TGF-b1 Signaling and Tissue Fibrosis

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TGF-b1 Signaling and Tissue Fibrosis Kevin K. Kim, 1 Dean Sheppard, 2 and Harold A. Chapman 2 1 Department of Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan 48109 2 Department of Medicine, Cardiovascular Research Institute, and Lung Biology Center, Universityof California, San Francisco, San Francisco, California 94143 Correspondence: [email protected] Activation of TGF-b1 initiates a program of temporary collagen accumulation important to wound repair in many organs. However, the outcome of temporary extracellular matrix strengthening all too frequently morphs into progressive fibrosis, contributing to morbidity and mortality worldwide. To avoid this maladaptive outcome, TGF-b1 signaling is regulated at numerous levels and intimately connected to feedback signals that limit accumulation. Here, we examine the current understanding of the core functions of TGF-b1 in promoting collagen accumulation, parallel pathways that promote physiological repair, and patholog- ical triggers that tip the balance toward progressive fibrosis. Implicit in better understanding of these processes is the identification of therapeutic opportunities that will need to be further advanced to limit or reverse organ fibrosis. C ommonly affecting many organs including lung, heart, kidney, and liver, tissue fibrosis is a leading cause of morbidity and mortality worldwide (Friedman et al. 2013). Yet the ca- pacity for rapid wound repair with collagen accumulation facilitates restoration of tissue strength as well as tissue integrity in many inju- ry situations. For example, early cardiac fibrosis after myocardial infarction is important for protection against fatal cardiac rupture (Hof- mann et al. 2012). In broader terms, the capac- ity of cells to secrete and adhere to a collagenous extracellular matrix (ECM) must be a strong evolutionary force for advantage because virtu- ally all metazoans express genes for fibrillar col- lagens (Hynes 2012). In mammals, the potential role of transforming growth factor (TGF)-b1 in physiological repair and collagen accumulation was recognized soon after the discovery of TGF-b1 expression in cancer cell lines (Moses et al. 2016). TGF-b1 was found to promote fibronectin and collagen production by both epithelial and mesenchymal cells in culture, and to do so by transcriptional activation of the relevant genes (Ignotz and Massague ´ 1986). Additionally, subcutaneous injection of TGF-b1 was seen to strongly promote collagen accumu- lation and a fibrotic tissue response (Roberts et al. 1986). These observations and several sub- sequent lines of evidence emerged solidifying the role of TGF-b1 as a master regulator of ECM accumulation and consequently a potential key driver of fibrosis (Bartram and Speer 2004). TGF-b1 is also intricately involved in regulating inflammation (Shull et al. 1992; Kulkarni et al. 1993). Deletion of the Tgfb1 gene in mice results Editors: Rik Derynck and Kohei Miyazono Additional Perspectives on The Biology of the TGF-b Family available at www.cshperspectives.org Copyright # 2018 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a022293 Cite this article as Cold Spring Harb Perspect Biol 2018;10:a022293 1 on October 18, 2021 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/ Downloaded from

Transcript of TGF-b1 Signaling and Tissue Fibrosis

TGF-b1 Signaling and Tissue Fibrosis

Kevin K. Kim,1 Dean Sheppard,2 and Harold A. Chapman2

1Department of Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan 481092Department of Medicine, Cardiovascular Research Institute, and Lung Biology Center, University of California,San Francisco, San Francisco, California 94143

Correspondence: [email protected]

Activation of TGF-b1 initiates a program of temporary collagen accumulation important towound repair in many organs. However, the outcome of temporary extracellular matrixstrengthening all too frequently morphs into progressive fibrosis, contributing to morbidityand mortality worldwide. To avoid this maladaptive outcome, TGF-b1 signaling is regulatedat numerous levels and intimately connected to feedback signals that limit accumulation.Here, we examine the current understanding of the core functions of TGF-b1 in promotingcollagen accumulation, parallel pathways that promote physiological repair, and patholog-ical triggers that tip the balance toward progressive fibrosis. Implicit in better understandingof these processes is the identification of therapeutic opportunities that will need to be furtheradvanced to limit or reverse organ fibrosis.

Commonly affecting many organs includinglung, heart, kidney, and liver, tissue fibrosis

is a leading cause of morbidity and mortalityworldwide (Friedman et al. 2013). Yet the ca-pacity for rapid wound repair with collagenaccumulation facilitates restoration of tissuestrength as well as tissue integrity in many inju-ry situations. For example, early cardiac fibrosisafter myocardial infarction is important forprotection against fatal cardiac rupture (Hof-mann et al. 2012). In broader terms, the capac-ity of cells to secrete and adhere to a collagenousextracellular matrix (ECM) must be a strongevolutionary force for advantage because virtu-ally all metazoans express genes for fibrillar col-lagens (Hynes 2012). In mammals, the potentialrole of transforming growth factor (TGF)-b1 inphysiological repair and collagen accumulation

was recognized soon after the discovery ofTGF-b1 expression in cancer cell lines (Moseset al. 2016). TGF-b1 was found to promotefibronectin and collagen production by bothepithelial and mesenchymal cells in culture,and to do so by transcriptional activation ofthe relevant genes (Ignotz and Massague 1986).Additionally, subcutaneous injection of TGF-b1was seen to strongly promote collagen accumu-lation and a fibrotic tissue response (Robertset al. 1986). These observations and several sub-sequent lines of evidence emerged solidifying therole of TGF-b1 as a master regulator of ECMaccumulation and consequently a potential keydriver of fibrosis (Bartram and Speer 2004).TGF-b1 is also intricately involved in regulatinginflammation (Shull et al. 1992; Kulkarni et al.1993). Deletion of the Tgfb1 gene in mice results

Editors: Rik Derynck and Kohei Miyazono

Additional Perspectives on The Biology of the TGF-b Family available at www.cshperspectives.org

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in rapid demise after birth because of over-whelming, systemic inflammation (Shull et al.1992). Based on this and many other observa-tions (Travis and Sheppard 2014), TGF-b1 isgenerally considered as a suppressor of excessiveinflammation. Regulated inflammation itselfis an important determinant of tissue repair,as resolution of provisional fibronectin-richECMs and elaboration of mediators that limitcollagen accumulation after injury limit fibro-sis. Inflammatory cells, especially macrophages,are important initiators of inflammation andmediators of inflammation resolution (Vannellaand Wynn 2016; Minutti et al. 2017). This reviewwill examine our current understanding ofthe core functions of TGF-b1 in promotingcollagen accumulation and parallel pathwaysthat help determine whether there is physiolog-ical repair or progressive fibrosis. Althoughincreased levels of TGF-b2 and TGF-b3 havebeen observed in fibrotic disease (Burke et al.2016), there is little evidence implicating eitherTGF-b2 or TGF-b3 with progressive tissuefibrosis, and therefore these TGF-b isoformswill not be discussed further here.

INTERPLAY BETWEEN INTEGRINS AND ECMSTIFFNESS IN TGF-b1 ACTIVATION ANDSTROMAL EXPANSION

Newly translated TGF-b is processed by cleav-age of an amino-terminal fragment, called thelatency-associatedpeptide(LAP), fromthepoly-peptide that homodimerizes to form the maturecytokine (Munger et al. 1997; Moses et al. 2016;Robertson and Rifkin 2016). The cleaved LAPalso homodimerizes and associates noncova-lently with mature TGF-b in a complex thatprevents access of TGF-b to its receptors. Inmost cells, this “small latent complex” is disul-fide linked to either a member of a family oflatent TGF-b binding proteins (LTBPs) andstored in the ECM or to the transmembraneprotein glycoprotein A repetitions predominant(GARP) for presentation on the cell surface(Stockis et al. 2009). Most organs of healthyadult mammals contain substantially morelatent TGF-b than would be required, whenactivated, to cause extensive tissue fibrosis.

Therefore, much of the regulation of TGF-bfunction in fibrotic diseases depends on theregulation of TGF-b activation rather than syn-thesis or secretion (Robertson and Rifkin 2016).

In vitro it is relatively easy to induce confor-mational change in latent TGF-b that leads torelease of the active cytokine (i.e., TGF-b acti-vation). For example, purified latent TGF-b canbe activated by acidic or basic pH, heat denatu-ration, and oxidation by reactive oxygen species(ROS), incubation with thrombospondin 1, ora wide variety of proteases (Munger et al. 1997;Crawford et al. 1998). However, with the excep-tion of thrombospondin 1, for which geneinactivation has identified some phenotypicfeatures consistent with loss of TGF-b activa-tion, and ROS in the setting of mammary glandirradiation (Barcellos-Hoff et al. 1994), the rolesof these activation pathways in vivo have notbeen established. The most intensely studiedactivation mechanism with demonstrated rele-vance in vivo has been activation by interactionwith a subset of integrins (Munger et al. 1999;Annes et al. 2002; Mu et al. 2002; Jaramillo et al.2003; Henderson et al. 2013; Hinck et al. 2016).

The first indication that integrins could par-ticipate in the activation of TGF-b came fromobservations of the phenotype of mice lackingthe b6 subunit of the epithelially restricted avb6

integrin (Munger et al. 1999). These mice showexaggerated inflammatory responses to normal-ly trivial injurious stimuli in multiple epithelialorgans (Huang et al. 1996). Despite these exag-gerated responses, Itgb62/2 mice are dramati-cally protected from tissue fibrosis (Mungeret al. 1999; Hahm et al. 2007). Tgfb12/2 micehave even more exaggerated tissue inflamma-tion, reflecting the more limited tissue expres-sion of Itgb6 relative to Tgfb1 (Kulkarni et al.1993). Biochemical evidence has shown directbinding of the avb6 integrin to an arginine-glycine-aspartic acid (RGD) tripeptide withinTGF-b1 LAP (Munger et al. 1999), and cellculture assays have shown that epithelial cellscan use this integrin to bind and activateTGF-b1 and -b3 (Munger et al. 1999; Anneset al. 2002). Since these initial observations,several studies have shown reduced TGF-b ac-tivation in epithelial organs of Itgb62/2 mice,

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by cells lacking the avb6 integrin, and in re-sponse to blocking antibodies targeting thisintegrin (Munger et al. 1999; Weinreb et al.2004; Jenkins et al. 2006; Aluwihare et al.2009; Giacomini et al. 2012).

Because the inflammatory phenotype ofmice lacking even one of the three isoforms ofTGF-b (e.g., TGF-b1) is much more severe thanthe phenotype of mice lacking the avb6 integ-rin, additional mechanisms must regulate acti-vation of TGF-b1 and -b3 in vivo. It is now clearthat additional RGD-binding integrins areimportant contributors to this process. Cell cul-ture studies with epithelial cells showed that theclosely related integrin, avb8, can also potentlyactivate TGF-b (Mu et al. 2002). Mice that lackintegrin b8 also recapitulate some features ofmice lacking TGF-b1, such as defects in vascu-lar development, or TGF-b3 (e.g., cleft palate)(Zhu et al. 2002). When Itgb82/2 mice arecrossed to an outbred genetic background thatallows some mice to survive into early adult-hood, treatment of these mice at birth withblocking antibody to the avb6 integrin resultsin a complete phenocopy of all developmentaleffects of loss of TGF-b1 and TGF-b3 (Aluwi-hare et al. 2009), suggesting that avb6 and avb8

together contribute to activation of all ofthe TGF-b1 and TGF-b3 required for normaldevelopment.

Additional RGD-binding integrins mayplay important roles in activating TGF-b todrive tissue pathology in adults. For example,Cre recombinase-mediated, tissue-restricteddeletion of the RGD-binding integrins thatshare the av subunit in cells expressing plate-let-derived growth factor b (PDGFb) protectsmice from pathologic fibrosis in the lungs, liverand kidney (Henderson et al. 2013). Fibroblastsderived from av-null mice following Pdgfb-Cre-mediated gene deletion have an impaired abilityto bind and activate TGF-b1, and these miceshow reduced evidence of TGF-b signalingin models of pathologic tissue fibrosis. The cellstargeted by Pdgfb-Cre do not express the avb6

integrin, but express avb1, avb3, avb5, andavb8. However, global deletion of avb3 and/or avb5, by specific deletion of their b chains,has no effect on induced tissue fibrosis, and

conditional deletion of avb8 in the same cellshas no effect on liver fibrosis. These results sug-gest that one or moreav integrins, in addition toavb8 and avb6, can contribute to pathologicTGF-b activation in vivo. Accordingly, a smallmolecule inhibitor of the avb1 integrin that hasminimal effects on other RGD-binding integ-rins was shown to inhibit TGF-b activation byprimary lung and liver fibroblasts (Reed et al.2015). Continuous subcutaneous administra-tion of this inhibitor during the late phasesof bleomycin-induced pulmonary fibrosis orcarbon tetrachloride (CCl4)-induced hepaticfibrosis results in protection that is similar tothat by a small molecule inhibitor of all av in-tegrins (Henderson et al. 2013). These datasuggest that the fibroblast integrin responsiblefor TGF-b activation and progression of lungand liver fibrosis is avb1.

