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LIM-domain proteins in transforming growth factor β-induced epithelial-to-mesenchymal transition and myofibroblast differentiation
Cellular Signalling 24 (2012) 819–825
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Review
LIM-domain proteins in transforming growth factor β-induced epithelial-to-mesenchymal transition and myofibroblast differentiation Päivi M. Järvinen a, Marikki Laiho a, b,⁎ a b
Molecular Cancer Biology Program and Haartman Institute, University of Helsinki, Helsinki, Finland Department of Radiation Oncology and Molecular Radiation Sciences and Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
a r t i c l e
i n f o
Article history: Received 14 October 2011 Received in revised form 15 November 2011 Accepted 4 December 2011 Available online 11 December 2011 Keywords: Transforming growth factor β Myofibroblast Epithelial to mesenchymal transition LIM-domain protein Cancer associated fibroblast Fibrosis
a b s t r a c t Epithelial to mesenchymal transition (EMT) is a process during which junctions of the cell–cell contacts are dissolved, actin cytoskeleton is deformed, apical–basolateral cell polarity is lost and cell motility is increased. EMT is needed during normal embryonal development and wound healing, but may also lead to pathogenic transformation and formation of myofibroblasts. Transforming growth factor β (TGFβ) is a multifunctional cytokine promoting EMT and myofibroblast differentiation, and its dysregulation is involved in pathological disorders like cancer and fibrosis. Lin11, Isl-1 and Mec-3 (LIM) domain proteins are associated with actin cytoskeleton and linked to regulation of cell growth, damage signaling, cell fate determination and signal transduction. LIM-domain proteins generally do not bind DNA, but are more likely to function via protein–protein interactions. Despite being a disparate group of proteins, similarities in their functions are observed. In this review we will discuss the role of LIM-domain proteins in TGFβ-signaling pathway and in EMT-driven processes. LIM-domain proteins regulate TGFβ-induced actin cytoskeleton reorganization, motility and adhesion, but also dissolution of cell–cell junctions during EMT. Finally, the role of LIM-domain proteins in myofibroblasts found in fibrotic foci and tumor stroma will be discussed. © 2012 Elsevier Inc. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. TGFβ induced EMT . . . . . . . . . . . . . . . . . . . 1.2. TGFβ induced myofibroblast differentiation . . . . . . . 1.3. TGFβ at the juncture of wound healing and fibrotic diseases 1.4. TGFβ in formation of idiopathic pulmonary fibrosis (IPF) . 1.5. TGFβ and cancer associated fibroblast (CAFs) . . . . . . 2. LIM-domain proteins . . . . . . . . . . . . . . . . . . . . . 2.1. TGFβ regulates expression of several LIM-domain proteins 2.2. LIM-domain proteins regulate TGFβ signaling . . . . . . 2.3. LIM-domain proteins in EMT and migration . . . . . . . 2.4. Expression of LIM-domain proteins in CAFs . . . . . . . 2.5. LIM-domain proteins in fibrotic diseases . . . . . . . . . 3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: CAF, cancer associated fibroblast; CRP, cysteine rich protein; ECM, extracellular matrix; EMT, epithelial to mesenchymal transition; ERK, extracellular signalregulated kinases; FHL, four and half LIM protein; HIC-5, hydrogen peroxide-inducible gene 5; IPF, idiopathic pulmonary fibrosis; JNK, jun N-terminal protein kinase; LIM, Lin11, Isl-1 & Mec-3; LMO, LIM only protein; MAPK, mitogen activated protein kinase; NOX-4, NAPDH/oxidase 4; PINCH, particularly interesting new cysteine-histidine rich protein; αSMA, α smooth muscle actin; TGFβ, transforming growth factor β. ⁎ Corresponding author at: Molecular Cancer Biology Program, University of Helsinki, PO Box 63, FIN-00014 Helsinki, Finland. Tel.: + 358 9 19125542; fax: + 358 9 19125554. E-mail address: [email protected] (M. Laiho). 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.12.004
