Blois et al. 2013). However, Osteoclasts reduce extracellular pH to 4.5 both the molecular mechanism and in vivo sig- during bone resorption (Teitelbaum 2000), nificance of this activator remain unknown. Te- and these cells activate latent TGF-b (Oreffo nascin-X has also been reported to promote et al. 1989). Tumor cells also significantly lower epithelial to mesenchymal transition by activat- the pH of their microenvironment (Jullien et al. ing TGF-b through interactions between its 1989). However, whether the drop in pH is both fibrinogen-like domain and the SLC. These in- necessary and sufficient for latent TGF-b acti- teractions may deform LAP and also rely on cell vation in this later context has not been clearly adhesion via integrin a11b1 to promote activa- shown. Lactic acid also promotes TGF-b activ- tion (Alcaraz et al. 2014). ity at physiological concentrations and may be a player in idiopathic pulmonary fibrosis (Kott- mann et al. 2012). But, although the ability of Activation by Physicochemical Factors lactate to up-regulate a-smooth muscle actin Latent TGF-b can be activated by exposure to was dependent on TGF-b signaling and the specific physical or chemical conditions, in- lowered pH of the medium, studies of lactic

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I.B. Robertson and D.B. Rifkin

acid in other contexts have shown that lactate in the most evolutionary distant TGF-b and up-regulates genes that may affect TGF-b bio- LTBPsequences, including those in sea urchins, availability (Seliger et al. 2013). The effects of lancelets, and acorn worms (Robertson et al. physiological levels of lactic acid on latent TGF- 2011; Robertson and Rifkin 2013). Additional- b in a cell free system have not yet been reported. ly, integrin-binding RGD motifs are also present Physical shear or stirring can activate a sig- in these evolutionary distant TGF-b sequences nificant percentage of the latent TGF-b present and are highly conserved through to humans in platelet releasate (Ahamed et al. 2008). This (except in TGF-b2, where they are consistently reaction is dependent on LTBP binding to LAP, absent). as activation is not seen with the releasate from These facts suggest that all elements re- C33S TGF-b1 mice (Ahamed et al. 2008). The quired for traction-induced latent TGF-b acti- full activation mechanism is not known, but it is vation by integrins were present soon after inhibited by the thiol isomerase-binding pep- “true” TGF-b diverged from the rest of the tide mastoparan and appears to involve disul- TGF-b family. If integrins were initially the fide exchange (Brophy et al. 2013). main activators of latent TGF-b, this would be Although there is compelling evidence for consistent with their continued in vivo signifi- many physicochemical factors playing impor- cance, as observed in mouse models (Yang et al. tant roles in TGF-b biology, the current lack 2007; Aluwihare et al. 2009). On the other hand, of detailed activation mechanisms and specific the evolutionary data do not support an early residues to target for knockin studies prevent us evolving function for TSP-1, plasmin, or many from testing their true significance in vivo. other specific proteases, which in many cases do not seem to emerge until well after the LTBP– TGF-b complex first appeared (Robertson and ACTIVATION OF TGF-b—AN Rifkin 2013). EVOLUTIONARY PERSPECTIVE If we make the assumption that the earliest The range of latent TGF-b activation mecha- evolving latent TGF-b activators have remained nisms may seem daunting, especially when try- important throughout evolution, traction-me- ing to consider what the trigger for TGF-b ac- diated activation by integrins may continue to tivity may be in different tissue contexts. In vivo be an important mechanism for releasing TGF- studies knocking out specific activators or key b in many tissue contexts. Therefore, we pro- activation sequences cast some light on their pose that future studies examining the role of relative significance. However, these experi- TGF-b in development and homeostasis should ments are difficult and time consuming and so give careful consideration the dynamic relation- there remain many potential activators whose ship between cells and their biomechanical en- significance has not yet been tested. vironment. An alternative approach to qualitatively gauge the significance of various latent TGF-b activators is to examine the evolutionary time POSTACTIVATION BIOAVAILABILITY OF TGF-b AND OTHER TGF-b FAMILY frames in which they emerge and to analyze the MEMBERS conservation of residues critical to specific acti- vation mechanisms. One generalization that distinguishes bona fide This evolutionary analysis has been per- TGF-b from many other members of the family, formed for a variety of established latent TGF- such as the BMPs and GDFs, is that TGF-b bio- b activators and has provided a number of in- availability is primarily regulated by binding sights (Fig. 4) (Robertson et al. 2011; Robertson and release from LAP, which locks up TGF-b and Rifkin 2013). One interesting observation is before it leaves the cell. However, the biological that bona fide TGF-b seems to have coevolved regulators characterized for BMPs and GDFs with LTBPs and that the key sequence features generally bind to the active forms of these for the attachment of LTBPand LAP are present growth factors after they have been secreted.

