Signaling in fibrosis: TGF-β, WNT, and YAP/TAZ converge

REVIEW published: 03 September 2015 doi: 10.3389/fmed.2015.00059 Signaling in fibrosis: TGF-β, WNT, and YAP/TAZ converge Bram Piersma *, Ruud A. Bank and Miriam Boersema Matrix Research Group, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands Edited by: Achim Weber, University of Zurich, Switzerland Reviewed by: Francesco Trapasso, University “Magna Græcia” of Catanzaro, Italy Kenji Notohara, Kurashiki Central Hospital, Japan *Correspondence: Bram Piersma, Matrix Research Group, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, Groningen 9728HR, Netherlands b.piersma@umcg.nl Specialty section: This article was submitted to Pathology, a section of the journal Frontiers in Medicine Received: 26 May 2015 Accepted: 13 August 2015 Published: 03 September 2015 Citation: Piersma B, Bank RA and Boersema M (2015) Signaling in ibrosis: TGF-β, WNT, and YAP/TAZ converge. Front. Med. 2:59. doi: 10.3389/fmed.2015.00059 Frontiers in Medicine | www.frontiersin.org Chronic organ injury leads to ibrosis and eventually organ failure. Fibrosis is characterized by excessive synthesis, remodeling, and contraction of extracellular matrix produced by myoibroblasts. Myoibroblasts are the key cells in the pathophysiology of ibrotic disorders and their differentiation can be triggered by multiple stimuli. To develop anti-ibrotic therapies, it is of paramount importance to understand the molecular basis of the signaling pathways contributing to the activation and maintenance of myoibroblasts. Several signal transduction pathways, such as transforming growth factor (TGF)-β, Wingless/Int (WNT), and more recently yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling, have been linked to the pathophysiology of ibrosis. Activation of the TGF-β1-induced SMAD complex results in the upregulation of genes important for myoibroblast function. Similarly, WNT-stabilized β-catenin translocates to the nucleus and initiates transcription of its target genes. YAP and TAZ are two transcriptional co-activators from the Hippo signaling pathway that also rely on nuclear translocation for their functioning. These three signal transduction pathways have little molecular similarity but do share one principle: the cytosolic/nuclear regulation of its transcriptional activators. Past research on these pathways often focused on the isolated cascades without taking other signaling pathways into account. Recent developments show that parts of these pathways converge into an intricate network that governs the activation and maintenance of the myoibroblast phenotype. In this review, we discuss the current understanding on the signal integration between the TGF-β, WNT, and YAP/ TAZ pathways in the development of organ ibrosis. Taking a network-wide view on signal transduction will provide a better understanding on the complex and versatile processes that underlie the pathophysiology of ibrotic disorders. Keywords: fibrosis, myofibroblast, TGF-β, WNT, YAP/TAZ, Hippo, signaling Introduction Regardless of the initial trigger, chronic organ injury disturbs the cellular and molecular processes of normal wound healing, resulting in organ ibrosis and eventually organ failure (1). Chronic injury causes prolonged activation of efector cells, such as ibroblasts (2), pericytes (3–5), bone marrow-derived cells (6–8), and possibly cells from epithelial (9) or endothelial origin (10, 11), which diferentiate toward myoibroblasts. In normal granulation tissue, myoibroblasts are essential for the deposition, contraction, and remodeling of the extracellular matrix (ECM) and thereby promote wound healing (12). However, aberrant wound healing results in increased proliferation 1 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge and attenuated apoptosis of myoibroblasts. he well-developed cytoskeletal apparatus of myoibroblasts contains actin and myosin, which are linked to the so-called supermature focal adhesions that connect the cells actin ilaments to the ECM (13). his allows myoibroblasts to contract the ECM around them and create contractures that impede organ function (14). he de novo expression of smooth muscle α-actin (αSMA), an isoform usually expressed in smooth muscle cells, further enhances their contractile capabilities (15, 16). Moreover, myoibroblasts are notorious producers of ECM components, such as collagens, glycoproteins, and proteoglycans, resulting in the formation of ibrous scar tissue. Cross-linking of collagen in ibrous scar tissue makes it highly resistant to protease degradation and results in irreversible scarring and destruction of the tissue architecture (17). Although a large body of knowledge exists on myoibroblast biology, as of to date, no approved therapies are available that can reverse ibrosis (18, 19). hus, understanding the molecular mechanisms that govern the diferentiation and maintenance of myoibroblasts in ibrotic diseases is of paramount importance. he diferentiation of myoibroblasts is governed by an interplay between diferent mechanisms. Under increased tissue stifness and mechanical strain, ibroblasts become activated and show increased β- and γ-actin and αSMA-containing stress ibers, linked to focal adhesions (15). hey also start to express the ED-A splice variant of cellular ibronectin – crucial for myoibroblast diferentiation – at the plasma membrane (20, 21). Membrane protruding integrin molecules connect the ECM components to the actin ibers, which allows for the conversion of mechanical into biochemical cues that are relayed to the nucleus. Alternatively, myoibroblast diferentiation is driven by biochemical signaling of extracellular growth factors. Many growth factor families have been studied extensively in the context of organ ibrosis, with an emphasis on the transforming growth factor (TGF)-β and Wingless/Int (WNT) signaling pathways as key mediators [reviewed in Ref. (22, 23)]. heir mode of action describes the production of soluble growth factor ligands by a variety of cell types. he growth factors are stored in the ECM, until they are activated and released by mechanical tension or proteolytic cleavage, which enables these ligands to engage their membrane-bound