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1. Introduction
1.1. TGFβ induced EMT
TGFβ signaling pathway regulates growth, apoptosis, differentiation, adhesion, invasion and extracellular matrix production [1,2]. TGFβ is a pleiotropic cytokine ubiquitously expressed by most cells and tissues in the body, essential for embryogenesis and maintenance of tissue homeostasis. The effect of TGFβ is context dependent; broadly, in epithelial cells it acts as a growth inhibitor, whereas in fibroblasts it stimulates proliferation. Dysregulation of TGFβ signaling pathway is involved in pathological disorders like cancer and fibrosis [3]. TGFβ ligands recognize and bind TGFβ type II receptor. Binding of the ligand causes the activation of intracellular serine/threonine kinase domain of the type II receptor, which phosphorylates the cytoplasmic tail of type I receptor and activates its respective serine/ threonine kinase domain. The activated heterotetrameric receptor complex regulates downstream Smad and non-Smad pathways [4]. Receptor activation will ultimately lead to the phosphorylation of receptor-regulated Smads 2 and 3, and heteromeric complex formation with Smad4, nuclear translocation, and activation of transcription [4]. Besides the canonical Smad-pathway, the functional receptor complex also activates so-called non-Smad mediated pathways (Fig. 1). These include MAP kinases (ERK, JNK, p38), Rho-like GTPase signaling and phosphatidylinositol-3-kinase/AKT pathways, which are especially relevant in EMT [5].
Epithelial cells are immobile and polar cells, and are characterized by strong adhesions between neighboring cells. These cell-cell contacts include tight and adherens junctions and desmosomes, which are connected to the circumferential actin belt. E-cadherin is a key component of the adherens junctions and is vital for initiating and maintaining epithelial architecture in vitro and in vivo [6]. During EMT, epithelial junctions are disintegrated and cells become more mobile and apolar fibroblast-cell like. EMT is a normal process during embryonal development and wound healing, but occurs also during pathological conditions like carcinogenesis and in fibrotic diseases. Molecular characteristics of EMT are loss of epithelial markers such as E-cadherin, zona occludens-1, and an increase in mesenchymal markers like N-cadherin, α smooth muscle actin (αSMA), matrix metalloproteinases and extracellular matrix (ECM) components including collagen and fibronectin [2]. Especially as about 90% of the cancers are of epithelial origin, EMT is of high relevance during cancer progression [7]. TGFβ is a key regulator of EMT and drives it both by Smaddependent and independent manner, and also by regulating the expression of well known EMT-inducer transcription factor families Snail, ZEB and bHLH [8]. 1.2. TGFβ induced myofibroblast differentiation Myofibroblasts are cells with features of both fibroblasts and smooth muscle cells. They may differentiate from several origins; mesenchymal cells such as fibroblasts, hepatic stellate or smooth muscle cells, epithelial or endothelial cells through EMT, bone marrow cells and from fibrocytes [9]. Myofibroblasts are identified by the expression of αSMA, contractility and production of specific ECM proteins, and are needed for normal wound repair. TGFβ drives the transdifferentiation of fibroblasts to myofibroblasts in vitro and in vivo [10,11]. TGFβ induces the myofibroblast differentiation of hepatic stellate cells [12], and causes myofibroblast differentiation through EMT [13]. Furthermore, myofibroblast marker αSMA is a direct target of TGFβ [14]. 1.3. TGFβ at the juncture of wound healing and fibrotic diseases
Fig. 1. Several LIM-domain proteins are activated by TGFβ either via Smad or nonSmad pathways. TGFβ is required to maintain normal tissue homeostasis, but may also promote tumorigenesis or fibrotic processes.