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Evolutionary history of TGF-β and its regulatory proteins H. sapiens Mammals (Human) –LTBP3+4 G. gallus (Chicken) Birds Kallekreins A. carolinensis Reptiles –LTBP2 (Lizard) GARP TGF-β3 TSP1 with RFK motif β X. tropicalis Divergence of TGF- 1, 2, and 3 Amphibians Divergence of LTBP1, 2, 3, and 4 (Frog)

Neuropilin L. chalumnae Duplication of TGF-β and LTBP (Coelacanth) Lobe-finned fishes –LTBP2 Fibronectin D. rerio MMP2/9 (Zebra fish) Fishes

P. marinus Lampreys Plasmin ? (Lamprey) –LTBP? C. intestinalis Tunicates Bona fide TGF-β (Sea Squirt) LTBP B. floridae Lancelets (Lancelet)

S. purpuratus Echinoderms (Urchin)

S. kowalevskii Hemichordates Fibrillin (Acorn worm)

Lophotrochozoa Ecdysozoa Follistatin (molluscs and + (arthropods and F-spondin annelids) nematodes)

N. vectensis Cnidarians (Anemone)

T. Adhaerens Placazoa (Trichoplax)

A. Queenslandica Porifera (Sponge) TGF-β family, α and β integrins, Tolloid-like proteases, MMPs emerge with or before the evolution of metazoa (animals)

Figure 4. Evolution of transforming growth factor b (TGF-b) and its extracellular regulators. Phylogenetic tree highlighting the evolutionary relationships between a selection of organisms with sequenced genomes. Green arrows show where specific proteins appear to have evolved based on their presence or absence from available genome and other sequence libraries. Red crosses indicate where specific genes appear to have been lost. The variable quality of some shotgun genome sequences means that the absence of a gene from a specific species is not always definitive, as some genes may be missed or fragmented in the available sequence data. This introduces some uncertainty to the evolutionary story, but ambiguity is kept to a minimum by searching through the many different genomes available. GARP,Glycoprotein A repetitions predominant protein; TSP1, thrombospondin 1; LTBP, TGF-b-binding protein; MMP, matrix metalloproteinase; H. sapiens, Homo sapiens; G. gallus, Gallus gallus; A. carolinensis, Anolis carolinensis; X. tropicalis, Xenopus tropicalis; L. chalumnae, Latimeria chalumnae; D. rerio, Danio rerio; P. marinus, Perkinsus marinus; C. intestinalis, Ciona intestinalis; B. floridae, Branchiostoma floridae; S. purpuratus, Strongylocentrotus purpuratus; S. kowalevskii, Saccoglossus kowalevskii; N. vectensis, Ne- matostella vectensis; T. Adhaerens, Trichoplax Adhaerens; A. Queenslandica, Amphimedon Queenslandica.