receptors. he receptors relay the biochemical signal inwards, via kinase complexes, to the nucleus. Nuclear transcriptional modulators then act on the chromatin complex in order to change the transcriptional landscape, and thereby promote or repress transcription of target genes. Recently, in ibrosis research the attention shited toward a relatively new signaling cascade: yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling. Interestingly, the three mentioned signal transduction pathways have but little molecular similarity but do share one principle: the cytosolic/ nuclear regulation of their transcriptional modulators. In the past, signaling cascades were oten studied in isolation, i.e., a ligand signals through its receptor and mediates the nuclear accumulation of one or several transcription factors to modulate target gene expression. his view changed since recent advances suggest that these cascades are in fact organized into complex signaling networks which, dependent on the cellular and environmental context, govern cell function and fate in Frontiers in Medicine | www.frontiersin.org ibrotic disorders. his inter-pathway communication allows for increased versatility and ine tuning of cellular responses, which may explain the variety of phenotypes found in ibrotic disorders. he aim of this review is to discuss the current understanding on the signal integration between the TGF-β, WNT, and YAP/TAZ pathways in the development of ibrosis. We will start with a short overview of the three pathways, and extend our discussion with a detailed view on how these pathways connect at multiple levels of signal transduction in the context of myoibroblast function and ibrosis. Finally, we touch upon the challenges and considerations in the design of anti-ibrotic therapies, with focus on the crosstalk between the three signal transduction cascades. Canonical TGF-β Signaling he TGF-β superfamily of growth factors consists of multiple proteins that govern a wide range of physiological processes, such as stem cell pluripotency, cell fate determination, proliferation, and diferentiation. In humans, over 30 members of the TGF-β superfamily have been documented, including TGF-βs, activins, inhibins, nodal, growth/diferentiation factors (GDFs), and bone morphogenetic proteins (BMPs). In this review, we focus mainly on the canonical signaling through TGF-β1, since considerable evidence exists for its role in ibrosis (22) (Figure 1). In homeostatic conditions, TGF-β is trapped in the ECM together with latency-associated peptides (LAPs) and latent TGF-β-binding proteins (LTBPs) in the so-called large latent complex (LLC) (24). Upon injury, proteolytic cleavage of the LAP (25), or binding of integrins together with increased mechanical forces (26–28), cause release of TGF-β from the LLC allowing it to engage its receptors. Signaling propagation occurs when a TGF-β homodimer interacts with two type I and two type II receptors. Ligand binding initiates the phosphorylation of the SGSGSG domain on the type I receptor by the type II receptor (29, 30). Subsequently, the activated type I receptor is now able to bind and phosphorylate Smad proteins, the central modulators of canonical TGF-β signaling. here are three classes of Smad proteins: regulatory (R)-Smads, co-activator (Co)-Smads, and inhibitory (I)-Smads. R-Smads (Smad2 and Smad3) are phosphorylated by the type I receptor and form heteromeric complexes with the Co-Smad, Smad4. Both R-Smads and Smad4 consist of a N-terminal MH1 and a C-terminal MH2 domain connected by a linker region. Upon phosphorylation of the MH2 domain, Smad complexes shuttle to the nucleus and together with DNA-binding proteins (31–33) localize to speciic CAGAC motifs, the so-called Smad-binding elements (SBE), to regulate transcription of target genes (34). here is ample evidence that TGF-β signaling is a key regulator of myoibroblast biology in the heart, lungs, liver, kidneys, and skin (35–49). TGF-β levels are elevated in ibrotic tissues and myoibroblasts display nuclear accumulation of Smads in vivo accompanied with an increased expression of TGF-β target genes and decreased levels of the inhibitory Smad6 and Smad7. Despite a tremendous body of experimental work, the mechanisms underlying Smadinduced ibroblast activation are incompletely understood, as both activation and inhibition of Smads can promote ibrogenesis, dependent on the context (37, 47, 50–55). Furthermore, inhibition 2 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge (D) The activated Smad complex forms a transcriptional module with several transcription factors, co-factors such as p300 and Creb binding protein (CBP) to promote transcription of target genes (e.g., PAI1, COL1A1, CCN2). (E) Dephosphorylated Smad proteins continuously shuttle between the nucleus and the cytoplasm. In the cytoplasm, they can be targeted for degradation by ubiquitin ligases. (F) Consecutive phosphorylation by CDK8/9 and GSK3 in the nucleus recruits the ubiquitin ligase Nedd4L that target Smad proteins for proteasomal degradation in the cytoplasm, and possibly the nucleus. FIGURE 1 | Canonical TGF-β signaling in fibrosis. (A) Transforming growth factor-β (TGF-β) homodimers engage the type II receptors, which phosphorylate and form a heterotetrameric complex with two type I receptors and additional Smad-binding proteins such as SARA. The signaling domain of the type I receptor mediates phosphorylation and activation of Smad proteins. (B) Smad4 associates with phosphorylated Smads to form an active heterotrimeric complex. (C) The inhibitory Smad7 together with Smad-speciic E3 ubiquitin protein ligases (Smurf) inhibits the receptor complex by targeting it for ubiquitination and proteasomal degradation. of the Smad signaling cascade does not completely attenuate the ibrotic response, which suggests that several other signaling cascades are involved in activating the transcriptional program of myoibroblasts. It has become evident that transcriptional output of Smad signaling is tightly controlled by the interplay with a variety of master transcription factors, DNA-binding (co)factors, repressors, and chromatin readers, and writers (33, 56). named Frizzled (Fz). A single WNT ligand can interact with several Fz receptors, and vice versa (59). In a WNT-of state, the concentration of endogenous WNT antagonists outweighs that of WNT ligands, which results in the phosphorylation of cytoplasmic β-catenin by two subunits from the β-catenin destruction complex, glycogen synthase kinase (GSK)3, and casein kinase (CK)1. hese phosphorylation events trigger subsequent ubiquitination and proteasomal degradation of β-catenin (60). In a WNT-on state, ligands engage the Fz receptors which function together with the low-density-lipoprotein-receptor-related proteins (LRP)5 and LRP6 co-receptors to activate the downstream signaling cascade. LRP is phosphorylated in its cytoplasmic tail by GSK3 and CK1 proteins (61–63). he activated Fz/LRP complex interacts with Disheveled (DVL), Axin, and GSK3 through Pro-Pro-Pro-(Ser/Tyr)-Pro repeats (62, 63). Axin functions as a scafold for the destruction complex, as it directly interacts with β-catenin, GSK3, CK1, the tumor suppressor protein adenomatous polyposis coli (APC), and the ubiquitin ligase β-TrCP. As Axin and GSK3 are sequestered to the plasma membrane by Canonical WNT Signaling Discovered in Sophophora (Drosophila) as Wingless and in the mouse as Int1, together termed WNT in mammals, canonical WNT signaling comprises the molecular interactions leading to the nuclear translocation of β-catenin [reviewed in Ref. (23, 57)] (Figure 2). WNTs have primarily been studied in fetal development as they are responsible for the formation and polarity of the primary body axis (58), but it has become evident that they are versatile growth factors in both homeostasis and disease. Soluble WNT ligands bind to a family of seven transmembrane receptors Frontiers in Medicine | www.frontiersin.org 3 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge FIGURE 2 | Active canonical WNT signaling in fibrosis. Simpliied scheme showing the activated canonical WNT cascade and translocation of β-catenin. (A) WNT ligands bind to a frizzled receptor and form a complex with the co-receptor Lipoprotein-related-receptor protein (LRP). (B) The ubiquitination activity of the destruction complex [comprised of disheveled (DVL) Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3), and casein kinase 1(CK1)] is inhibited causing β-catenin to accumulate in the cytoplasm. (C) As later event, clusters of receptor complexes are internalized into endosomes, which triggers the sequestering of the destruction complex, and subsequent inhibition of GSK3. (D) GSK3 is then transported to multivesicular bodies where it cannot interact with cytoplasmic β-catenin, and thus protects β-catenin from proteasomal degradation. (E) Newly synthesized β-catenin translocates to the nucleus to interact with LEF/TCF transcription factors and other co-factors, such as p300 and CBP. (F) Termination of the WNT/β-catenin signaling cycle may occur through phosphorylation of β-catenin by protein kinase C (PKC)δ and subsequent ubiquitination by tripartite motif (TRIM)33. These steps target β-catenin for proteasomal degradation in the nucleus. (G) Another possible route for the termination of β-catenin activation is the cytoplasmic sequestering by 14-3-3ζ and Chibby (Cby). have emphasized a key role for canonical WNT signaling in ibrogenesis of the heart, lungs, kidneys, and several ibrotic disorders of the skin (70–86). Aberrant activation of WNT signaling can be caused by increased expression of WNT agonists (87), or by silencing of endogenous WNT antagonists, such as proteins from the Dikkopf (DKK) and secreted frizzled-related protein (sFRP) families (77, 86, 88, 89). Experimental models that use exogenous overexpression of WNT ligands or sustained nuclear accumulation of β-catenin suggest that canonical WNT signaling is enough to trigger the expression of a ibrogenic program in ibroblasts (85, 86). However, depletion of β-catenin in the same model could not completely prevent the development of ibrosis, suggesting that β-catenin functions in concert with other pro-ibrotic signals (86). Similar indings come from pulmonary ibroblasts, in which the Fz/LRP complex, β-TrCP is excluded from the destruction complex, limiting β-catenin ubiquitination and degradation (64). An alternative route of WNT activation describes the formation of LRP5/6 aggregates that are internalized together with the destruction complex in the so-called multivesicular bodies. Once inside the multivesicular bodies, a large portion of β-catenin cannot interact with the destruction complex and thus escapes ubiquitination (65, 66). Stabilized β-catenin now accumulates in the nucleus where it associates with T-cell factor/lymphoid enhancer-binding factor-1 (TCF/Lef-1) transcription factors and several co-factors like p300 and CREB binding protein (CBP) to regulate transcription of target genes (67, 68). About 10 years ago, the irst evidence of WNT involvement in myoibroblast biology was found (69). Since then, many studies Frontiers in Medicine | www.frontiersin.org 4 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge β-catenin stabilization was not suicient for the upregulation of myoibroblasts-speciic genes (74). he discrepancy between the diferent studies may be due to diferences in the constructs used to stabilize β-catenin. Taken together, it has become evident that regulation of β-catenin cytoplasmic/nuclear shuttling is an intricate process, evidenced by the complex expression pattern of WNT ligands in the course of ibrogenesis (71, 77, 82). YAP/TAZ Signaling YAP and TAZ are regarded as the main output of the Hippo pathway (Figure 3). YAP and TAZ have been extensively studied in relation to the Hippo core kinase complex, and its role in organ size control, stem cell fate, and cancer (90–92). Both YAP and TAZ contain a WW domain that binds to Pro–Pro–X–Tyr motifs of associated proteins (93–95). he Hippo signaling cascade consists of the Ser/hr kinases MST1 and MST2, which are orthologs of the Drosophila Hippo kinase (96). MST1/2 binds to Salvador (SAV)/WW45 to form an active enzyme complex that phosphorylates the MOB1A/B subunits of LATS1/2 (97). he activated LATS1/2–MOB1A/B complex in turn phosphorylates YAP and TAZ. he primary