During wound healing, damaged epithelial or endothelial cells produce inflammatory mediators that activate inflammatory cells and fibroblasts, which start the excess deposition of ECM. Myofibroblasts, epithelial and endothelial cells produce matrix metalloproteinases that degrade the basement membrane and thus enhance the migration of inflammatory cells to the site of the injury. Macrophages and neutrophils engulf the tissue debris, dead cells and pathogens and secrete cytokines and chemokines, which invite endothelial cells to surround the injured site and to form new blood vessels. Furthermore, lymphocytes and other cells become activated and start to secrete profibrotic cytokines and growth factors such as TGFβ, IL-13 and platelet derived growth factor, which further activate macrophages and fibroblasts. Activated myofibroblasts help the wound closure through contraction [15]. The wound healing process is completed by multiplication of epithelial and endothelial cells, which migrate over the basal layer to replace the injured tissue. While myofibroblasts disappear after successful wound healing, in fibrotic lesions these cells persist, and are responsible for scarring of tissue and excessive production of ECM altering the tissue architecture, and in severe cases, lead to the organ failure [9]. Fibrosis is a pathological condition that occurs in various organs, such as lung, liver, kidney and the cardiovascular system, and is characterized by overgrowth, stiffening, scarring and excess production of ECM. Fibrosis is also caused by chronic inflammatory reactions induced by various stimuli including persistent infection, autoimmune
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reaction, allergic response, chemical insult, radiation, and tissue injury [15]. TGFβ has been implicated as a major inducer of fibrosis in many tissues, including the lung [11]. 1.4. TGFβ in formation of idiopathic pulmonary fibrosis (IPF) IPF is an interstitial lung disease and an example of TGFβ-induced fibrotic diseases. Chronic inflammation and accumulation of myofibroblasts in fibroblastic foci is observed in the lung leading to decreased alveolar gas exchange and pulmonary volume restriction [16]. IPF has a poor prognosis and the 5-year survival is only 20% [17]. Although the etiology of IPF is largely unknown, persistent lung injury, inflammation and inefficient wound repair contribute to the disease. Furthermore, acquired or hereditary genetic alterations, including telomerase mutations, predispose to IPF [18]. A key process of repairing lung injury is the activation of TGFβ, and its uncontrolled activity contributes to IPF. An emerging concept is that IPF is a disease of deregulated EMT crosstalk [19]. IPF can be triggered by alveolar injury that leads to the activation of TGFβ and alveolar basement membrane disruption. Subsequently, TGFβ may promote enhanced epithelial apoptosis and EMT, as well as fibroblast and fibrocyte differentiation into myofibroblasts. Deposition of excess extracellular matrix by the myofibroblasts disposes to the development of IPF [19]. Elevated levels of TGFβ have been detected in IPF lung specimens as compared to the controls [20,21], and the presence of TGFβ1 in the lung epithelium indicates chronic injury [22]. Polymorphism in the TGFB1 gene has been found in codons 10 and 25, and while they do not predispose to the development of IPF, they may affect disease progression [23]. Moreover, specific inhibitors of the type I receptor reduce myofibroblast transformation and collagen gel contraction in a rat bleomycin-induced lung fibrosis model [24]. Several genes are reported to mediate TGFβ-induced effects in IPF. NAPDH/oxidase 4 (NOX-4) belongs to a group of enzymes that catalyze the formation of reactive oxygen species and is required for TGFβinduced myofibroblast differentiation, production and contractility [25]. Furthermore, silencing or pharmacological targeting of NOX-4 abrogated the formation of fibrosis in two murine models with lung injury [25]. Peroxisome proliferator-activated receptor-γ has been shown to repress TGFβ-induced myofibroblast differentiation via Smadindependent manner by affecting two TGFβ-dependent pro-survival pathways involved in myofibroblast differentiation [26].