The release of certain BMPs and GDFs by spe- the extracellular space. These regulation mech- cific cells can generate morphogen gradients, anisms have been discussed in more detail else- and their antagonists help to localize and con- where (Hinck et al. 2016; Kim et al. 2016), but fine the activity of these growth factors once in they are mentioned here in passing to highlight

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key conceptual differences between signaling by proliferation (Bi et al. 2005). Other molecules bona fide TGF-b and signaling by other mem- that may interact with active TGF-b include bers of the family. collagen IV in the basement membrane (Paral- The simplified concepts of growth-factor kar et al. 1991), human IgG (Stach and Rowley signaling by localized activation or morphogen 1993; Bouchard et al. 1995), and the cell-surface gradients are shown in Figure 5. In a morpho- proteins b glycan (also known as TGF-b type III gen gradient, the active growth factor is released receptor) and endoglin, which may play a role as by a specific cellular source and its concentra- TGF-b co-receptors. tion reduces as it diffuses away from that source. It is important to consider that TGF-b is a Further control may also be introduced by an- “sticky” molecule that associates nonspecifical- tagonists that prevent the diffusing growth ly with hydrophobic surfaces. TGF-b is active at factors from binding their receptors and add very low concentrations and copurifies with a further 3D structure to the growth-factor gra- number of His-tagged proteins when they are dient. The active concentration of a growth expressed and purified from HEK293 cells factor in any given region of the morphogen (Kaur and Reinhardt 2012), as well as with gel- gradient can provide individual cells with an atin-purified fibronectins (Fava and McClure indication of their relative position within a de- 1987). These properties further complicate in- veloping organism, which may help define their terpretation of in vitro experiments, and under- niches within a tissue. In the localized activation line the importance of in vivo studies to confirm model, on the other hand, the growth factor is proposed biochemical mechanisms. predistributed throughout the ECM in its latent To ensure that the TGF-b signal does not state. Its local concentration is determined by persist after activation, mature TGF-b is rapidly the rate of activation rather than its rate of re- cleared from the extracellular space. a2-Macro- lease by cells and its diffusion from a central globulin binds TGF-b and may mediate its en- source. One might speculate that this mode of docytosis (LaMarre et al. 1990, 1991). However, activation may allow bona fide TGF-b to act as a a2-macroglobulin knockout mice have no clear more direct readout of local extracellular events defects to suggest excessive accumulation of ac- rather than a long distance signal of develop- tive TGF-b in tissues (Umans et al. 1995), and mental status. so the full mechanisms by which active TGF-b is Although we currently consider factors that cleared from the ECM remain a mystery. regulate TGF-b latency and activation to be the Although the biological data on how active key controllers of its signaling, there are also a TGF-b is regulated after its release from LAP variety of proteins that bind and regulate active remain patchy, antagonists of other members TGF-b after its release from LLC. For example, of the TGF-b family have been well described the proteoglycan decorin binds active TGF-b and shown to play significant roles in vivo. (Hildebrand et al. 1994; Schonherr et al. 1998) Myostatin (GDF-8) and activin, for example, and appears to inhibit TGF-b signaling in many are more closely related to TGF-b than many contexts (Isaka et al. 1996; Teicher et al. 1997), other TGF-b family members (Burt and Law although early studies of decorin suggested that 1994; Herpin et al. 2004), and although not it could enhance TGF-b bioactivity in the bone much is known about the significance of their matrix (Takeuchi et al. 1994). Decorin can pro-peptides in their regulation, follistatin and inhibit TGF-b signaling through calcium-de- follistatin-related proteins play a significant role pendent phosphorylation of Smad2 at Ser240 in controlling these two growth factors by bind- (Abdel-Wahab et al. 2002), and may interface ing and blocking their interaction with recep- with TGF-b signaling on a more complex level tors (Walton et al. 2011). The interplay of my- than just binding the active growth factor. In the ostatin and follistatin has been particularly well bone marrow stroma, both decorin and bigly- examined in the context of muscle growth, can appear to work together to inhibit TGF-b where loss of myostatin causes widespread mus- signaling and maintain sufficient osteoblast cle hypertrophy and hyperplasia (McPherron

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A Morphogenic gradient Growth factor diffuses Cells expressing antagonist help away from source control and shape gradient