ROLE OF MECHANICAL FORCE ININTEGRIN-MEDIATED TGF-b ACTIVATION

In the original description of avb6-mediatedTGF-b activation, it was shown that sequenceswithin the b6 subunit cytoplasmic domainrequired to link the integrin to the actincytoskeleton were essential for TGF-b activa-tion (Munger et al. 1999). Activation was alsocompletely inhibited by cytochalasin D, aninhibitor of actin polymerization. These obser-vations provided the first evidence that integ-rin-mediated TGF-b activation requires linkageto and active re-organization of the actincytoskeleton. Subsequently, cell culture studiesshowed that contractile fibroblasts can use in-tegrins to activate latent TGF-b, and that thisprocess depends on actin–myosin interactionand generation of mechanical force (Wipffet al. 2007). Similar requirements were shownfor avb6-mediated TGF-b activation by epithe-lial cells (Giacomini et al. 2012), although therole, if any, of mechanical force in TGF-bactivation by avb8 is less clear (Mu et al.2002). avb6-mediated TGF-b1 activation wasalso shown to require covalent disulfide linkageof LTBP-1 to TGF-b1 LAP (Annes et al. 2004).The effect of deletion of Ltbp1 could be rescuedby expression of a short fusion protein com-

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posed of the region containing the cysteine res-idues responsible for disulfide linkage of LTBP-1to LAP and the region required for direct bind-ing to fibronectin in the ECM. These observa-tions suggest that the requirement for LTBP-1is explained by its role in physically tetheringlatent TGF-b.

The important role of mechanical force inintegrin-mediated TGF-b activation fits verywell with the crystal structures of latent TGF-b1 and of cocrystals of the avb6 integrin withTGF-b1 LAP (Shi et al. 2011; Dong et al. 2014,2017; Hinck et al. 2016). The latent TGF-b1structure showed a 180˚ axis between theexposed loop in LAP containing the integrinbinding RGD sequence and the cysteine resi-dues that form the disulfide linkage to LTBP-1,identifying how the force of cellular contractioncould be transmitted through avb6 to deformphysically constrained latent TGF-b. Further-more, a single unstructured loop, called thelatency lasso, was shown to make all of the crit-ical contacts with active TGF-bwithin the latentcomplex and to be the likely weakest link thatwould be unfolded by mechanical force, therebyreleasing free TGF-b from the complex. Theavb6 structure identified critical residues, espe-cially a hydrophobic pocket that provide a dock-ing site for hydrophobic residues adjacent to theRGD sequence in TGF-b1 and -b3 LAP thathelp to explain the unusually high affinity ofthe interaction between avb6 and LAP.

To exert physical force on tethered latentTGF-b, cells must themselves be firmly attachedto a relatively stiff substrate. Indeed, one of theprincipal methods used to show a requirementfor physical force in integrin-mediated TGF-bactivation has been plating cells on polyacryl-amide gels of varying stiffness, and showing thatcells plated on more deformable substrates haveprogressively impaired ability to activate TGF-bas the substrate deformability increases (Wipffet al. 2007; Giacomini et al. 2012). Integrins areone of the main families of receptors cells usedto adhere tightly to components of the ECM, soit seems likely that inhibitors targeting integrinscould inhibit TGF-b activation, even if the in-hibited integrins do not directly bind tightly toTGF-b LAP. For example, a blocking antibody

targeting the avb3 integrin can inhibit TGF-bactivation by cultured renal fibroblasts buthas no effect on adhesion of these cells to LAP.Rather, this inhibitory effect is completely de-pendent on the substrate on which these cellsare plated, with marked inhibition when thecells are plated on the avb3 ligand or vitronec-tin, but no inhibition when the cells are platedon an irrelevant ligand, collagen 1 (Chang et al.2017). Thus, integrin interactions with theECM are intricately involved in how the ECMimpacts TGF-b1 activation.

TGF-b1 SIGNALING AS A DRIVER OFCOLLAGEN ACCUMULATION

TGF-b1 signals through its specific heterotetra-meric receptor complex comprised of a pair ofTbRI receptors that phosphorylate the receptoractivated-Smads (R-Smads), termed Smad2and Smad3, and a pair of TbRII, which containthe initial binding site for TGF-b1. These recep-tors are expressed on most cells in culture andtheir roles in TGF-b signaling have beendiscussed elsewhere (Feng and Derynck 2005;Heldin and Moustakas 2016). Cell-specificgene deletion by Cre-mediated recombinationof either Tgfbr1 or Tgfbr2 revealed that TGF-b1signaling, rather than TGF-b1 expression, inboth epithelial cells and mesenchymal cells isrequired for bleomycin-induced lung fibrosis(Huang et al. 2002).

These findings indicate that TGF-b1-medi-ated signaling in both epithelial and fibroblasticcells is a common and critical feature of fibro-genesis. These findings also support the conceptthat TGF-b1 mediates fibrosis through multiplecell types and multiple interacting signalingpathways. The latter include not only coregula-tors of R-Smad-dependent transcription ofTGF-b1 target genes such as AP-1 and Egr-1,specific negative regulators of R-Smad functionsuch as Smad7, but also early response genessuch as Nox4 and Tgfb1 itself further promoteTGF-b1 signaling (Derynck and Zhang 2003;Feng and Derynck 2005). An altered profile ofmicroRNAs (miRNAs) induced or suppressedin response to TGF-b1 also acts to stabilizetranslation of mesenchymal genes important

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to fibrosis and minimize antifibrotic pathways(Bowen et al. 2013). Finally, numerous othersignaling pathways provide input that allowsintegration of Smad and non-Smad signalingimmediately downstream from TGF-b receptoractivation with contextual cues from the micro-environment that favor expansion of the ECM(Fig. 1). Integrins, in addition to mediatinglatent TGF-b1 activation, activate prosurvivaland adhesive signals as a function of ECMcomposition and stiffness. Receptor tyrosinekinases respond to ligands after injury and pro-mote proliferation and survival of fibroblasts.Hypoxia, acting through hypoxia inducible fac-tors (HIFs), promotes epithelial–mesenchymaltransition (EMT) and/or mesenchymal expan-

sion with enhanced collagen expression (Fa-langa et al. 2002; Zhou et al. 2009; Chapman2010; Kottmann et al. 2012). Several morpho-gen pathways, especially those induced by Wntand Notch ligands, are activated in response toinjury and have been implicated in promotingtissue fibrosis in multiple organs. The elaboratecellular response of stromal cells within injuredtissues, directed in part by the influx of inflam-matory cells, appears heavily oriented towardtransient ECM accumulation accompanied byproliferation and migration of epithelial andstromal elements, followed by stabilization ofthe collagenous components of the ECM, andfinally fibroblast apoptosis and removal ofexcess collagen to restore normal architecture

Growth factors(PDGF, FGF)

Growthfactors (E)MT

transcription factors

SMA

Smad2/3

Rhop

ECM

ECM(Col, Fn)

Latent TGF-β1

Active TGF-β1

IntegrinRTK

MAPKPI3K-Akt

Coactivators(β-catenin)

Figure 1. Epithelial and mesenchymal cell fibrogenic activation by TGF-b1: costimulation by other signalinginputs and opportunities for feed-forward activation. Latent TGF-b is activated by av integrins. Active TGF-bbinds to its receptors leading to activation of canonical Smad signaling and Smad-independent signalingpathways, including MAPK pathways and small GTPases such as RhoA. Inputs from other signaling pathwaysconverge on these pathways to regulate the TGF-b response. These are activated by other growth factors, such asPDGF, which generally signal through RTKs and through MAPK, integrin-mediated mechanotransductionmediated by Rho family GTPases, and coactivators such as b-catenin. These signaling factors lead to expressionof profibrotic genes such as those encoding a-SMA, ECM proteins, and secreted cytokines and growth factors,which further modify the fibrogenic effector cell response. The expression of transcription factors involved inepithelial or mesenchymal cell transition into an activated state is also induced. PDGF, platelet-derived growthfactor; FGF, fibroblast growth factor; RTK, receptor tyrosine kinase; MAPK, mitogen-activated protein kinase;(E)MT, (epithelial–) mesenchymal transition; Col, collagen; Fn, fibronectin; SMA, a-smooth muscle actin;ECM, extracellular matrix.

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(Beers and Morrisey 2011). TGF-b1 is a keycytokine participating in virtually all elementsof the early tissue response to injury.

Collagens are comprised of three polypep-tide chains highly organized into a triple helicalconformation. Collagen monomers are not verysoluble and, as discussed below, numerous post-translational modifications are necessary forproper procollagen folding and secretion. Thereare 28 collagen proteins in mammals, the mostabundant being collagen I, a major componentof bone, most musculoskeletal structures,eye, lung, and the vasculature (Hulmes 2008).Collagen I belongs to the group of fibrillar col-lagens, including collagens I, II, III, and VI, thatare largely triple helical structures and thusbecome organized into fibrils with very defined“packing” dependent on extensive covalentcross-linking of carboxyl terminus to aminoterminus of adjacent collagen monomers. Thiselaborate organization results in high tensilestrength for the collagen fibrils. This is incontrast to the much less extensively organizednonfibrillar collagens, such as collagen IV that isthe major basement membrane collagen. Afterinjury, collagens I and III are the dominantcollagens whose expression is induced to restoretensile strength and tissue integrity. TGF-b1 is arequired cytokine for the induction of thesecollagens after tissue injury.

The intricate nature of TGF-b1 signaling infibrogenesis is highlighted by the mechanismsengaged in expression and tissue accumulationof type I collagen. Type I procollagen is com-prised of two polypeptide chains, pro-a1 andpro-a2, assembled in a 2:1 ratio (2a1/a2) lead-ing to a heterotrimeric three-dimensional he-lical structure (Fig. 2). Following activation oflatent TGF-b1 and TGF-b receptor engage-ment, initial transcription of COL1A2 gene(encoding type I collagen a2) is dependent onthe nuclear translocation of Smad3 and Smad4,the DNA-binding transcription factors Sp1 andAP-1 (c-Jun/c-Fos), as well as CREB-bindingprotein (CBP) or p300 that act as coactivatorsand histone acetylases to facilitate chromatinrelaxation and access of the transcription factorsto the COL1A2 promoter (Zhang et al. 1998;Ghosh et al. 2000). The appearance of AP-1 in

the nucleus in turn depends on activation ofSmad-independent Erk or c-Jun amino-termi-nal kinase (JNK) mitogen-activated protein ki-nase (MAPK) signaling pathways by TGF-b1.Similar mechanisms operate to induce an earlyresponse of Egr-1 that has been shown to becritical for collagen I expression by fibroblastsin response to TGF-b1 (Chen et al. 2006). Nu-merous other factors coordinate with Smad2/3signaling in the nucleus to promote COL1A1and COL1A2 transcription. Once transcribed,the translation of collagen I mRNAs is alsoheavily regulated, in no small part by TGF-b1-induced repression of the expression of miRNAsknown to target collagen mRNAs or mRNAfor proteins required for collagen expression(Bowen et al. 2013). miR-29, for example, isknown to repress the expression of heat shockprotein 47 (HSP47) and lysyl oxidase like-2(LOXL2), which both participate in the highlyorganized process of stable collagen accumula-tion in the ECM (discussed below). The expres-sion of the miR-29 family is down-regulatedin response to TGF-b1 in fibroblasts, and thisrepression corresponds to increased fibroblastcollagen expression. Mice treated with miR-29mimics have attenuated lung fibrotic responsesto bleomycin (Yang et al. 2013). miR-96 andmiR-130b also attenuate collagen accumulationand their expression is repressed in response toTGF-b1. Similarly, miR-326 has been shownto attenuate TGF-b1 translation (Das et al.2014). Levels of miR-326 were found to be lowin the bleomycin mouse model and in lungsof patients with idiopathic pulmonary fibrosis(IPF). Additionally, mice treated intranasallywith a miR-326 mimic have attenuated fibrosisin this model. Collectively, the changes inmiRNA expression by TGF-b1 signaling act tostabilize collagen protein expression, secretion,and stabilization in the ECM (Fig. 2).