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proteins have been suggested in development, gene expression, cytoarchitecture, cell adhesion and motility, signal transduction, cell fate determination and tumor formation [32,33]. These activities are mediated by protein–protein interactions, and depending on their interacting partners, a LIM-domain protein can have several different functions [33]. These include their actions as adaptors, competitors, autoinhibitors or localizers of other proteins. LIM-domain proteins have been categorized into four main families based on their functional domains and localization [33]. Nterminal tandem LIM-domain proteins such as LHX and nuclear LIM only protein (LMO) proteins are found in the nucleus and act as a transcription factors or cofactors. LIM-only proteins are detected both in the nucleus and cytoplasm, and include cysteine rich protein (CRP), four and half LIM (FHL) and particularly interesting new cysteine–histidine rich protein (PINCH) protein families. LIMdomain proteins that have additional protein–protein interaction motifs (PDZ, leucine-aspartate repeats, or actin-target domains) are represented by zyxin, EPLIN, and ABLIM protein families. Lastly, LIM-domain proteins LIMK and MICAL contain additional functional domains such as mono-oxygenase or kinase catalytic motifs [33]. Most LIM-domain proteins are associated with actin cytoskeleton in the cytoplasm. Several shuttle between cytoplasm and nucleus. For example, translocation of zyxin is triggered by extracellular stimulation by UV [34]. Furthermore, zyxin responds to cell stretching, and translocates from focal adhesions into nucleus to regulate gene expression [35]. Thus, LIM-domain proteins are involved in mediating signals between the nucleus and cytoplasm [32]. CRP family consists of three members: CRP1, CRP2 and CRP3/MLP [36]. CRPs are small proteins, 22 kDa of size, and comprise of two functional LIM-domains. Three-dimensional structure analyses of CRPs predict that these proteins may act as molecular adapters [37]. CRPs exhibit a differential expression pattern in chicken; CRP1 expression is detected in most tissues, and is especially enriched in smooth muscle cells, whereas CRP2 is expressed in smooth muscle cells especially in vasculature and in fibroblasts, and CRP3/mlp in striated muscle [38]. CRP1 exhibits dual localization pattern in cell; it is found both in the nucleus and in cytoplasm. In nucleus, CRP1 functions as a transcriptional co-regulator [39]. It also directly interacts with actin. CRP1 may play a role in actin cytoskeleton remodeling by bundling the stress fibers by crossing actin filaments and by stabilizing the interactions of α-actinin with actin filament bundles [40,41].
1.5. TGFβ and cancer associated fibroblast (CAFs) 2.1. TGFβ regulates expression of several LIM-domain proteins Tumor microenvironment has become a subject of intensive research. During tumor development, tissue homeostasis between different cell types is disturbed. The invasive nature of tumor cells is an end result of interaction between tumor stroma and the epithelial cells. The tumor stroma includes cancer associated fibroblasts (CAFs), immune cells, vasculature and extracellular matrix [27]. CAFs are often present in carcinomas, and they play a role in tumorigenesis by secreting paracrine growth factors and in this way promote tumor growth, angiogenesis and invasion [28]. TGFβ significantly increases the percentage of myofibroblasts in CAF population [29]. Several studies show that CAFs produce TGFβ at higher level than normal fibroblasts. TGFβ and SDF1 are released in cell autonomous fashion during tumorigenesis by stromal fibroblasts leading to CAF differentiation in the breast [30]. Similarly, CAFs isolated from prostate show increased TGFβ immunoreactivity as compared to normal human prostate fibroblasts [31]. 2. LIM-domain proteins LIM-domains consist of two zinc fingers and have been found in a wide variety of eukaryotes. So far, 135 LIM-domains have been identified within 58 human genes [32]. Diverse roles for LIM-domain