phosphorylation of YAP and TAZ triggers subsequent phosphorylation by CK1 kinases. his generates a “phosphodegron” recognized by β-TrCP, leading to YAP and TAZ polyubiquitination and subsequent proteasomal degradation (98). he Serine residues relevant for the inactivation of YAP and TAZ are Ser127 (Ser89 in TAZ) and S381 (S311 in TAZ) (99, 100). When the Hippo pathway is inactive, YAP and TAZ are dephosphorylated and translocate to the nucleus, where they associate with transcription factors and other DNAbinding proteins to modulate target gene transcription. Despite a similar mechanism of activation, YAP and TAZ can bind diferent transcription factors, but also display overlap as seen with the association with TEA DNA-binding domain (TEAD) transcription factors (101–104). his suggests that their functions only partially overlap, but do share redundancy in some biological contexts. he capabilities of YAP and TAZ to regulate organ growth and size are striking, but at the same time incompletely understood. It is however clear that they perform these functions, at least in part, as mechanical rheostats independent of the core kinase complex. his becomes evident in cells cultured in vitro, which show strong nuclear localization of YAP/TAZ in semi-conluent cultures, but when reaching conluence YAP and TAZ translocate to the cytoplasm (97). A similar biomechanical program can be observed in cells grown on pathologically stif substrates or substrates that allow cell spreading, as they display predominantly nuclear accumulation of YAP and TAZ and increased transcription of their target genes. By contrast, cells grown on compliant substrates or space limiting substrates display cytoplasmic localization of YAP and TAZ (105–107). F-actin polymerization determines cell morphology and increases in cells cultured in sub-conluence or on stif substrates. Indeed, F-actin polymerization proves to be the link between cell spreading and YAP and TAZ nuclear translocation (106). he mechanical properties of YAP and TAZ were recently translated to myoibroblast activation and the induction of ibrosis. In biopsies from idiopathic pulmonary ibrosis, both Frontiers in Medicine | www.frontiersin.org FIGURE 3 | YAP and TAZ signaling. Simpliied scheme showing activation of YAP and TAZ. (A) When the Hippo kinase complex [comprised of Serine/ threonine-protein kinases (MST1/2), MOB kinase activator 1 (MOB1), Salvador (SAV), and serine/threonine-protein kinases (LATS1/2)] is active, YAP and TAZ become phosphorylated on multiple sites, creating a so-called phosphodegron. (B) Both YAP and TAZ are then sequestered in the cytoplasm by 14-3-3 proteins or targeted for degradation by β-TrCP. (C) Polymerization of the F-actin cytoskeleton inhibits the activity of MST1/2, rendering the core kinase complex inactive (several other upstream activators of the core kinase complex are not shown). (D) YAP and TAZ now translocate to the nucleus where they associate with transcription factors such as Runt-related transcription factor (RUNX) and TEA domain family member (TEAD) to modulate transcription. YAP and TAZ levels are elevated, and display a predominantly nuclear localization, which suggests increased transcriptional activity (108). Moreover, YAP and TAZ knockdown in mouse lung and liver ibroblasts cultured on stif substrates reduces the levels of proteins associated with myoibroblast diferentiation such as pro-collagen, αSMA, and plasminogen activator inhibitor (PAI)1 (108, 109). Adding to this, mice heterozygous for TAZ show a remarkable resilience against bleomycin-induced pulmonary ibrosis, possibly due to reduced levels of CCN2 (CTGF), one of the YAP and TAZ target genes (110). Also in cardiac ibrosis, YAP and TAZ have been a topic of investigation, but whether YAP and TAZ promote ibrogenesis remains elusive, and is probably dependent on the context of injury (111, 112). 5 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge Cytoplasmic Retention and Degradation of Transcriptional Modulators Taken together, these indings suggest that the nuclear translocation of YAP and TAZ is the sum of multiple tiers of regulation acting in concert. Because YAP and TAZ activity is not only controlled by Hippo signaling but also by mechanical signals and other signaling cascades, we refer to YAP and TAZ signaling in its broader context. TGF-β, WNT, and YAP/TAZ output activity all rely on a general mechanism: the nuclear translocation of its transcriptional modulators. To prevent continuous activation, the cell has several means to prevent spontaneous nuclear entry or binding to the DNA. For instance, Smad proteins continuously shuttle between the cytoplasm and the nucleus (33). Without tail-phosphorylation by the TGF-β type I receptor, Smads cannot interact with Smad4 and are unable to engage the DNA. Instead, they are phosphorylated in the linker region by CDK8/9 and GSK3 which renders them susceptible for polyubiquitination and degradation (31). By contrast, β-catenin and YAP and TAZ are sequestered in protein complexes with E-cadherin or 14-3-3 proteins, respectively, or directly targeted for proteasomal degradation by associating with β-TrCP (60, 94, 97). Recent studies highlighted that extensive cross-talk occurs on the level of cytoplasmic retention and degradation. One example of cross-talk between the YAP and TAZ, and TGF-β pathways is through the interaction with Smad7. As mentioned above, activated Smad proteins need to form a complex with Smad4 in order to become transcriptionally active modules. To regulate the Smad activation cycle, the TGF-β pathway uses Smad7 to form a negative feedback loop via various mechanisms. First, Smad7 can associate with the TGF-β type I receptor. Consequently, R-Smad phosphorylation and complex formation between R-Smads and Smad4 are inhibited (117). Smad7 also recruits E3 ubiquitin ligases, such as Smurf1 and Smurf2, to initiate receptor ubiquitination and degradation of the receptor complex. his self-regulating layer of the TGF-β signaling cascade can be linked to YAP and TAZ signaling, as YAP was found to associate with Smad7 at the type I receptor (118). By binding to YAP, Smad7 has a higher ainity for the type I receptor