Actual assembly and cellular traffickingof collagen I polypeptides through the cellsecretory machinery is problematic because ofthe large size and tenuous solubility of the col-lagen chains. Extensive posttranslational modi-fications are required for the proper assemblyof procollagens into very well ordered helicalstructures early in the secretory process (Fig. 2)

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TGF-β1Collagen α1, 2

Procollagen LOXLOXL1-4

PAI-1TIMP1, 3

BiglycanOsteopontinPeriostin

Fibronectin

HSP47

Meprin α/β

HSP47FKBP10

PLOD2HSP47P4HA3FKBP10

BiP/GRP78gal-transferaseglc-transferase

BMP-1

MAPK

AP-1 CBP/pSmad

Nucleus

Ribosome

ER

N

PDI

C

Helicaltrimer

Lysyl oxidases

Collagenturnover

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Organized collagen fibrils

Collagenmicrofibrils

Tropocollagen

gal

gal

gal

OH

OH

OHOH

Collagen-regulatingmicroRNAs

2129

32620019696

130b

pSmad

Figure 2. TGF-b1-induced collagen expression program. The schematic illustrates the proteins and microRNAsthat are coordinately regulated by TGF-b1 to effect tissue collagen accumulation. Proteins whose expression isactivated by TGF-b1 are highlighted in red, indicating that the expression of more than a dozen proteins isinduced along with that of collagen and is required for substantial collagen expression and tissue accumulation.These proteins act at nearly every stage of collagen processing, from posttranslational proline and lysine hy-droxylations by procollagen-lysine 2-oxogluterate 5-deoxygenase (PLOD2) and prolyl-4-hydroxylase (P4HA3),and glycosylation in the endoplasmic reticulum (ER), to required chaperones (HSP47 and FKBP10) to sustainthe trimeric collagen structure and prevent premature fibril formation during passage through the secretorymachinery. Additional, constitutively expressed proteins, such as protein disulfide isomerase (PDI), and qualitycontrol sensors, such as BiP/GRP78, are also important for collagen folding and subsequent expression. Oncesecreted, procollagen is proteolytically processed to generate tropocollagen that then spontaneously begins toself-associate into microfibrils. The final physical form and extent of stable, matrix fibrillar collagens dependheavily on additional TGF-b1-induced proteins such as fibronectin, the lysyl oxidases, and inhibitors of collagenturnover, plasminogen activator inhibitor 1 (PAI-1) and tissue inhibitor of metalloproteinases 1 and 3 (TIMP1,TIMP3). Finally, proteins such as biglycan and periostin associate with mature fibrils and control packing andorganization. Collectively, this reprogramming of cells can be considered as the TGF-b1-induced collagenexpression program.

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(Hulmes et al. 2002; Bourhis et al. 2012; Ishikawaand Bachinger 2013). The primary amino acidsequences of fibrillar collagens underlie the pro-pensity of these collagens to form a helical struc-ture, with approximately 300 repeats of Gly-X-Ymotifs providing sufficient glycines for tightchain folding. HSP47, whose expression isstrongly induced in response to TGF-b1 in nearlyall cells that express collagen I, acts as a key chap-erone in collagen assembly, binding to collagenand promoting stability of both the unfoldedcollagen chains in the endoplasmic reticulum(ER) and the more assembled structures traffick-ing through the Golgi. Assembly into a properlyfolded procollagen also requires many distinctmodifications. Immediately after synthesis inthe rough ER, and possibly bound to its innermembrane, the unfolded collagen chains begin toundergo lysine and proline hydroxylations. Thetwo key enzymes in this process are procollagen-lysine 2-oxogluterate 5-deoxygenase (PLOD2 orLH2) and prolyl-4-hydroxylase (P4HA3). Theexpression of both enzymes is strongly inducedin response to TGF-b1. Avariable fraction of thehydroxy-lysines are then glycosylated, and theremainder is further processed to very stable col-lagen cross-links once collagen is secreted. Likeglycines, the hydroxyprolines are required fortight helical folding as the three collagen chainsbegin to assemble. The modified collagen chainsare also substrate for a constitutively expressedprotein disulfide isomerase (PDI) that mediatesdisulfide formation in the carboxy-terminal re-gion. This is thought to place the chains in “reg-ister” for subsequent helical folding, beginning atthe carboxyl terminus and proceeding toward theamino terminus, and full assembly of the procol-lagen molecule. Once the ordered, helical procol-lagen molecule assembles, there is a critical needfor additional proteins to chaperone the traffick-ing of procollagen through the Golgi and secre-tory process without denaturation or prematurefibril formation. HSP47 and a FK506 bindingprotein 10 (FKBP10) serve these functions. Theirexpression is strongly induced by TGF-b1 in fi-broblasts (Ishida and Nagata 2011; Staab-Weij-nitz et al. 2015).

Collagen is finally secreted as a procollagenwith the extended carboxy- and amino-termi-

nal nonhelical peptide chains (carboxy- andamino-terminal telopeptide extensions) thatare then removed by proteolysis to generatethe helical structure, tropocollagen (Fig. 2). Anumber of proteases appear to be capable ofamino- and carboxy-terminal processing ofprocollagen and it is unclear if these are inducedby TGF-b. The earliest described carboxy-ter-minal processing protease is bone morphoge-netic protein-1 (BMP-1), a metalloproteinasethat is critical to collagen maturation in bone(Li et al. 1996; Prockop et al. 1998; Asharaniet al. 2012). Recently, meprin a and b metal-loproteinases were shown to cleave both theamino- and carboxy-terminal peptides of pro-collagen, leading to fibril formation (Prox et al.2015). These meprin levels are high at sites ofcollagen accumulation and fibrosis (Broderet al. 2013). Meprin expression is inducedby pathways that lead to activation of nuclearAP-1, and thus may be induced by TGF-b1(Biasin et al. 2014). Tropocollagen is thoughtto spontaneously form a polymeric collagenfibril, but collagen stabilization as a fibril (con-taining hundreds of tropocollagen molecules)requires lysyl (or hydroxyl-lysyl) oxidation toan aldehyde that is both necessary and sufficientfor irreversible collagen cross-linking. Lysyloxidases, a set of key enzymes that mediatecollagen cross-linking, are all strongly inducedby both TGF-b1 and HIF1a (Erler et al. 2006;Blaauboer et al. 2013). In addition to supportingfibril assembly, TGF-b1 concurrently inducesthe expression of protease inhibitors such asplasminogen activator inhibitor 1 (PAI-1) andtissue inhibitor of metalloproteinase 3 (TIMP3)that attenuate the breakdown of newly depos-ited tropocollagens that are vulnerable to pro-teases. The pattern of deposition of collagen isstrikingly similar, and follows temporally that offibronectin secretion, a prominent mesenchy-mal protein that is induced by TGF-b1 signaling(Hernnas et al. 1992; Muro et al. 2008). Extra-cellular fibronectin assembly, itself a complicat-ed and integrin-dependent process, providesthe suitable extracellular fibrillar substrate fororganized deposition of collagen fibrils. Thefurther organization of fibrils, depending per-haps on the needed tensile strength, is promot-

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ed by yet additional TGF-b1 target genes thatencode collagen-interacting proteins such asosteopontin, periostin, and the proteoglycanbiglycan (Oku et al. 2008; Farhat et al. 2012).Transglutaminase and nonenzymatic interac-tions among these proteins are thought to fur-ther enhance tissue stiffness over time (Cox andErler 2011).

The evolutionary expansion of collagensfrom early metazoans to mammals has beenaccompanied by expansion of the family ofmonoamine oxidases, lysyl oxidases, needed tocross-link and stabilize ECM collagen (Kaganand Trackman 1991). The parent enzyme,LOX, is critically important to normal muscu-loskeletal development as deletion of LOX inmice is perinatal lethal with aortic aneurysms,defective skeletal structures, and defective air-way development (Trackman 2005). In additionto LOX, there are four LOX-like enzymes(LOXL1–4) that share highly conserved catalyt-ic domains but also are comprised of additionalprotein interaction domains. LOX and LOXL1are closely related and expressed in a similarpattern. LOXL2, 3, and 4 have similar structuresand, among these, LOXL2 is the most studiedand widely expressed, although primary lungfibroblasts in culture express all of the LOX-like enzymes at variable levels. The expressionof LOXL2 is strongly induced by TGF-b1 andhypoxia, and is reported to not only functionextracellularly in collagen cross-linking butalso intracellularly to promote Snail1 proteinaccumulation. Because of the strong positiveimpact of Snail1 on the expression of fibrillarcollagens, the induction of LOXL2 expression inresponse to TGF-b1 can be viewed as a positivefeedback loop to support ECM expansion afterinjury. Thus, LOXL2 levels are strongly linked tostable collagen accumulation, ECM stiffness,and fibrosis progression (Barry-Hamiltonet al. 2010; Adamali and Maher 2012).

As might be predicted from the elaboraterequirement for multiple posttranslational mod-ifications to support fibrillar collagen foldingand solubility in the ER, high-level expressionof collagen renders the cell susceptible to ERstress from misfolded protein. Accordingly,TGF-b1 induces the expression of proteins

that are linked to protection from the unfoldedprotein response (UPR) (e.g., BiP/GRP78, andHSP47) in parallel to expression of fibrillar col-lagens and modifiers of its primary structure(Fig. 2). Nonetheless, it has been reported thatinositol-requiring enzyme-1a (IRE-1a), a keymediator of the UPR, is activated after TGF-b1-induced collagen expression in fibroblastsand in fibroblasts from patients with fibroticdisease (Heindryckx et al. 2016). The investiga-tors implicated both increased X-box bindingprotein 1 (XBP-1) signaling, resulting in ERexpansion and cleavage of miR-150 by theRNAase activity of activated IRE-1 in alteringcollagen expression, and fibrogenesis. ElevatedmiR-150 levels have been linked to protectionfrom fibrosis (Honda et al. 2013). Hence, some-what paradoxically, the activation of IRE-1,indicative of an activated UPR, promotes ratherthan suppresses collagen expression. Inhibitionof IRE-1 in cell culture and in vivo attenuatesthe fibrotic response to TGF-b1 in liver and skinfibrosis models. These findings underscore thepotential pathological operation of the UPR inthe context of ER stress with a negative impacton tissue integrity (Fig. 3). This conclusion issupported by the findings that mutations insurfactant protein (SP) A2, a cause of familialpulmonary fibrosis, leads to type II alveolarepithelial cell ER stress accompanied by in-creased TGF-b1 secretion (Wang et al. 2009).

Collectively, it is clear that TGF-b1 drives alarge, multifaceted reprogramming of both ep-ithelial and mesenchymal cells to favor expres-sion and extracellular accumulation of matrixproteins that provide rapid restoration of tissueintegrity in the setting of injury and/or disrup-tion of tissue barriers (Fig. 2). The phylogeneticorigin of fibrillar collagen, and its complicatedassembly process, dates to the earliest metazoansand has been highly conserved and further am-plified for increased structural strength through-out the vertebrate family. The importance ofcollagen and numerous other ECM proteins inthe injury response is highlighted not only bythe highly coordinated induction by TGF-b1 ofa large set of ECM structural proteins but alsothe parallel induction of numerous enzymes andchaperones that promote proper folding, secre-

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tion, and extracellular stabilization of collagen aswell as down-regulation of miRNAs that specif-ically attenuate collagen expression and accu-mulation. It is hardly surprising then that sucha robust and complicated cellular response sys-tem, driven after injury largely by a single masterregulator, is subject to breakdown and disor-dered function leading to disease.