B Latent cytokine activation Localized activator ECM

Figure 5. Conceptual mechanisms for the extracellular regulation of transforming growth factor b (TGF-b) and bone morphogenetic protein (BMP)/growth and differentiation factor (GDF) compared. Growth factors may convey information to cells in a number of ways. Shown here are some hypothetical schemes for ways in which active growth factors may be distributed in the extracellular matrix (ECM). Cells are colored purple to indicate their responsiveness to growth-factor signaling, or light blue if unresponsive. (A) Here, cells are provided with a measure of their position relative to the source of the growth factor, as its concentration will decrease when diffusing away from the site of release. Antagonists further control this gradient, which becomes even more relevant when considering the three dimensions of a developing tissue. Growth factors are light green and antagonists light brown. (B) Here, the growth factor does not come from a localized source but is distributed through the matrix in a latent state. The local concentration of growth factor is determined by the availability of activators to release it from this state. Also, depending on how quickly the growth factor is cleared from the extracellular space, it may not have time to diffuse far, which could limit gradient formation. Growth factors are red, whereas the ECM and proteins that anchor them are dark green. These different concepts could be important when considering growth-factor signaling from a systems biology perspective.

et al. 1997). Transgenic overexpression of folli- 2006). A number of BMPantagonists share little statin in skeletal muscle causes similar muscle clear sequence homology, but several contain a overgrowth (Lee and McPherron 2001), but fol- “cystine-knot” structural motif, which is a high- listatin also stimulates muscle hypertrophy ly versatile fold that often forms dimers and is through myostatin-independent mechanisms found in a diverse selection of ECM proteins. (Winbanks et al. 2012). Follistatin-null mice For example, cystine-knot motifs also comprise display multiple developmental defects, reflect- the growth factor domains of the TGF-b family ing its important role in regulating bioavailabil- (Vitt et al. 2001), suggesting a common, if ex- ity of both activin and myostatin (Matzuk et al. tremely distant, evolutionary ancestor for both 1995). the TGF-b family and many of its antagonists. A diverse array of other proteins is hypoth- Noggin, chordin, and DAN family proteins all esized to regulate the bioavailability of TGF-b interact with multiple members of the BMP family members by binding and sequestration. family and are crucial in the biology of these Unlike TGF-b, many BMPs are biologically ac- signaling proteins. tive when they are secreted from cells, and form There are clearly a large number of both morphogenic gradients (Gazzerro and Canalis TGF-b family members and their antagonists.

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I.B. Robertson and D.B. Rifkin