and increases its repressive efects on TGF-β signaling. Another line of evidence revealed that Smad7 interacts with β-catenin to promote Smurf2induced mediated ubiquitination and degradation, attenuating WNT activity in the skin (119). By contrast, in cancer epithelial cells it was found that Smad7 promotes the stability of β-catenin by enhancing its association with E-cadherin at the plasma membrane (120). Moreover, upon TGF-β stimulation, the WNT scafold protein Axin can form a complex with Smad7 and the E3 ubiquitin ligase Arkadia to promote Smad7 degradation (121). hese conlicting reports underline that Smad7 may act as repressor or enhancer of cellular signaling depending on the cell type and environmental context. As mentioned above, the type I receptor initiates phosphorylation of R-Smads, a process that is regulated through the interaction with several adaptor proteins. Interestingly, in unstimulated ibroblasts, Axin facilitates the binding of Smad3 with the type I receptor, independent from the adapter protein SARA (122). Upon TGF-β simulation, Axin promotes the tail-phosphorylation of Smad3 and subsequently dissociates from the type I receptor. Depletion of Axin results in decreased expression of TGF-β responsive genes such as PAI1, suggesting that Axin mediates cytoplasmic cross-talk between the TGF-β and WNT pathways which promotes the transcription of pro-ibrotic genes. Signaling Cascades Converge to Control Fibrotic Processes In ibrosis, it is becoming clear that the TGF-β, WNT, and YAP and TAZ signaling pathways work in concert, instead of being isolated entities. Several studies have hinted at the inter-pathway cross-talk in the diferentiation of myoibroblasts. For instance, in lung ibrosis, protein levels of both YAP and TAZ are increased and have increased nuclear localization (108). his corresponds to the increased levels of nuclear β-catenin and phosphorylated R-Smads found in ibrotic tissues. During skin wound healing in mice, both YAP and TAZ are increased upon injury and translocated to the nucleus. Moreover, TGF-β1 levels are also increased in the dermis, suggesting a link between activation of YAP and TAZ and the production of TGF-β1 (113). Adding to this, YAP- and TAZ-deicient ibroblasts are less reactive to TGFβ stimulation in vitro, produced less ECM, have lower expression of myoibroblast markers PAI1 and αSMA, and show lower contractile capabilities (108). he mechanisms through which these pathways communicate are diverse and range from modulating the availability of growth factors and the availability of membrane bound receptors to nuclear entry and activation of transcription factors (Figure 4). Pathways Govern Agonist and Antagonist Expression of Other Pathways he most straightforward form of cross-talk between signaling cascades occurs when activity of one pathway enhances the production of agonists or antagonists of the second. his type of cross-talk can create a feed-forward or feedback loop to enhance or attenuate, respectively, the transcriptional activity of another signaling cascade. For instance, stimulation of ibroblasts with WNT3a enhances the expression of TGF-β1 and subsequent phosphorylation of the MH2 domain of Smad2 (114). Consistently, absence of WNT signaling through LRP5 in bleomycin-induced lung ibrosis decreases the expression of TGF-β1 and attenuates the ibrotic response (115). Reconstitution of active TGF-β1 signaling in LRP5-deicient mice overrides the protective efects of abrogated WNT signaling. Moreover, it was found that TGF-β also enhances WNT signaling through the inhibition of DKK1 (116). Reduced expression of DKK1 enhances the stability and nuclear accumulation of β-catenin in both epithelial cells and ibroblasts, whereas reconstitution of DDK1 in vivo attenuates TGF-β-induced ibrosis. his allows cells to communicate over a certain distance and inluence the microenvironment of its neighboring cells. Despite the strong efects of altered growth factor signaling, this is oten not enough to modulate complex ibrogenic responses. To achieve this kind of complexity, direct interaction between signaling components is required. Frontiers in Medicine | www.frontiersin.org 6 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge Phosphorylated YAP also associates with β-catenin to inhibit its nuclear translocation and promote its degradation. (F) Upon WNT activation, the destruction complex is inhibited because YAP and TAZ dissociate from the complex. As later event, the destruction complex is sequestered by the LRP/ Frizzled/DVL receptor complex and targeted for degradation in the microvascular bodies. (G) F-actin polymerization inactivates the core kinase complex, causing YAP and TAZ to be dephosphorylated. Concurrently, β-catenin is not degraded by the inactive destruction complex, so that newly synthesized β-catenin accumulates in the cytoplasm. (H) The activated Smad complex associates with YAP or TAZ and translocates to the nucleus. Free and stabilized β-catenin also translocates to the nucleus. (I) In the nucleus, the transcription factors may co-localize at the chromatin depending on the context to govern transcription of myoibroblast related genes. (J) At the end of the transcription cycle, transcription factors are degraded in the nucleus, or translocate back to the cytoplasm for either degradation or a new round of activation. FIGURE 4 | The TGF-β, WNT, and YAP/TAZ signaling pathways converge. Schematic overview of the molecular cross-talk between components of the TGFβ, WNT, and YAP/TAZ pathways. (A) Upon TGF-β stimulation, Axin promotes the tail-phosphorylation of Smad3. (B) Axin also promotes the degradation of inhibitory Smad7, thereby further enhancing the TGF-β signal. Smad7 can associate with both YAP and β-catenin. Binding of YAP to Smad7 increases the afinity for the type I receptor and increases the repressive effects on TGF-β signaling. Smad7 binding with β-catenin can mediate both degradation and stabilization of β-catenin. (C) TAZ inhibits the phosphorylation of disheveled (DVL) by casein kinase (CK)1, providing either positive or negative feedback depending on the WNT ligand present. (D) The active Hippo core kinase complex phosphorylates