CONTRIBUTIONS OF INTEGRINS ANDTGF-b1 TO TISSUE STIFFNESS ONPROGRESSION OF FIBROSIS

It is now abundantly clear that tissue stiffnesscan exert profound effects on cell differentiationand behavior. For example, mesenchymal stro-

mal cells differentiate into fat cells, muscle cells,or bone cells when they are plated on substrateswith stiffness mimicking that normally found infat, muscle, or bone, respectively (Engler et al.2006). Stiffness also profoundly affects the be-havior of tissue fibroblasts, with increased stiff-ness favoring increased collagen production andexpression of contractile proteins such as a-smooth muscle actin (SMA), and decreasedstiffness favoring a more quiescent and noncon-tractile phenotype (Liu et al. 2010). As a conse-quence, the process of tissue fibrosis, which lo-cally increases tissue stiffness, participates in afeed-forward loop, where the presence of localfibrosis increases fibroblast collagen productionand force generation, further accelerating fibro-

Oxidantstress

DNA damagereponse

Misfoldedprotein Pathological

triggers

Profibroticpathways

Cell states

TGF-β1

SENESCENCE EMT

COLLAGEN ACCUMULATIONAND ECM EXPANSION

p16INK4ap53NF-κB

Smad2/3HIFMAPK

XBP-1IRE-1HIF

ER STRESS

Hypoxia

Alteredimmunity andM2 macrophages

↑Mesenchymalproteins

Fibroblastactivation

Pro-fibrotic secretory phenotypes

Figure 3. Diverse pathways of epithelial responses to stress converge on profibrotic secretory phenotypessupporting extracellular matrix (ECM) expansion. The figure highlights three major epithelial states resultingfrom different pathological triggers: epithelial–mesenchymal transition (EMT), senescence, and endoplasmicreticulum (ER) stress with unfolded protein responses (UPRs). Each epithelial state has its characteristictranscriptional drivers. All of these states elicit secretory responses that share the potential to promote collagenaccumulation. Distinctions among the secretory responses are still not completely defined and represent op-portunities to better understand the connections between epithelial and mesenchymal biology in the context ofcell stress. As discussed, TGF-b1 is a major driver of fibroblast activation and collagen secretion. Like epithelialcells, fibroblasts also respond to many of the same pathological triggers with activation of senescent or UPRsignaling pathways, potentially further promoting fibrosis.

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sis. One of the mechanisms underlying thisfeed-forward loop is integrin-mediated TGF-bactivation, because, as noted above, this processdepends on cell contraction and is enhancedwhen cells are tethered to a stiff substrate. How-ever, increased TGF-b activation does not fullyexplain this feed-forward loop. For example,tissue stiffness increases the sensitivity of cellsto already activated TGF-b (Liu et al. 2010).

EPITHELIAL–MESENCHYMAL TRANSITION

An important potential contribution of EMT totissue fibrosis was first proposed as early as themid 1990s (Strutz et al. 1995). More insight hasemerged with greater usage of fibrotic humantissue samples for biomedical research, the de-velopment of novel techniques for genetic mod-ifications to mice, and a greater understandingof some of the prominent EMT signaling path-ways (Kalluri and Neilson 2003; Teng et al. 2007;Kalluri and Weinberg 2009; Thiery et al. 2009;Flier et al. 2010; Lamouille et al. 2014). Howev-er, rather than establishing a definite role forEMT during fibrosis, these advances have ledto increased controversy regarding the role oreven the occurrence of EMT during fibrosis.Seemingly solid evidence both supports andrefutes EMT as having a major contribution tofibrosis. Some of the controversies regardingEMT point to broader uncertainties and con-troversies in the fibrosis field at large.

Definition of EMT

Epithelial cells are characterized by a relativelysessile state with a defined shape (e.g., cuboidal,columnar, or squamous) forming tight cell–cellcontacts, which establish an epithelial barrierthat is important for the function of manyorgans. Epithelial cells have an apical–basalpolarity and often produce factors that aresecreted from the apical surface into a luminalspace. In contrast, the mesenchymal cell pheno-type provides front–rear polarity and enablesmigratory behavior. The cell–cell contactsare often transient and the cell shape is moredynamic and less defined. EMT can thus bedefined as a process in which epithelial cells

lose epithelial cell–cell contacts, undergo cyto-skeletal changes and change polarity fromapical–basal to anterior–posterior, all favoringacquisition of migration and invasion. Thesechanges are accompanied by changes in geneand protein expression, including proteinsthat are involved in cell contact (e.g., loss ofE-cadherin and gain of N-cadherin) and cyto-skeletal proteins (e.g., loss of cytokeratin andgain of vimentin and SMA). There is also lossof proteins that are normally secreted into theluminal space (e.g., SPs, hormones) in favor ofproteases and ECM proteins that promotemigration and invasion. EMT can therefore beidentified and defined by a set of biomarkers,reflecting loss of epithelial and gain of mesen-chymal proteins (Zeisberg and Neilson 2009).EMT occurs during and is required for normalembryonic development. Roles for EMT duringcarcinogenesis and fibrosis have been suggested,but remain subjects of discussion and furtherresearch (Cardiff 2005; Tarin et al. 2005;Zeisberg and Duffield 2010). EMT during thesedifferent scenarios has overlapping features butalso important differences leading some to clas-sify EMT into type I (development and regen-eration), type II (fibrosis) and type III (cancer)EMT, dependent on the physiological andpathological context (Kalluri and Weinberg2009; Zeisberg and Neilson 2009). The transi-tion of endothelial or endodermal cells into amesenchymal phenotype has been labeled en-dothelial–mesenchymal transition (EndMT),but is often categorized within EMT, as theseprocesses are similarly regulated and havemany overlapping characteristics (Teng et al.2007; Kokudo et al. 2008; Kovacic et al. 2012).EMTmay simply represent a tunable, stereotyp-ical epithelial response to the demand for dy-namic cellular changes, as might occur duringembryonic development, response to injury,and carcinogenesis. Frequently, the acquisitionof such dynamic changes in epithelial behavioris reversible.

EMT Pathways

Features of EMT can be induced by many li-gand–cell surface receptor interactions. TGF-b

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is the most extensively studied inducer of EMT.TGF-b binding to its receptors, TbRI andTbRII, activates Smad-dependent and Smad-independent pathways that can directly activatemesenchymal gene and suppress epithelial geneexpression (Derynck et al. 2014; Lamouille et al.2014). TGF-b–Smad signaling can also activateEMT through induction of the expression ofseveral mesenchymal transcription factors,such as Snail1 and Twist, and these are thereforeoften referred to as EMT transcription factors.The TGF-b pathway intersects with a number ofother pathways that may augment or attenuatethe EMT response (Derynck et al. 2014). Hepa-tocyte growth factor (HGF), initially describedas a “scatter factor,” and fibroblast growth factor(FGF) were among the earliest examples of asingle factor capable of inducing EMT in cellculture (Stoker and Perryman 1985; Valleset al. 1990; Nusrat et al. 1994). Subsequently,many other factors have been shown to induceaspects of EMT through activation of cellsurface receptors that ultimately lead to tran-scriptional changes. Among the better studiedligand–receptor combinations are Wnt factorsthat act through Frizzled receptors, which leadsto stabilization of b-catenin, Sonic Hedgehog(Shh) ligand that acts through Patched recep-tors, which leads to Gli activation, and theDelta–Notch combination, which leads tocleavage and activation of the intracellularNotch domain. For example, b-catenin hasemerged as an important Smad coactivator, soin addition to activating its own target genesthrough interactions with T-cell factor (TCF)or lymphoid enhancer-binding factor (LEF),b-catenin can regulate the EMT responsethrough cooperation with the Smad complex.Among the most prominent Smad-indepen-dent EMT pathways is activation of Rho familyGTPases, including RhoA, Rac1, and Cdc42.Rho GTPases are important regulators of cellmorphology, adhesion, and movement. TGF-b signaling through the Rho GTPases allowsconvergence of signaling inputs from the ECMand cytoskeleton to control the EMT responseto TGF-b, and allows TGF-b to coordinatetranscriptional expression changes with cyto-skeletal changes involved in mesenchymal tran-

sition. Activation of these GTPases leads to lossof adherens junction complexes, breakdown ofthe apical–basal polarity, and cytoskeletal rear-rangement. Activation of small GTPases alsopromotes transcriptional changes through fac-tors such as myocardin-related transcriptionfactor (MRTF). Inhibition of several mediatorsof the Rho GTPase signaling pathway, includingRho kinase and MRTF, in genetically modifiedmice or with chemical inhibitors has beenshown to attenuate EMTand fibrosis (Theriaultet al. 2007; Bendris et al. 2012; Harris et al.2013; Okada et al. 2015; Sisson et al. 2015).Other prominent pathways activated by TGF-b are the MAP kinase pathways and the phos-phoinositide-3 kinase (PI3K)–Akt pathway.These pathways again allow for interactionand input from a number of different factors,such as FGF, epidermal growth factor (EGF),and tumor necrosis factor (TNF)-a, and corre-spondingly activated receptors. The extensivecross-talk between intracellular pathways directlyactivated by TGF-b and secondary pathwaysthat modulate impact of proximal TGF-b sig-nals on gene expression is discussed extensivelyin other reviews (Derynck et al. 2014).

EMT in Development

The role of EMT during development is dis-cussed at length in many other reviews (Thieryet al. 2009), but is briefly mentioned here toprovide context to studies exploring the poten-tial role of EMT in fibrosis. Embryonic devel-opment has been proposed to involve multipledifferent EMTevents that have been extensivelystudied (Acloque et al. 2009). EMT occursearly in embryogenesis during gastrulation, asepiblasts migrate to form the primitive streak,move to the interior of the blastocyte, and formthe mesodermal germ layer (Nieto et al. 1994;Viebahn 1995; Viebahn et al. 1995; Nieto 2011).Later, a subset of neural tube cells detach andundergo EMT to form the neural crest cellsthat migrate and eventually give rise to manystructures of mesenchymal origin (Dubandet al. 1995; Sauka-Spengler and Bronner-Fraser2008). EMT is also prominent at later stages ofembryogenesis during organogenesis. Among

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the most studied are cardiac valve formation(Nakajima et al. 2000), palate closure (Fitchettand Hay 1989; Griffith and Hay 1992), andpancreatic development. During organogensis,EMT is often followed by a reversal of EMTor mesenchymal–epithelial transition (MET),highlighting the plasticity of cells during devel-opment (Davies 1996; Li et al. 2014). For exam-ple, a subset of cells from the developingpancreatic bud undergo EMT, dissociate, andmigrate into the interstitium where they under-go MET to form the islets of Langerhans(Johansson and Grapin-Botton 2002). Moredurable or permanent EMT also occurs duringorganogenesis in later embryonic development.Recent studies of lung and gut developmentsuggest that serosal mesothelial cells, whichare squamous epithelial cells, are a major sourceof vascular smooth muscle cells, which aremesenchymal cells (Wilm et al. 2005; Dixitet al. 2013). Notably, there is no common epi-thelial cell progenitor during embryogenesis,as epithelial cells are derived from all threeembryonic primordial germ layers. For exam-ple, lung airway and alveolar epithelial cells arederived from the endoderm, renal tubularepithelial cells are derived from mesoderm,and epidermal skin cells are derived from theectoderm. Similarly, mesenchymal cells canderive from ectoderm (e.g., facial cartilage)and mesoderm (e.g., skeletal muscle) (Acloqueet al. 2009; Thiery et al. 2009).