The full significance of this diversity is far from absence of thrombospondin-1. J Biol Chem 275: 17933– well understood, and the ability of multiple 17936. Abdel-Wahab N, Wicks SJ, Mason RM, Chantry A. 2002. TGF-b family members to bind multiple antag- Decorin suppresses transforming growth factor-b-in- onists and receptors introduces significant po- duced expression of plasminogen activator inhibitor-1 tential for redundancy, multifunctionality, and in human mesangial cells through a mechanism that in- volves Ca2þ-dependent phosphorylation of Smad2 at ser- the formation of complex protein gradients in a ine-240. Biochem J 362: 643–649. developing organism. This complexity may, in Abe M, Oda N, Sato Y. 1998. Cell-associated activation of turn, play an important role in directing the latent transforming growth factor-b by calpain. J Cell elaborate patterns adopted by our organs and Physiol 174: 186–193. tissues during development. Ahamed J, Burg N, Yoshinaga K, Janczak CA, Rifkin DB, Coller BS. 2008. In vitro and in vivo evidence for shear- induced activation of latent transforming growth factor- b1. Blood 112: 3650–3660. CONCLUDING REMARKS Akita K, Okuno M, Enya M, Imai S, Moriwaki H, Kawada N, The bioavailability of TGF-b and TGF-b family Suzuki Y, Kojima S. 2002. Impaired liver regeneration in mice by lipopolysaccharide via TNF-a/kallikrein-medi- members is regulated in complex and diverse ated activation of latent TGF-b. Gastroenterology 123: ways. It is rarely appropriate to consider these 352–364. growth factors as simple autocrine or paracrine Alcaraz LB, Exposito JY,Chuvin N, Pommier RM, Cluzel C, Martel S, Sentis S, Bartholin L, Lethias C, Valcourt U. signals sent from one cell to another. Instead, 2014. Tenascin-X promotes epithelial-to-mesenchymal they must be considered in the broader struc- transition by activating latent TGF-b. J Cell Biol 205: tural context of the ECM, both in terms of how 409–428. they may be sequestered, stored, and concen- Aluwihare P, Mu Z, Zhao Z, Yu D, Weinreb PH, Horan GS, Violette SM, Munger JS. 2009. Mice that lack activity of trated although interactions with insoluble ma- avb6- and avb8-integrins reproduce the abnormalities trix components, and also how these growth of Tgfb1- and Tgfb3-null mice. J Cell Sci 122: 227–232. factors will be regulated by gradients and local- Amarnath S, Dong L, Li J, Wu Y,Chen W.2007. Endogenous ization of specific inhibitors and activators. TGF-b activation by reactive oxygen species is key to Foxp3 induction in TCR-stimulated and HIV-1-infected This field poses many complex challenges human CD4þCD252 T cells. Retrovirology 4: 57. for future study, which will require extensive Annes JP, Rifkin DB, Munger JS. 2002. The integrin aVb6 and coordinated efforts to decipher the key binds and activates latent TGFb3. FEBS Lett 511: 65–68. mechanisms at work and their significance in Annes JP, Munger JS, Rifkin DB. 2003. Making sense of health and disease. However, the considerable latent TGF-b activation. J Cell Sci 116: 217–224. b Annes JP, Chen Y, Munger JS, Rifkin DB. 2004. Integrin therapeutic potential of modulating TGF- aVb6-mediated activation of latent TGF-b requires the family signaling in an extracellular context latent TGF-b-binding protein-1. J Cell Biol 165: 723– makes this research a particularly pressing en- 734. deavor. Anscher MS, Thrasher B, Rabbani Z, Teicher B, Vujaskovic Z. 2006. Antitransforming growth factor-b antibody 1D11 ameliorates normal tissue damage caused by high-dose radiation. Int J Radiat Oncol Biol Phys 65: ACKNOWLEDGMENTS 876–881. Weacknowledge B. Dabovic, M. Horiguchi, and Antonelli-Orlidge A, Saunders KB, Smith SR, D’Amore PA. 1989. An activated form of transforming growth factor b L. Zilberberg for their insightful comments, and is produced by cocultures of endothelial cells and peri- funding from National Institutes of Health cytes. Proc Natl Acad Sci 86: 4544–4548. (NIH) Grants R01 CA034282 (Regulation of Arteaga-Solis E, Gayraud B, Lee SY, Shum L, Sakai L, Ra- TGF-b Activity in the Lung by LTBP-4) and mirez F. 2001. Regulation of limb patterning by extracel- lular microfibrils. J Cell Biol 154: 275–281. P01 AR49698 (Cell Signaling in Marfan Syn- Attur MG, Palmer GD, Al-Mussawir HE, Dave M, Teixeira drome) to D.B.R. CC, Rifkin DB, Appleton CT,Beier F,Abramson SB. 2009. F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via REFERENCES TGF-b activation. FASEB J 23: 79–89. Bailly S, Brand C, Chambaz EM, Feige JJ. 1997. Analysis of Abdelouahed M, Ludlow A, Brunner G, Lawler J. 2000. Ac- small latent transforming growth factor-b complex for- tivation of platelet-transforming growth factor b-1 in the mation and dissociation by surface plasmon resonance.

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Regulation of the Bioavailability of TGF-β and TGF-β-Related Proteins

Ian B. Robertson and Daniel B. Rifkin

Cold Spring Harb Perspect Biol 2016; doi: 10.1101/cshperspect.a021907

Subject Collection The Biology of the TGF-β Family

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