both YAP and TAZ creating a phosphodegron. Phosphorylated YAP and TAZ are either sequestered by 14-3-3 proteins or associate with the β-catenin destruction complex. In the destruction complex, YAP and TAZ are necessary for docking of β-TrCP to the complex. (E) Recently it was found that TAZ also communicates with WNT signaling through the interaction of TAZ and β-catenin in the cytoplasm (123). In a WNT-of state, both β-catenin and TAZ associate with β-TrCP and are ubiquitinated and degraded in the Frontiers in Medicine | www.frontiersin.org proteasome. his process requires active GSK3 phosphorylation of the β-catenin phosphodegron. Upon WNT stimulation, GSK3 dissociates from the destruction complex, β-catenin is dephosphorylated and unable to bind to TAZ. hus, WNT stimulation 7 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge induces the stability and nuclear localization of its own transcriptional modulator, β-catenin, as well as TAZ. Furthermore, inactive YAP and TAZ form a complex together with β-catenin, GSK3, and Axin1 (124). Cytoplasmic YAP/TAZ speciically bind Axin in absence of WNT signals. In this case, Axin facilitates the function of cytoplasmic anchor, as Axin depletion results in a rapid nuclear accumulation of YAP/TAZ. Indeed, stimulation by WNT3a causes dissociation of YAP/TAZ from the destruction complex ater which they translocate to the nucleus, and modulate transcription of TEAD target genes. Vice versa, as part of the destruction complex, YAP/TAZ are needed for the docking of β-TrCP to the destruction complex. By releasing YAP/TAZ upon WNT stimulation, β-TrCP cannot bind to the destruction complex and ubiquitinate β-catenin. Furthermore, it was found that protein kinase C zeta (PKCζ) associates with the destruction complex and can phosphorylate YAP and β-catenin on several residues, adding to quick proteasomal degradation (125). Next to its function in the destruction complex, it was found that TAZ binds to the PY and PDZ domains of DVL2 upon stimulation with WNT3a (126). TAZ binding inhibits the phosphorylation of DVL2 by CK1, which results in reduced β-cateninmediated activity of LEF/TCF transcription. Interestingly, WNT3a and WNT5a – which in part have opposite functions in β-catenin stabilization – both induce the phosphorylation of CK1, suggesting that TAZ binding to DVL2 may have diferent outcomes depending on the WNT isoform and receptor pair present (127). Furthermore, it was found that YAP too fulills multiple roles in YAP/WNT cross-talk. By directly binding to β-catenin, phosphorylated YAP prevents nuclear translocation of β-catenin and subsequent transcription of LEF/TCF target genes (128). his process is dependent on the activity of the Hippo core kinase complex, as increased Hippo activity induces phosphorylation of YAP and concomitantly reduces levels of β-catenin in the nucleus. Evidence thus shows that the transcriptional modulators of the TGF-β, WNT, and YAP/TAZ pathways are integral factors in the cross regulation between these pathways. Cytoplasmic retention of transcription factors and transcriptional activators proves to be an ingenious system through which the three diferent pathways tightly regulate their own and each other’s activity. WNT signaling, β-catenin levels are maintained low due to degradation in absence of WNT signals, although β-catenin can be observed in both the cytoplasm and the nucleus. Recent developments reveal that the fold-change in β-catenin levels ater WNT stimulation is more important for transcriptional modulation than the absolute levels of β-catenin (130). his inding suggests that even in cells with low basal β-catenin levels, slight changes in nuclear β-catenin are suicient to initiate transcriptional changes. Nuclear accumulation of YAP and TAZ is governed by the activity of the Hippo signaling cascade as well as biomechanical signals that are relayed from outside the cell. It is becoming clear that ine tuning of nucleocytoplasmic shuttling is not just mediated by a single signaling pathway, but rather by the cross-talk of several components of the TGF-β, WNT, and YAP/TAZ cascades. In ibrosis, epithelial to mesenchymal transition (EMT) describes the process of epithelial cells that undergo transdiferentiating toward a myoibroblast-like phenotype, a phenomenon observed in both cancer metastasis and ibrosis (9, 131, 132). Upon injury, epithelial cells lose their characteristic cellular junctions and acquire a spindle-like morphology. Cells undergoing EMT oten show increased motility, de novo expression of αSMA, and elevated expression of ECM components, such as collagens and ibronectin. Also during EMT, several studies have provided evidence that TGF-β, WNT, and YAP/TAZ interact with each other to drive the transformation toward a mesenchymal-like cell type. One of the irst studies, describing the integration of YAP/TAZ and TGF-β signaling in nucleocytoplasmic shuttling, found that TAZ interacts with Smad2/4 and Smad3/4 complexes in epithelial cells (133). he coiled-coil domain in the C-terminal region of TAZ binds to the MH1 domain of Smad2/3 and thereby promotes the nuclear accumulation of Smad2/3 and increases their transcriptional activity on target genes such as PAI1 and SMAD7. Interestingly, low levels of TAZ promote nuclear accumulation, but when the concentration of TAZ increased, it is predominantly located in the cytoplasm and nuclear localization of Smad2/3 is blocked. his suggests that Smad accumulation is strongly dependent on the expression levels and activation status of TAZ. Similar indings were obtained for YAP, which forms a complex with Smad3, TEAD, and p300 on the CCN2 promoter in mesothelioma cells (134). Knock down of YAP results in attenuated expression of endothelin1 (ET1) and CCN2, whereas no immediate diferences are seen in the expression of ibronectin and collagens, suggesting that YAP controls the expression of a subset of TGF-β responsive genes. Moreover, levels of metalloproteinase 2 (MMP2) are increased upon YAP knock down, strengthening the hypothesis that YAP, together with Smads, governs a pro-ibrotic phenotype. hese indings were corroborated in mammary epithelial cells, as YAP/TAZ associates with Smad2/3 