EMT in Fibrosis

The rationale for considering EMT during fi-brosis arose from similarities in prominentEMT and fibrotic signaling pathways. TGF-bis the best defined profibrotic factor and, asmentioned above, a prominent inducer ofEMT (Bartram and Speer 2004). Identificationof aberrant Wnt/b-catenin signaling in IPFlung samples prompted interest in the possibil-ity of EMT in lung fibrosis (Chilosi et al. 2003).Subsequently, almost every cytokine or intracel-lular signaling pathway that has been implicatedin fibrosis or fibroblast activation has beenshown to activate EMT in cell culture. Thus,the cytokine milieu in fibrotic tissue suggests a

pro-EMT stimulus. Given the abundance ofepithelial cells in tissues like the lung and kid-ney, EMT of even a small fraction of epithelialcells could potentially account for a significantnumber of activated fibroblasts. Further, theproposed myofibroblast function may promotefurther activation of other fibroblasts throughthe production of profibrotic factors and induc-tion of mechanical forces. Thus, even transientEMT could initiate a process that leads tomore sustained and robust fibroblast responses.As with cancer, the identification of cells thathave undergone EMT in human disease invivo has been challenging. Many studies haverelied on co-immunostaining for epithelialand mesenchymal markers. Identification ofcostaining cells is suggestive of EMT, but hasinherent limitations (Kakugawa et al. 2005;Willis et al. 2005; Kim et al. 2006). The co-immunostaining approach is at best descriptive,potentially identifying expression of two ormore markers within a cell but lacking anassessment of the functional contribution ofthat cell. Even if an epithelial cell expresses pro-collagen I or SMA, to what extent is it contrib-uting to fibrogenesis? The co-immunostainingapproach likely underrepresents EMT becausemost studies of EMT show simultaneous de-crease in epithelial markers and up-regulationof mesenchymal markers. EMT cells in earlytransition may have weak expression of mesen-chymal markers, and EMT cells in late transi-tion may have weak expression of epithelialmarkers. Co-immunostaining is also beset bydifficulties in achieving specificity of antibodiesand by the potential for artifactual staining ofoverlapping cells giving the appearance of a co-stained cell. Microscopic approaches, such asconfocal or deconvolution microscopy, havebeen used to overcome this limitation (Williset al. 2005; Rock et al. 2011). To partially addressthese issues, several groups have isolated cells byflow cytometry to unequivocally show expres-sion of mesenchymal proteins in epithelial cellsand expression of epithelial cell proteins infibroblasts (Larsson et al. 2008; Marmai et al.2011). To more directly track EMT during fibro-genesis, the alternative approach of fate map-ping individual cells has emerged.

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Cell Fate Mapping

The Cre-recombinase/lox P sequence systemin mice is a powerful way to map and trace thefate of specific cell types in vivo (Akagi et al.1997). The system involves a reporter gene(e.g., green fluorescent protein [GFP] or b-ga-lactosidase) regulated by a strong constitutivelyactive promoter but its expression is disruptedby a short sequence containing a stop codon.This sequence is flanked by loxP sites, enablingit to be permanently removed by Cre recombi-nase. A second transgene encodes the Cre re-combinase under the control of a cell type–specific promoter, enabling cell type–specificremoval of the stop codon sequence, and label-ing those cells by expression of the reporter.Importantly, the labeling occurs at the level ofthe genomic DNA and is thus permanent. Thus,the Cre/lox system has been proposed to enablecell type–specific and permanent labeling ofcells, making it an attractive way to map thefate of cells in response to injury, and to identifythe origin of cells of interest, such as a myofi-broblasts in fibrosis. A limitation of this ap-proach is the potential for off-target expressionof a constitutive Cre recombinase allele, eithertransiently during organ development or inresponse to new signals appearing after tissueinjury. For example, a promoter specific to anadult epithelial cell may have been transientlyexpressed in a mesenchymal cell during organdevelop, or only after tissue injury, resulting inspurious conclusions on the extent or even pres-ence of EMT. To obviate this limitation, Crerecombinase alleles have been constructed thatenable temporal as well as spatial control of Creactivity. Most early versions of this methodolo-gy used cell type–specific expression of a reversetetracycline transactivator (rtTA) to regulateexpression of Cre recombinase by a tetracy-clin-response element (TRE), but this sufferedfrom the requirement of three transgenes for afully operational system and the problem thatthe rtTA function was not completely doxycy-cline-dependent. More recently, Cre recombi-nase activity has been temporally regulatedthrough the use of a fusion protein comprisedof Cre recombinase and a modified estrogen

receptor “knocked in” to a cell-specific promot-er. The estrogen receptor has been further mod-ified such that it is much more sensitive totamoxifen than estrogen (e.g., ERT2) (Feilet al. 2009, 2014). Virtually all expressed Crerecombinase remains in the cytoplasm unlesstamoxifen is present to empower nuclear trans-location and DNA recombination. Various Cre-dependent strategies have been used extensivelyto map the fate of epithelial cells in fibrosis, butwith conflicting outcomes. In lung fibrosis, atleast four different groups have identified sig-nificant numbers of lung epithelial–derivedcells that coexpress mesenchymal markers inmodels of fibrosis (Table 1) (Kim et al. 2006,2009; Tanjore et al. 2009; DeMaio et al. 2012;Balli et al. 2013). These groups used differentpromoters to drive expression of Cre (Nkx2.1and surfactant protein C [SPC]), different re-porter genes (GFP/LacZ), different techniquesto quantify colabeled cells (immunohistochem-istry, single cell suspension with cell sorting,cytospin staining, RNA quantification, and im-munoblotting), and several models of fibrosis(bleomycin, overexpression of TGF-b, and ra-diation) (Kim et al. 2006, 2009; Balli et al. 2013).However, one report, also using the bleomycinmodel of fibrosis, found no evidence of EMT inmice using Sftpc promoter-driven CreERT2 ex-pression to fate-map type II alveolar epithelialcells (Rock et al. 2011). Contradictory results onEMT cell fate mapping are not limited to lungfibrosis. Similar cell fate mapping studies inmodels of kidney and liver fibrosis found sub-stantial evidence or lack of evidence of EMT.Contradictory results in cell fate mapping arenot limited to EMT. Pericytes and fibrocytes,which have been proposed as sources of activat-ed myofibroblasts, have cell fate mappingstudies in favor of or against them serving assignificant sources of myofibroblasts. For peri-cytes, the conflict is even more stark. For exam-ple, in the bleomycin model of lung fibrosis, onereport concludes that more than two thirds ofthe myofibroblasts are derived from pericytes(Hung et al. 2013). In contrast, that same reportconcluded that there is no evidence of EMTand excludes pericytes and two epithelial cellpopulations at the origin of myofibroblasts

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Table 1. Reports on fate mapping during fibrosis

Report Fate mapping system Model EMT

Lung fibrosisKim et al. 2006 Sftpc-rtTA/tetO-Cre/RosaLacZ AdTGF-b YesKim et al. 2009 Sftpc-rtTA/tetO-Cre/ZEG Bleo YesTanjore et al. 2009 Sftpc-Cre/RosaLacZ Bleo YesHashimoto et al. 2010 Tek-Cre/RosaLacZ Bleo YesDeMaio et al. 2012 Nkx2-1-Cre/RosaLacZ Bleo YesRock et al. 2011 Sftpc-CreER/dTomato Bleo NoBalli et al. 2013 Sftpc-rtTA/tetO-Cre/RosaLacZ XRT Yes

Kidney fibrosisIwano et al. 2002 Ggt1-Cre/RosaLacZ UUO YesZeisberg et al. 2008 Tek-Cre/RosaYFP UUO YesHumphreys et al. 2010 Six2-Cre/RosaLacZ UUO NoLeBleu et al. 2013 Ggt1-Cre/RosaLacZ UUO Yes

Cdh5-Cre/RosaYFP UUO Yes

Gastrointestinal and liver fibrosisZeisberg et al. 2007b Alb-Cre/RosaLacZ CCl4 YesFlier et al. 2010 Vil1-Cre/RosaLacZ TNBS YesTaura et al. 2010 Alb-Cre/RosaLacZ CCl4 NoChu et al. 2011 Alb-Afp-Cre/RosaYFP CCl4 No

Cardiac infarctZhou and Pu 2011 Wt1-CreER/tmGFP Art ligation YesZeisberg et al. 2007a Tie1-Cre/Rosa26 Ao banding Yes

Pericyte!Myofib

Lung fibrosisRock et al. 2011 Cspg4-CreER/fGFP Bleo NoHung et al. 2013 Foxd1-Cre/dTomato Bleo Yes

Kidney fibrosisHumphreys et al. 2010 Foxd1-Cre/Rosa26 UUO YesLeBleu et al. 2013 Cspg4-Cre/RosaYFP UUO No

Pdgfrb-Cre/RosaYFP UUO No

BM!Myofib

Lung fibrosisHashimoto et al. 2004 GFP BMT Bleo NoSchmidt et al. 2003 ex vivo labeling OVA YesMadala et al. 2014 GFP BMT TGF-a NoKidney FibrosisIwano et al. 2002 S100a4-GFP BMT UUO YesLeBleu et al. 2013 Acta2-RFP BMT UUO Yes

Liver fibrosisHigashiyama et al. 2009 Col1a2-GFP BMT CCl4 NoKisseleva et al. 2006 Col1a1-GFP BMT BDL No

EMT, epithelial–mesenchymal transition; AdTGF-b, adenovirus encoding active TGF-b1; bleo, bleomycin; XRT, X-ray

treatment; OVA, ovalbumin; UUO, unilateral ureteral obstruction; TNBS, trinitrobenzene sulfonic acid; Pericyte!myofib,

pericyte to myofibroblast transition; BM!myofib, bone marrow–derived cell to myofibroblast transition; art, arterial; Ao,

aortic; BMT, bone marrow transplant; CCl4, carbon tetrachloride; BDL, bile duct ligation; GFP, green fluorescent protein.

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(Rock et al. 2011). Thus, those favoring oropposing the possibility of EMT can point tocell fate-mapping publications supporting theirconclusion.

Despite the promise of cell fate mapping,this approach has failed to resolve the contro-versy regarding the origin of activated myofi-broblasts during fibrosis. This may result fromsome of the same limitations as referred to forthe analysis of costained tissues. In addition,the Cre recombinase system has particularlimitations. Cre-mediated recombination maybe overly sensitive because of weak and transientactivation of the cell-specific promoter in off-target cells leading to sufficient expressionof Cre recombinase to permanently activatereporter gene expression, thus potentially con-fusing the cellular origin of the labeled cell(Vaughan et al. 2015). Additionally, tamoxifen,which is used to activate estrogen receptorfusion proteins, can persist in tissues or cellsfor much longer than previously appreciated(Reinert et al. 2012). Typical tamoxifen dosingand “chase” periods intended to allow loss oftamoxifen before injury may fail to eliminatefunctional tamoxifen levels during experimen-tal injury and this may also result in spuriousconclusions on the cells of origin of expandedpopulations after injury (Vaughan et al. 2015).Ultimately, new methods such as DNA barcod-ing will have to be applied to studies of cell fateto resolve these uncertainties. Even extremelyaccurate cell fate mapping may not reveal thefunctional contributions of subpopulations ofepithelial or mesenchymal cells to fibrosis.

Definition of EMT Revisited

Much of the controversy regarding EMT in can-cer, fibrosis, and even development stems fromdifferences in the definition of EMT, and somehave suggested the need to broaden beyond theoriginal narrow definition of EMT (Nieto et al.2016). On one extreme, epithelial cells might bedefined, in part, as being derived from a parentepithelial cell, and mesenchymal cells as beingderived from a parent mesenchymal cell. EMTis then defined as a process in which a fullydifferentiated epithelial cell loses all epithelial

cell characteristics and gains all mesenchymalcharacteristics. This definition virtually ex-cludes the possibility of EMT that is seen invivo. On the other extreme, EMT might be de-fined as any deviation from a classic epithelialcell phenotype and protein expression patterntoward any acquisition of mesenchymal celltraits or proteins. By this loose definition, typeII alveolar epithelial cell differentiation to type Ialveolar epithelial cell, or epithelial cell migra-tion to re-epithelialize after injury might beregarded as EMT. Indeed, in some circum-stances EMT transcription factors are activatedin re-epithelialization after injury (Savagner et al.2005) in a way that is reminiscent of develop-mental organogenesis, in which ductal epithe-lial cells undergo EMT, migrate and invade, andthen revert back to an epithelial cell phenotypethrough MET. Whether this is EMT or simplya transient gain of migratory behavior with ac-companying changes in a few proteins is a focusof debate. There is increasing use of the terms“partial” and “full” EMT. But most, if not all,transcriptional responses and other cellular pro-cesses are partial. Furthermore, there is increas-ing recognition of unique functions attributedto the hybrid epithelial–mesenchymal pheno-type (Jolly et al. 2016). Defining EMT duringfibrosis has relied on lists of epithelial and mes-enchymal markers (Zeisberg and Neilson 2009),but it remains unclear at what threshold loss ofepithelial markers or gain of mesenchymalmarkers would be sufficient to achieve at leastpartial EMT and what threshold is required forfull EMT. An alternative approach is to focus onthe function of the cells of different origin. Thisis a departure from defining epithelial and mes-enchymal cells as different lineages, but rather afocus on cell phenotype and function.

Similar to the controversy on defining EMT,there is a controversy on the definition of my-ofibroblasts. Some have argued that expressionof SMA itself is not sufficient or required todefine a myofibroblast but rather requires stressfiber formation and a contractile phenotype.Given their heterogeneity, some have suggestedthat the term myofibroblast may better define aphenotype rather than a specific cell type (Hinz2013). Furthermore, although myofibroblasts

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are important fibrogenic effector cells, fibrosis isclearly a multicellular process. Also, myofibro-blasts are an important source of type I collagenduring fibrosis, but type I collagen expression isclearly not limited to myofibroblasts (Zhanget al. 1994). Furthermore, cell type–specific ex-pression of a cytotoxic protein that can be acti-vated, such as thymidine kinase or the diptheriatoxin receptor, enables deletion of specific celltypes and shows that M2 macrophages and my-ofibroblasts are necessary for fibrogenesis (Duf-field et al. 2005; LeBleu et al. 2013). Furtherevidence shows a critical role for M2 polarizedmacrophages during fibrosis (Gibbons et al.2011; Osterholzer et al. 2013).

Deletion of the TGF-b receptor in fibro-blasts, epithelial cells, or bone marrow cells re-sults in abrogated fibrosis (Hoyles et al. 2011; Liet al. 2011; LeBleu et al. 2013). TGF-b clearlyelicits a phenotypic response in many cell typesthat might be viewed as an “activated” state(Katsuno et al. 2013; Derynck et al. 2014; La-mouille et al. 2014). Although the epithelialresponse to TGF-b might result in profibroticeffects other than EMT, epithelial-specific dele-tion of a gene encoding an EMT transcriptionfactor leads to attenuated fibrosis. For example,hepatocyte-specific deletion of Snai1 attenuatesfibrosis in the CCl4 model of liver fibrosis, anddeletion of the gene encoding Forkhead box M1(FoxM1) in alveolar epithelial cells blunts lungfibrosis after bleomycin or radiation (Rowe et al.2011; Balli et al. 2013). In models of kidneyfibrosis, epithelial-specific deletion of the genesencoding the EMT transcription factor Snail1or Twist leads to attenuated fibrosis (Grandeet al. 2015; Lovisa et al. 2015). These studiessupport a complex role of the EMT programbeyond a simplistic view of EMT-derived cellscontributing to a pool of activated myofibro-blasts. Rather, cells in partial EMT may play aunique role necessary for fibrogenesis, throughregulation of TGF-b-induced cell-cycle arrestand cross-talk with other cell types.

Some studies show that fibrosis can beattenuated by epithelial cell-specific deletion ofsecreted proteins that have been attributed to thefunction of activated fibroblasts. For example,connective tissue growth factor (CTGF) is a pro-

fibrotic matricellular protein, whose expression isinduced by TGF-b. CTGF expression has beenused as a marker of activated fibrotic fibroblasts,but several studies indicate that epithelial cellsmay be the major source of CTGF during fibrosisand deletion of CTGF within epithelial cellsblocks fibrosis (Leask and Abraham 2003; Ma-kino et al. 2013; Al-Alawi et al. 2014). Finally,production of type I collagen may be the mostbasic function of fibrogenic effector cells and de-letion of the Col1a1 gene within lung epithelialcells leads to significant reduction in bleomycin-induced lung fibrosis (Rhim et al. 2014).

Thus, using a loss-of-function approach,there is strong evidence that epithelial cells con-tribute to fibrosis in ways that overlap with thoseof other fibrogenic effector cells. Whether this issufficient to be labeled EMT depends on theprecise definition of EMT. A broader questionis whether fibrogenesis requires a differentiationevent or whether cells can undergo significantphenotypic activation without necessarily dif-ferentiating into another cell type. There is in-creasing evidence that fibrosis is a multicellularprocess. The overlapping versus nonredundantfunctional contributions of these cell typesduring fibrosis remains unclear. Finally, atten-tion to how activation, proliferation, and apo-ptosis of different cell types are orchestratedin response to injury or other fibrotic stimulusmight reveal novel targets for intervention, withbetter effects and fewer side effects than target-ing single cell types.

TGF-b REGULATION OF CELLPROLIFERATION AND APOPTOSIS

TGF-b activates the expression of a number ofproteins involved in cell-cycle control, includ-ing regulators of cyclin-dependent kinases, andsignaling by the MAP kinase and PI3K–Aktsignaling pathways that promote cell prolifera-tion. Depending on the context and cell type,TGF-b can have dramatic effects on cell prolif-eration, cell-cycle arrest, senescence, or apopto-sis. Although these effects have mainly beenstudied in the context of cancer biology, thesechanges can have important consequences onthe progression of fibrosis.

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Regulation of Fibroblast Proliferation andApoptosis

Fibrosis is characterized by accumulation offibroblasts and myofibroblasts. The roles ofTGF-b in fibroblast activation to a profibroticphenotype are well described; however, the roleof TGF-b in fibroblast proliferation as a way toaccount for the abundance of these cells remainsunresolved (Bartram and Speer 2004). TGF-bwas initially identified as an extracellular growthfactor that enables anchorage-independent fi-broblast proliferation (Roberts et al. 1980).Many early studies show TGF-b-induced pro-liferation of fibroblasts from multiple tissues(Hill et al. 1986; Moses et al. 1987, 1994; Schre-ier et al. 1993). However, other studies havefound either no effect on proliferation (Fineand Goldstein 1987) or inhibition of fibroblastproliferation by TGF-b in cell culture and invivo (Moses et al. 1990; McAnulty et al. 1997;Keerthisingam et al. 2001; Hostettler et al. 2008.Furthermore, immunostaining tissue sectionsfrom animal models of fibrosis or from lungsof patients with IPF for proliferation markers,such as Ki67, shows substantial proliferation ofepithelial cells but seldom of myofibroblastsof fibroblast foci (El-Zammar et al. 2009; Lomaset al. 2012. These conflicting reports on theability of TGF-b to induce fibroblast prolifera-tion might be resolved by understanding thecostimulatory activation of other signalingpathways that affect the cellular response toTGF-b (Grotendorst et al. 2004). TGF-b caninduce the activation of several signaling path-ways that promote cell proliferation (e.g.,MAPK, PI3K, and Rho signaling). However,the activation of these pathways is not uniqueto TGF-b, and many other mitogens also pro-mote cell proliferation. Therefore, the microen-vironment with the cytokines that stimulatefibroblast proliferation may determine theproliferative response in fibrosis. Importantly,TGF-b can stimulate the expression and secre-tion of a number of growth factors and cyto-kines by fibroblasts or other cell types. Many ofthese TGF-b-induced factors, such as FGF2,PDGF, and CTGF, may secondarily act in auto-crine or paracrine fashion to induce fibroblast

proliferation (Kay et al. 1998; Strutz et al. 2001;Bartram and Speer 2004; Leask and Abraham2004; Leask 2009; Xiao et al. 2012). Some stud-ies argue that the TGF-b responses of fibroblaststoward activation and myofibroblast differenti-ation versus proliferation are mutually exclusive(Grotendorst et al. 2004).

Finally, there is evidence that fibroblastsfrom fibrotic tissue are resistant to apoptosis,and that TGF-b may confer resistance to apo-ptosis by classic death pathways such as theFas-caspase cascade (Zhang and Phan 1999;Chodon et al. 2000; Tanaka et al. 2002; Than-nickal and Horowitz 2006). However, the mech-anism for their resistance to apoptosis ispoorly characterized. It may be mediatedthrough activation of signaling pathways, suchas p38 MAPK and PI3K–Akt (Kulasekaran et al.2009), and involve regulation of “inhibitor ofapoptosis” (IAP) family members (Horowitzet al. 2012; Sisson et al. 2012). TGF-b mayalso promote fibroblast resistance to apoptosisthrough cell-cycle regulators such as p14ARF

(Cisneros et al. 2012). Inhibition of fibroblastand myofibroblast apoptosis through thesepathways may account for some of the accumu-lation of these cells during fibrosis.

TGF-b Regulation of Cell Senescence andApoptosis

The response to TGF-b by epithelial and endo-thelial cells includes inhibition of growth, cell-cycle arrest, senescence, or apoptosis (Tuckeret al. 1984; Heimark et al. 1986; Takehara et al.1987). Various insults can lead to damaged cellsor undamaged cells that have become essentiallynonfunctional within the context of a damagedtissue, promoting either epithelial cell senes-cence or apoptosis with resulting initiation offibrosis (Fig. 3).

Cellular senescence can be broadly definedas a process by which cells irreversibly ceaseto proliferate and typically acquire altered cellshapes and secretory profiles (Kuilman et al.2010; Campisi 2013; Munoz-Espin and Serrano2014) whereas apoptosis results from a processof programmed cell death (Campisi 2003). Sen-escence and apoptosis are adaptive necessary

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processes in response to injury. Senescence ischaracterized by the absence of markers of pro-liferation and increased expression of tumorsuppressor proteins that function as cyclin-de-pendent kinase inhibitors including p16INK4a

and p21Cip1. Strong mitogenic activators suchas activated Ras or growth factor receptor acti-vation can provoke a shut down in the cell cyclewith induction of senescence (Blagosklonny2003; Senturk et al. 2010).

Apoptosis can be initiated through an ex-trinsic or intrinsic pathway (Kuwano et al. 1998,2004; Kuwano 2007). The extrinsic pathway in-volves engagement of an extracellular ligand to acell surface death receptor. Awell-studied inter-action in this context is the activation of Fas byFas ligand (FasL), but other ligands such asTNF-a can also initiate an apoptotic pathway.The intrinsic or mitochondrial pathway is ini-tiated by cell stresses, such as oxidative stress. Asnoted above, this stress can lead to senescence orapoptosis through activation of pro-apoptoticBcl-2 family members, or release of cytochromeC. The role of p21Cip1 may be particularly im-portant in determining the epithelial cell re-sponse to stress toward senescence or apoptosis(Zhang et al. 2005).

TGF-b signaling through Smad-dependentand -independent pathways intersects withmany senescence and apoptotic pathways (Mu-noz-Espin et al. 2013; Munoz-Espin and Ser-rano 2014). Smad complexes can directlyactivate transciption of p21Cip1 (Datto et al.1995), which, as discussed above, is a criticalinducer of cellular senescence (Munoz-Espinet al. 2013). Transcription of the gene encodingp21Cip1 is also regulated in response to PI3K andROS, which are both downstream mediatorsfrom TGF-b signaling. ROS generation canalso lead to activation of latent TGF-b poten-tially perpetuating senescence (Yu et al. 2009).In a more delayed fashion, TGF-b can alsosuppress the expression of transcription factors,such as Id (inhibitor of differentiation or DNA-binding) family members, which have beenassociated with cell proliferation and inhibitionof senescence (Shibanuma et al. 1994; Di et al.2006). Finally, TGF-b-induced secreted factorscould indirectly contribute to epithelial cell

senescence and apoptosis. As mentioned, ROSgeneration in response to TGF-b can initiatecell-cycle arrest or apoptosis. TGF-b can alsoinduce expression and secretion of pro-apopto-tic factors, including FasL and angiotensin(Wang et al. 1999). As with effects on prolifer-ation, concurrent stimulation or activation byother signaling pathways has the potential totune the degree of this TGF-b response.

Early studies in cell culture indicate that ep-ithelial and endothelial cells undergo apoptosisin response to TGF-b. These observations cor-relate well with the increased epithelial cell ap-optosis in fibrotic tissue and in animal modelsof fibrosis (Uhal et al. 1998; Barbas-Filho et al.2001). Indeed, expression of activated TGF-b1using transgenic approaches or adenoviral genetransfer is sufficient to induce epithelial cell ap-optosis and subsequent fibrosis (Lee et al. 2004,2006; Ask et al. 2008). Importantly, TGF-b1-induced fibrosis is attenuated by treating micewith a pan-caspase inhibitor, and in mice withinactivation of BH3-interacting domain deathagonist (BID) or BCL-2-associated (BAX) ex-pression, suggesting that apoptosis is critical forfibrosis (Lee et al. 2004; Kang et al. 2007).