and TEADs (135). A question that then arises is whether transcriptional modulators only need each other for nuclear entry, or also associate with each other at speciic promoter or enhancer regions to modulate transcription. Interestingly, during EMT in alveolar epithelial cells, simultaneous stimulation by TGF-β and WNT ligands has synergistic efects on the expression of αSMA as well as the activity of LEF/TCF responsive elements (136). TGF-β alone induces nuclear translocation of β-catenin by inactivating Nuclear Shuttling and Transcriptional Modulation he original view on growth factor signaling described the nuclear accumulation of transcriptional modulators solely as a consequence of ligand-mediated activation. In the absence of a growth factor ligand, Smads and β-catenin were thought to reside exclusively in the cytoplasm and translocate only to the nucleus upon receptor activation. We now know that transcription factor shuttling is not as black and white as once proposed. Without stimulation, R-Smad proteins continuously shuttle between the nucleus and cytoplasm, but display a signiicant higher concentration in the cytoplasm (129). It is thought that R-Smads reside in the cytoplasm, until TGF-β stimulation releases them for nuclear translocation, enhances their ainity for nuclear importin proteins, and induces nuclear anchoring. In the case of canonical Frontiers in Medicine | www.frontiersin.org 8 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge on a series of phosphorylation and dephosphorylation events by multiple kinases and phosphatases [reviewed in Ref. (141)]. he variety of kinases and phosphatases introduces another level of complexity in the regulation of Smad action, and is greatly dependent on signaling through other pathways at a speciic place and time. One of the examples, through which other pathways interact with Smad recycling describes the temporal regulation by CDK8/9 and GSK3. First CDK8/9 phosphorylate Smad1 on Ser206 and Ser214, which allows binding with YAP and simultaneously triggers phosphorylation by GSK3 on hr202 and Ser210. he latter phosphorylation events cause YAP to dissociate and Smurf2 to bind with Smad1, and initiate ubiquitination (32). Although Smad1 is not activated by TGF-β but rather by BMPs, one can envision identical mechanisms in the recycling of canonical R-Smads by Nedd4L (31, 142). Whether the association of TAZ with Smad2/3 has similar efects on their recycling remains to be determined. As mentioned above, YAP is able to enhance the repressive functions of Smad7 at the type I receptor. Interestingly, Smad7 has also been found to inhibit TGF-β signaling in the nucleus, where it can use its MH2 domain to bind to DNA sequences containing SBEs (143). DNA bound Smad7 competes with Smad2/Smad4 complexes, thus, directly impairing the transcription of TGF-β responsive genes such as PAI1. Whether the interactions between YAP and Smad7 are of importance in this process remain to be elucidated. GSK3-mediated degradation, which is further enhanced by WNT stimulation. Delicate ChIP-re-ChIP experiments revealed that β-catenin and Smad3 co-localize at the SBE1 containing region of the αSMA promoter, in a CBP-dependent fashion. hese indings are supported by co-localization of β-catenin, Smad3, and CBP in nuclei of epithelial cells in idiopathic pulmonary ibrosis biopsies. Other reports contradict these indings and propose that β-catenin induces the expression of αSMA through interaction with myocardin-related transcription factor (MRTF), a process inhibited by Smad3 (52, 137). One explanation for these conlicting results may be the diferences in experimental setup and the diferent species studied. Smads and β-catenin were also found to interact on other genes involved in ibrogenesis. As proof of principle, TGF-β and WNT3a synergistically enhanced the promoter activity of sequences containing both SBE and LEF/TCF responsive elements (138). Co-stimulation resulted in a unique expression proile distinct from that seen ater stimulation with single growth factors. Interestingly, recent developments describe how YAP can compete with Smad2/3 for promoter occupancy in the transcription of genes involved in mesendoderm diferentiation. Gene transcription strongly depends on the phosphorylation status of RNA polymerase II (RNAPII) [reviewed in Ref. (139)]. Briely, phosphorylation of RNAPII on Ser5 is important for the initiation of transcription, whereas subsequent phosphorylation on Ser2 and Ser7 are crucial for the elongation steps of transcription. β-catenin and LEF-1 associate with enhancer regions of mesendodermal genes such as MIXL1 and EOMES and recruit Ser5 phosphorylated RNAPII to initiate transcription (140). Upon activin stimulation, Smad2/3 localize to the promoter region of these genes to enhance the phosphorylation of Ser2 and Ser7 on RNAPII and thereby promote elongation of transcription. YAP was found to actively inhibit this process by recruitment of the negative elongation factor NELF. Knockdown of YAP reduces the occupancy of NELF and enhances the phosphorylation on Ser2 and Ser7 at target genes, which promotes transcription. Although these results do not directly link to myoibroblast function, they have signiicant implications on the mechanism by which YAP regulates gene transcription. Future research will reveal if similar mechanisms apply to the regulation of myoibroblast-related genes. Taken together, TGF-β, WNT, and YAP/TAZ signals converge by modulating the nuclear accumulation and transcriptional activity of their transcription factors. Furthermore, the outcome of this type of cross-talk is not only dependent on the concentration of transcription factors but also on the availability of co-activators and co-repressors, chromatin conformation, and the phosphorylation status of RNAPII, which may vary from one cell type to another (31, 140). Therapeutic Targeting at the Cross-Roads Remarkable progress in both biology and pharmacology has led to advances in the development of anti-ibrotic therapies. Many of these therapies aim to target the usual suspects such as ligands and receptors of the TGF-β and WNT signaling cascades using antagonistic antibodies or small-molecule