The roles of senescence and apoptosis ininjury, repair, and fibrosis depend on the con-text. Transient apoptosis or senescence mayresult in removal of unwanted cells and cell de-bris, and facilitate repair to reinstate a homeo-static architecture and function. Conversely,sustained or abnormal senescence or apoptosiscould lead to a pathological wound repairresponse that progresses to fibrosis. Excessivesenescence or apoptosis could also lead to de-layed restoration of a damaged endothelial orepithelial barrier, thus promoting persistentleak of serum components into the lumenwith continued activation of damage responsepathways and sustained inflammation. Epithe-lial cell turnover and impaired proliferation mayalso delay production of antifibrotic factors,such as prostaglandin E2 (Moore et al. 2003).Activation of senescence leads to dramatic tran-scriptional changes that are mediated in largepart by responses to activation of the NF-kBand C/EBP-b transcription factors (Campisi2013; Campisi and Robert 2014). In particular,

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significant changes occur in the expression ofmany secreted factors, including cytokines,growth factors, and proteases in what has beencalled a “senescence-associated secretory phe-notype” (SASP). The SASP thus allows the sen-escent cell to become an abundant source ofinjury and inflammatory mediators with poten-tial important effects on promoting fibrosis(Fig. 3) (Aoshiba et al. 2003; Coppe et al.2008, 2010; Aoshiba et al. 2013). Thus, therole of TGF-b in regulating fibrosis is mediatedby divergent effects on both fibroblast and epi-thelial cell survival.

ROLE OF TGF-b1 IN DIVERSE FIBROTICDISEASES

Tissue fibrosis is a common consequence ofmany different acute and chronic insults, whichcan severely impair the function of nearly everyorgan system. Fibrosis can also occur as a pri-mary progressive process without an apparentinciting event. Collectively, fibrosis is a leadingcause of death and morbidity in the developedworld, accounting for up to 45% of all deaths bysome estimates (Wynn 2007). There is consid-erable overlap among the different fibroticconditions, with TGF-b1 signaling having aprominent role. Although progressive fibrosis,in general, is difficult to reverse or even slowdown, understanding the role of TGF-b1 sig-naling in this process offers insight into poten-tial novel therapeutic strategies. A review of thehistory and current thinking of the roles ofTGF-b1 in pulmonary fibrosis serves to high-light this principle.

Evidence Linking TGF-b1 to Fibrosis in theLung Parenchyma

As the promoting role of TGF-b1 in experimen-tal fibrosis was well described soon after discov-ery of TGF-b1 (Roberts et al. 1986), the role ofTGF-b1 in pulmonary fibrosis was examineddecades ago. TGF-b1 expression was found toappear early in the bleomycin-induced model ofpulmonary fibrosis, preceding collagen deposi-tion, and to localize to sites of subsequent col-lagen accumulation in both a mouse model and

in humans with fibrotic lung disease (Hoyt DG1988). Active TGF-b1, when overexpressed inthe lung using a viral vector, was found tobe sufficient to cause severe lung fibrosis inrodents, requiring signaling through Smad3(Sime et al. 1997; Gauldie et al. 1999). Interest-ingly, in an inducible model of TGF-b1 expres-sion in lungs, reversal of TGF-b1 expression ledto substantial resolution of the fibrotic process(Lee et al. 2004). TGF-b1 proved to be bothnecessary and sufficient for lung fibrosis. Asdiscussed above, activation of endogenous,latent TGF-b1, via the integrin avb6, was foundto be required for lung fibrosis in both bleomy-cin- and radiation-induced lung fibrosis models(Munger et al. 1999), as well as models of kidneyfibrosis. The critical role of TGF-b1 in fibrosiswas further validated by expression profiling ofhuman fibrotic lungs. Microarray profiles revealthat �80% of the genes activated in fibrotichuman lungs are known TGF-b1 target genes(Kaminski et al. 2000).

Epithelial Activation and Fibrogenesis

Genetic analysis of patients with familial inter-stitial pneumonitis point to type II alveolarepithelial dysfunction as a consequence of SPgene mutations and as a causal mechanism forpulmonary fibrosis (Nogee et al. 2001; Thomaset al. 2002; Wang et al. 2009; Garcia 2011).Mouse models expressing mutant SPs supportthis conclusion. This has led to the general con-cept that epithelial cells are both vulnerable tofurther injury and “activated” as a driver offibrosis. There is evidence to support the devel-opment of at least three distinct dysfunctionalstates in the context of chronic injury and/orstress to the epithelium: TGF-b1-induced mes-enchymal transition (EMT as defined above),senescence, and ER stress (Fig. 3). As discussedabove, TGF-b1 may itself contribute to bothsenescence and ER stress. Each of these dysfunc-tional states have distinct biological features butall show evidence of enhanced secretory activity,at least some of which consists of profibroticand/or pro-inflammatory cytokines and medi-ators. Frequently superimposed on these dys-functional states are hypoxia, enhanced oxidant

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production, and exposure to environmental tox-ins such as smoke and viruses, further contrib-uting to alveolar dysfunction. Collectively, thesestresses reprogram the epithelium toward a se-cretory phenotype altering the immune milieuand promoting mesenchymal expansion (Fig. 3).

Progressive dysfunction of the alveolar epi-thelium can lead to apoptosis and alveolar col-lapse. Collapse of small distal airways (,2 mmdiameter) and surrounding alveoli is prominentduring parenchymal lung injury and repair(Seeger et al. 1993; Lutz et al. 2015). Active sitesof fibrogenesis, termed fibrotic foci, in intersti-tial pneumonitis have been noted by many in-vestigators to contain basement membranewithin the interstitium, suggesting that the fo-cus of fibrogenesis may emanate from hypoxicareas of small airway and alveolar collapse withincorporation of epithelial lining and inflam-matory alveolar cells into a mesenchymal com-partment (Myers and Katzenstein 1988). Thisphenomenon has been termed induration fi-brosis and may be unique to lung architecturebecause incorporation of epithelial cells into amesenchymal compartment has not been ob-served during either liver or kidney fibrogenesis(Lutz et al. 2015). Airway and alveolar collapsemay promote direct epithelial–mesenchymalinteractions, including activation and signalingby TGF-b1. The ultimate fate of such epithelialcells is uncertain but could be either apoptosisand/or persistent EMT, as discussed above.Hypoxia is known to promote collagen expres-sion in fibroblasts and TGF-b1-dependentEMT in primary epithelial cells and within tu-mors (Falanga et al. 2002; Tzouvelekis et al.2007; Zhou et al. 2009; Kottmann et al. 2012).Alveolar collapse also likely contributes to in-creased tissue stiffness that itself can propagateTGF-b1 activation as discussed above, raisingthe possibility that such collapse is an impor-tant trigger for disease progression.

TGF-b1 Signaling in Airway Fibrosis:Integration of Immunity and Stromal Signaling

The major lung airways are a frequent site ofinjury from environmental exposures. Themost common chronic lung diseases, asthma

and chronic obstructive pulmonary disease(COPD), are airway-centric inflammatoryprocesses and in both cases there is repeatedepithelial dysfunction and disordered ECMremodeling with collagen accumulation in thesubmucosa of airways (Aoshiba and Nagai2004; Davies 2009; Hirota and Martin 2013).The development of fixed airway obstructionin some asthmatic patients over time is thoughtto result from airway wall remodeling in whichairway collagen accumulation is a common andprominent feature. In some studies, high-reso-lution imaging of asthmatic airway wall thick-ness correlates with lung function and fixedairflow obstruction, cardinal features of airwaydisease (de Jong et al. 2005; Bosse et al. 2008).Airway remodeling, however, is not simply fi-brotic tissue accumulation in the airway walls.Remodeling involves almost all elements of theairway wall: chronic inflammation involvinginnate and adaptive immune cells, epithelialgoblet cell and subepithelial smooth musclehyperplasia, as well as submucosal neovascula-rization in addition to thickening of the sube-pithelial lamina reticularis, one of the hallmarksof asthma histopathology (Postma and Timens2006; Hirota and Martin 2013). Lying immedi-ately below the basement membrane, the lami-na reticularis is a membranous, collagenousairway structure found in large conductingairways and thickened in asthmatics by the ac-cumulation of several ECM proteins, includingthe fibrillar collagens type I and type III. Colla-gen accumulation in the lamina reticularis inasthma is not as well organized into fibrillarbundles as fibrils in fibrotic tissues, but none-theless collagen fibrils are commonly observedthroughout these structures (Saglani et al.2006). In addition, the airway submucosal col-lagens surrounding fibroblasts are frequentlyexpanded in both animal models of chronicasthma and asthmatic patients. Similar changesin airway collagen accumulation occur to asomewhat lesser degree in COPD airways (Jef-fery 2001; Aoshiba and Nagai 2004; Postma andTimens 2006). Such airway accumulation offibrillar collagens may restrict airway lumensize, and this has been argued in rodent models.Because of the many elements involved in air-

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way remodeling, it has not been determined towhat degree collagen deposition per se contrib-utes to fixed airway obstruction in longstandingactive asthma. Nonetheless, airway remodelinginvolving accumulation of collagen is a promi-nent feature of asthma, and, as discussed below,accumulating evidence links this pathobiologyto TGF-b1 activation (Makinde et al. 2007).

The dynamic interplay between activatedepithelial cells and surrounding airway stromalelements in asthma has led to the concept of anepithelial–mesenchymal “trophic unit,” imply-ing that airway homeostasis is maintained by anongoing exchange of soluble mediators betweenthe epithelium and surrounding mesenchymalcells (Holgate et al. 2004). In contrast, there isno evidence that activated epithelial cells crossthe airway basement membranes to directlycontact mesenchymal cells or become mesen-chymal cells, in either asthma or COPD. Theimpact of signaling interactions between the ep-ithelial lining cells and underlying stromal ele-ments has largely been studied in the context ofperturbed epithelial and immune cell functionin inflammatory airway disease. Most studiespoint to mediators released by native and adap-tive immune cells that activate epithelial cellsto release cytokines impacting airway broncho-constriction and remodeling (Boxall et al. 2006;Doherty et al. 2011; Halwani et al. 2011; Hirotaand Martin 2013; Trian et al. 2015). Given thecollagen accumulation as a prominent part ofairway wall remodeling, it is not surprising thata major target of immune-mediated cytokinesignaling is increased TGF-b1 expression andactivation in both the epithelium and the airwaywalls of asthmatic and COPD patients. Epithe-lial–mesenchymal cross-talk has been studiedin COPD, focusing on the squamous metaplasiacommonly observed in large conducting air-ways of COPD patients (Araya et al. 2007).Squamous epithelial cells in culture, but notthe airway basal cells from which they are de-rived, release cytokines, especially IL-1b, whichthen induce TGF-b1 activation and myofibro-blast accumulation. Activated fibroblasts arethought to promote a positive feedback forthis process by further releasing TGF-b1 to im-pair epithelial proliferation and promote the

squamous phenotype. The squamous morphol-ogy of cultured bronchial epithelial cells and thehigh expression of involucrin and the cyclin-dependent kinase inhibitor p21Cip1 are sugges-tive of a senescent phenotype, a phenotypeknown to be promoted by stress and accompa-nied by release of pro-inflammatory cytokines,although this possibility has not been directlyaddressed in the lung (Rodier and Campisi2011). Similar pathways are likely activated inasthma. In this case, the production of Th2 cy-tokines by innate and adaptive immune cells isincreased, and their activities on epithelial cells,as well as epithelial injury, lead to the release ofcytokines including TGF-b1, PDGF, and insu-lin-like growth factors by the epithelial cells.These then promote proliferation of fibroblastsand smooth muscle cells that is characteristic ofairway remodeling in asthma (Holgate et al.2004; Boxall et al. 2006; Davies 2009). As dis-cussed below, a common and important ele-ment in altered fibroblast function in all thesesettings involves TGF-b1 activation, which isthe likely driver of submucosal airway collagenaccumulation.