inhibitors. Although the therapeutic eicacy in animal models proves promising (78), trials oten fail to achieve signiicance in clinical endpoints or sufer from severe adverse efects (73) with the exception of one recent study in systemic sclerosis (144). he discrepancy in eicacy between rodents and humans suggests that animal models poorly mimic the pathophysiology of human ibrotic disorders. As we gain insight in the molecular mechanisms that link the TGF-β, WNT, and YAP/TAZ cascades, we come to understand the challenges and pitfalls of targeting one speciic signaling pathway [thoroughly reviewed in Ref. (19, 145–147)]. We have seen that signaling cascades are complex and that many pathway components fulill multiple functions. Because TGF-β, WNT, and YAP/TAZ signals have distinct functions in diferent cell types and tissues, speciic targeting of the ibrotic lesion is crucial. he temporal properties of signal transduction in diferent phases of disease and homeostasis pose another diiculty in the administration of pathway-wide modulating agents. It is therefore not just a matter of up- or downregulation. For example, targeting of TGF-β or WNT signaling with neutralizing antibodies may have widespread efects on the functioning of several components of the TGF-β, WNT, and YAP and TAZ signaling pathways, as well as numerous other growth factor cascades. To circumvent the wide-spread efects of growth factor inhibition and limit Transcription Factor Recycling he inal stage of the signal transduction cascades involves the process of transcription factor recycling. In the case of Smad proteins, tail-phosphorylation of the MH2 domain induces nuclear accumulation. Whether nuclear R-Smads engage in transcription or are targeted for nuclear exit and proteasomal degradation relies Frontiers in Medicine | www.frontiersin.org 9 September 2015 | Volume 2 | Article 59 Piersma et al. Signaling in ibrosis: pathways converge adverse efects, we increasingly depend on the development of small-molecule intracellular inhibitors. he small-molecule intracellular inhibitor LY2157299 speciically targets the kinase pocket of the type I receptor of the TGF-β cascade without inhibiting the type II receptor and thereby attenuates Smad2/3-dependent transcription of target genes (145). However, LY2157299 has not been studied in clinical trials to halt or reverse ibrosis. Examples of small-molecule intracellular inhibitors for the WNT pathway, such as PKF115–584 and CGP049090 (148), act on the association between β-catenin and LEF/TCF transcriptions factors. Other strategies focus on inhibition of the PDZ domain of DVL or the transcriptional coactivators CBP and p300 (145). By inducing a shit from β-catenin association with p300 to CBP, genes such as COL1A1 may be negatively regulated (149). However, other studies report that inhibition of β-catenin–CBP also ameliorates ibrosis, suggesting that there is no such thing as pure “good” and “bad” β-catenin signaling (73, 78). As YAP and TAZ have but recently been linked to ibrogenesis, no clinical trials have been performed. hus, whether targeting of YAP and TAZ is a fruitful strategy against ibrosis progression remains to be elucidated. One of the challenges in the targeting of YAP and TAZ is that they do not possess catalytic domains, but rather depend on speciic protein binding domains for the interaction with their binding partners such as LATS1/2, Src family kinases, and TEADs. Nonetheless, a recent study described a potent inhibitor of YAP–TEAD: the benzoporphyrin derivative verteporin (150). Verteporin is currently used in the clinic for the treatment of macular degeneration, which makes it appealing for the use in clinical trials for ibrosis. Although inhibition of the YAP–TEAD complex seems a promising anti-ibrotic strategy, as several pro-ibrotic genes are not under control of TEADs, this may prove not to be the best approach. he disadvantages of pathway-wide molecular inhibitors challenge the scientiic community to develop speciic targeting strategies against intracellular processes and protein–protein interactions. he increasing insight in the molecular cross-talk between signaling cascades adds new possibilities in drug development. Additionally, by focusing on the elucidation of the crystal structures of protein complexes, we can pursue the rational design of novel small molecular inhibitors to interfere at the cross-roads of signal transduction cascades. Conclusion Recent advancements in the ield of TGF-β, WNT, and YAP/TAZ signaling have revealed that these signaling entities do not act alone. he notion that pathway components can have multiple and even opposed functions within one cell partly explains how the inhibition of a single molecular target oten does not result in the desired therapeutic efect. his does not only add to the mere understanding of ibrotic processes, but also promotes the necessity to develop highly speciic small-molecule intracellular inhibitors that act on protein–protein interactions at the crossroads of signaling cascades. It should be noted that several of the studies described in this review used artiicial ectopic expression of the proteins investigated. his may introduce artifacts that can inluence the activity and functionality of the signaling cascades involved. hus, more detailed studies in representative models for ibrosis focusing on endogenous proteins are required to completely understand the molecular cross-talk in vivo. Broadening our view on signal transduction will provide a better understanding of how a limited set of growth factors is able to govern the complex processes that underlie the physiology and pathology of ibrotic disorders. Author Contributions BP, RB, and MB designed the manuscript. BP and MB collected literature and BP drated the manuscript. All authors critically discussed and revised the content of the manuscript and had inal approval of the manuscript in its present form. 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Copyright © 2015 Piersma, Bank and Boersema. his is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). he use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 14 September 2015 | Volume 2 | Article 59