Stromal elements involved in remodelingof asthmatic airways show enhanced sensitivityto immune Th2 cytokine signals. Primary hu-man airway fibroblasts in culture respond toIL-13, a prominent Th2 cytokine implicatedin asthma pathobiology, with activation ofCOL1A2 mRNA expression and collagen pro-tein deposition. This response is greater inprimary cells isolated from asthmatic patientswhen compared with cells of healthy individu-als (Firszt et al. 2014). IL-13-induced collagenexpression was found to depend on TGF-b1activation and signaling by fibroblasts. IL-13also promotes TGF-b1 expression and releasefrom asthmatic epithelium. Earlier studiesexploring the response of airway fibroblasts toIL-4 report similar findings, suggesting that insome way the ongoing inflammation and stim-ulation of stromal elements primes these airwayfibroblasts for further response (Saito et al.2003). In addition, smooth muscle cell prolifer-ation has been reported to be hyperactive inasthmatic airways (Trian et al. 2015). Bronchialsmooth muscle cells from asthmatic large air-

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ways express higher levels of the cysteinyl leuko-triene receptor CysLTR1, a receptor for leuko-triene C4, and display an increased proliferativeresponse to the epithelial-derived leukotrieneC4 compared with smooth muscle cells fromnormal airways (Trian et al. 2015). These find-ings underscore the paradigm that the epitheli-al-derived signals are in part responsive to cy-tokine stimulation from immune cells, andactivate bidirectional signaling that leads to air-way remodeling with airway fibrosis in bothasthma and COPD.

The effector mechanisms of TGF-b1-de-pendent fibrogenesis in airways do not appeardifferent from other sites of fibrosis driven byTGF-b1, but the mechanisms of latent TGF-b1activation are distinct in the conducting air-ways. Whereas alveolar epithelial TGF-b1 acti-vation depends on integrinavb6, large conduct-ing airway epithelial TGF-b1 activation ismediated by both avb6 and avb8 integrins(Munger et al. 1999; Mu et al. 2002). The mech-anism of activation of TGF-b1 by avb8 requiresmetalloproteinase (MMP)-mediated cleavageof latent TGF-b1, in contrast to cytoskeleton-mediated conformational strain on integrins inavb6-mediated TGF-b1 activation. Dendriticcells also require avb8 to activate surface TGF-b1 that in turn promotes differentiation of CD4T cells to T cells that produce IL-17 along withIL-6, and to regulatory T cells (Tregs) (Travisand Sheppard 2014). Expansion of IL-17-pro-ducing CD4þ T cells enhances airway smoothmuscle contraction in response to binding ofIL-17 to its receptors that are expressed onsmooth muscle cells, extending the well-knownrole of IL-17 as a pro-inflammatory cytokine(Kudo et al. 2012). Mice that lack expressionof the dendritic cell integrin avb8, as a resultof Cre recombinase expression from the Itgaxpromoter and Cre-mediated deletion of b8,spontaneously develop an autoimmune colitisat about six months of age, consistent with thewell-known role of TGF-b1 in promoting dif-ferentiation of T cells toward an immunosup-pressive Treg lineage (Wan and Flavell 2007).The anti-inflammatory and immune regulatoryeffects of TGF-b1, mediated in large part bydendritic cells, are prominent in asthma and

are layered on top of the profibrotic pathwaysinitiated in activated epithelial cells and fibro-blasts. Hence, it may not be meaningful toattempt to correlate the overall levels of latentTGF-b1 expression or even TGF-b activityin the lung with the severity or progression ofasthma and COPD. For example, inhibition ofTGF-b1 signaling protects mice from airwayremodeling in an allergen-induced model, butwhat affected cell type accounts for this protec-tion is uncertain (McMillan et al. 2005). Micedeficient in periostin expression have enhancedbronchoconstrictor responses and higher aller-gen-induced IgE expression in an allergy model(Gordon et al. 2012). Periostin interacts withcollagen and promotes its organization intofibrils. In the absence of periostin, TGF-b1 ac-tivation is reduced resulting in decreased Tregcell differentiation with an enhanced immuneresponse. Thus, the pathogenic role of TGF-b1in asthma pathobiology, and peri-airway fibro-sis specifically, is complicated by the high acti-vation level of the mucosal immune system inasthma. Indirect effects through changes in im-mune cell function rather than directly alteredfibroblast collagen production could be a majormechanism for TGF-b1 effects in asthma andCOPD.

THERAPEUTIC IMPLICATIONS

Integrin-mediated TGF-b activation has beenshown to substantially contribute to tissuepathology in a number of disease models. Forexample, mice lacking theavb6 integrin are pro-tected in murine models of lung, kidney, andbiliary fibrosis (Munger et al. 1999; Jaramilloet al. 2003; Hahm et al. 2007). These mice arealso protected in models of acute lung injury(Pittet et al. 2001), allergic asthma (Sugimotoet al. 2012; Jolly et al. 2014), and severe influen-za infection (Jolly et al. 2014), and in each case,protection appeared to be explained by localinhibition of TGF-b activation. These results,together with evidence that avb6 integrin ex-pression is dramatically increased in dysfunc-tional epithelial cells overlying regions of pul-monary fibrosis in humans (Horan et al. 2008),suggest that this integrin could be a promising

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therapeutic target. The development of potentblocking monoclonal antibodies that inhibitavb6-mediated TGF-b1 activation in multiplespecies has made it possible to evaluate their usein disease models after tissue pathology hasalready been established. The success of theseantibodies in multiple disease models (Horanet al. 2008; Puthawala et al. 2008) has stimulatedthe development of a humanized monoclonalantibody that targets avb6 and is currently in aphase II clinical trial for treatment of IPF.

avb8-dependent TGF-b1 activation has alsoemerged as a promising therapeutic target. Micelacking this integrin on dendritic cells are dra-matically deficient in antigen-specific T helper17 (Th17) cells, and are protected in experimen-tal autoimmune encephalitis (Melton et al.2010) and allergic asthma (Kudo et al. 2012).Mice lacking avb8-mediated TGF-b1 activationin fibroblasts are protected in models of airwayinflammation, and studies in mice engineeredto replace murine b8 with its human orthologshow that a monoclonal antibody against hu-man avb8 that blocks TGF-b1 activation pro-tects against allergic airway inflammation andremodeling induced by exposure to cigarettesmoke (Minagawa et al. 2014).

As noted above, recent evidence suggeststhat the avb1 integrin on fibroblasts contributesto tissue fibrosis in multiple organs (Lawsonet al. 2008, 2011; Tanjore et al. 2011, 2012,2013; Torres-Gonzalez et al. 2012; Hendersonet al. 2013; Reed et al. 2015). Therapeutic inter-vention with a broadly active av integrin inhib-itor or a highly specific avb1 small moleculeinhibitor in mice with already established liveror pulmonary fibrosis causes a marked reduc-tion in collagen accumulation, suggesting thatavb1 could also be a therapeutic target for treat-ment of diseases characterized by excessive tis-sue fibrosis.

Besides the strong rationale for therapeuticagents directed at integrin-dependent TGF-b1activation, elements of TGF-b1 signaling andother aspects of the pathophysiology of fibro-genesis are also potential therapeutic targets. Asnoted above, a number of profibrotic cytokinessuch as CTGF, FGF, and PDGF have beenexplored as potential targets for antifibrotic

therapy, in large part because of their conver-gence with the TGF-b pathway. Studies of EMTin fibrosis began largely because of the strongparallels between signaling pathways of fibrosisand pathways of EMT in cell culture. One of theironies then about the possible roles of EMT infibrosis is that, from a clinical viewpoint, ther-apeutic agents directed at either pathways ofEMT or pathways of fibroblast expansion andECM secretion may almost completely overlap,making definitions and cells of origin of littleimportance. For example, the best-studied EMTtranscription factors are Snail1, Slug/Snail2,Twist, ZEB1, and Sip1/ZEB2. Their expressioncan be induced by TGF-b1 in both epithelialand mesenchymal cells. Understanding thesepathways and the epithelial cell response toTGF-b offers potential for therapeutic interven-tion in fibrosis, even if many of these pathwaysare not unique to EMT. In addition, expansionof the ECM impacts fibrogenesis downstreamfrom TGF-b1 activation by exerting strongprosurvival signals as a result of integrin activa-tion. Activated b1 integrins promote FAK andAkt activation leading to accumulation of anti-apoptotic factors that could counter the pro-apoptotic signaling in response to TGF-b1in the epithelium, ER stress, and senescence(Horowitz et al. 2007; Thannickal et al. 2014).Moreover, recent studies point to ECM stiffnessand integrin activation as a TGF-b1-indepen-dent pathway of epithelial Stat3 activation withsubsequent further fibroblast activation andECM accumulation (Prele et al. 2012; Duffield2016). These studies highlight some additionalsites of potential therapeutic interventiondownstream from TGF-b1 activation that arestudied in clinical and preclinical trials (Ahlu-walia et al. 2014; Blackwell et al. 2014). Thecurrent rationales and status of trials directedat TGF-b signaling in fibrosis have recentlybeen well summarized (Ahluwalia et al. 2014).

Emerging evidence indicating the promi-nence of ER stress and cell senescence in thepathobiology of fibrotic lung disease also offersnew therapeutic possibilities (Lawson et al.2008, 2011; Tanjore et al. 2011, 2012, 2013; Tor-res-Gonzalez et al. 2012). TGF-b1 signalingmay contribute to this pathophysiology indi-

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rectly by the induction of enhanced collagensecretion in cells that are already stressed bythe accumulation of misfolded proteins. In-deed, collagen itself is only marginally solubleand its complicated helical structure is prone tomisfolding. Whether and how the UPR can bemanipulated to attenuate the potentially profi-brotic and self-destructive elements in itsresponse to ER stress (Fig. 3) is an importantavenue for further investigation. Likewise, theaccumulation of senescent cells in the contextof chronic and recurring injury, as well as agingitself, may offer new insights (Sanders et al.2013; Thannickal 2013; Hecker and Thannickal2016). For example, mice lacking caveolin 1expression were found to be protected fromfibrosis through decreased epithelial senescenceafter bleomycin-induced injury (Shivshankaret al. 2012). Further studies of telomerase acti-vators have been suggested as a potential pointof therapeutic intervention in senescence-initi-ated fibrosis (Le Saux et al. 2013). Similarly,targeting nicotinamide adenine dinucleotidephosphate (NADPH) oxidase 4 (NOX4), aTGF-b1-responsive gene, attenuates lung fibro-sis at least in part through inhibition of NOX4-or ROS-mediated senescence (Hecker et al.2009). The potentially profibrotic and pro-in-flammatory character of the SASP secretomeand regulators of senescence during fibrogene-sis need better clarification (Kang et al. 2007).It remains unclear to what degree the SASPand the secretory profile of TGF-b1-inducedEMT and ER stress (Fig. 3) share expressionof common profibrotic mediators or exert theireffects on fibrosis through distinct pathways.These uncertainties remain important toclarify as attempts to develop new therapeuticscenter around the early events that lead to ep-ithelial dysfunction and a disordered repairprocess, not only in the lung but at all sites offibrogenesis.

ACKNOWLEDGMENTS

We thank Justin A. Klein, CMI, for assistancewith illustrations. This work was supported inpart by the National Institutes of Health (NIH)grant PO1 HL108794.

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21, 20172018; doi: 10.1101/cshperspect.a022293 originally published online AprilCold Spring Harb Perspect Biol 

 Kevin K. Kim, Dean Sheppard and Harold A. Chapman 

1 Signaling and Tissue FibrosisβTGF-

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Development Family Signaling in Early VertebrateβTGF-

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Family Signaling in MesenchymalβTGF-

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ApproachesBased Therapeutic−Bone Morphogenetic Protein

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1 Signaling and Tissue FibrosisβTGF-

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and Branching Morphogenesis Family Signaling in Ductal DifferentiationβTGF-

MoustakasKaoru Kahata, Varun Maturi and Aristidis

Homeostasis and DiseaseBone Morphogenetic Proteins in Vascular

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and Cancer Progression Family Signaling in Tumor SuppressionβTGF-

Joan Seoane and Roger R. GomisSkeletal Diseases

Family Signaling in Connective Tissue andβTGF-

Dietz, et al.Elena Gallo MacFarlane, Julia Haupt, Harry C.

Signaling for Therapeutic GainβTargeting TGF-Rosemary J. Akhurst

Family in the Reproductive TractβThe TGF-

A. PangasDiana Monsivais, Martin M. Matzuk and Stephanie

Family Signaling MoleculesβDisease by TGF-Regulation of Hematopoiesis and Hematological

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Drosophila Family Signaling in βTGF-

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Differentiation, Development, and Function Family Signaling in Neural and NeuronalβTGF-

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