LIM-domain proteins act both as downstream targets and modulators of TGFβ signaling. TGFβ positively regulates the expression of several LIM-domain proteins: CRP1 [42], CRP2 [43,44], LMO1 [45], zyxin [46] and hydrogen peroxide-inducible 5 (HIC-5) [47] (Fig. 1). In addition, the expression of paxillin is modulated by TGFβpathway, increasing paxillin phosphorylation on Tyr31 and Tyr118. Conversely, paxillin delta lacking these phosphorylation sites is downregulated during TGFβ-induced EMT [48]. Regulation of the LIM-domain proteins by TGFβ is mediated through the Smad-pathway leading to transcriptional activation of the respective targets, with one exception. Interestingly, the increase in CRP1 by TGFβ is independent of TGFβ-induced transcriptional regulation [42]. This is in contrast to the close relative, CRP2 [44], even though the kinetics of elevated protein expression of CRP1 and CRP2 post TGFβ-treatment are similar. Furthermore, the regulation of CRP1 by TGFβ appeared biphasic. TGFβ-treatment led to a rapid increase of CRP1 within one hour followed by a decrease. A sustained increase of CRP1 by TGFβ then ensued a few hours later and was stable for few days [42]. Although the sustained increase of CRP1 appeared dependent on type I receptor and activated Smadpathway, the rapid induction was independent of both [42]. However,
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both the rapid and sustained increases in CRP1 were dependent on p38 signaling pathway. Interestingly, p38/MAPKs have been reported to mediate signaling from TGFβ receptor III independently of Smads [49]. In addition, p38 MAPK has been shown to mediate TGFβ regulated apoptosis and EMT independently of Smads [50], and p38 MAPK pathway is activated by TGFβ type III receptor (betaglycan) [51,52]. Given that the type I receptor inhibitor used here was without effect on the rapid response, it is possible that the response is mediated via type III receptor and p38 MAPK signaling [42]. 2.2. LIM-domain proteins regulate TGFβ signaling Certain LIM-domain proteins modulate TGFβ signaling pathway. HIC-5 is a coactivator of androgen receptor and has four LIMdomains. HIC-5 modulates TGF-β-signaling by interaction with Smad3, Smad4 [53] and Smad7 [54], but does not interact with Smad2 [53]. HIC-5 suppresses Smad7 by physically interacting with Smad7 through its third LIM-domain causing loss of Smad7 [54]. Loss of Smad7, the negative regulator of Smads 2 and 3, leads to enhanced TGFβ signaling [54]. The third LIM-domain is also needed for interaction between HIC-5 and Smad3 [53]. The suppressive activity of HIC-5 on Smad3 and Smad7 enhances Smad2 activity thus suggesting that HIC-5 could be a factor involved in switch in TGFβ from being a tumor suppressor to being an oncogene during tumor progression [54]. Furthermore, FHL proteins (FHL1, FHL2, FHL3) modulate TGFβsignaling pathway via interaction with Smads (Smad2, Smad3, Smad4). FHL proteins are positive regulators of TGFβ-responsive transcription. They enhance phosphorylation of Smad2/3, increase interaction between Smad2/3 and Smad4 and their nuclear accumulation via casein kinase 1δ independently of TGFβ-receptor signaling. Interestingly, FHL proteins are often downregulated in hepatocellular carcinoma, and the levels correlate with decreased TGFβ-like responses in the clinical samples [55]. These findings suggest that LIM-domain proteins act both by activating and repressing TGFβ signaling, and contribute to both TGFβ physiological and pathophysiological regulation. 2.3. LIM-domain proteins in EMT and migration Given that LIM-domain proteins classically have been linked to actin cytoskeleton, their levels may be elevated simply due to actin cytoskeleton reorganization during TGFβ-induced EMT. Although we did not find evidence that CRP1 is involved in regulation of EMT [42], several LIM-domain proteins, like zyxin [56], HIC-5 [57], LMO2 [58] and LIMK [59], are required for TGFβ-driven stress fiber reorganization, cell motility and adhesion (Fig. 2). Zyxin is a target of TGFβ signaling cascade and positive regulator of EMT. It is required for stress fiber reorganization and cell migration during EMT in mouse mammary gland epithelial cells and in endocardial cells [56]. Furthermore, depletion of Hic-5 in TGFβ treated cells leads to decreased cell migration, actin stress fiber formation, retains the epithelial organization and thus inhibits EMT [57]. Conversely, expression of LMO2 protein in prostate cancer cells downregulates Ecadherin and promotes EMT by enhancing cell motility and invasiveness [58]. Using similar silencing approach towards LIMK, it was shown that while LIMK is not required for TGFβ-induced 2D motility, but it is needed for TGFβ-induced actin stress fiber reorganization and for cell invasion in matrix [59]. Although the expression of LIM-domain proteins in many cases is associated with mesenchymal cells, also LIM-domain proteins negatively regulating EMT exist. EPLIN (epithelial protein lost in neoplasm) is downregulated during EMT, and its depletion leads to the marked increase of mesenchymal markers. Downregulation of EPLIN was further demonstrated in lymph node metastases of human solid tumors suggesting that it may be a frequent event during tumor formation [60].
Fig. 2. LIM-domain proteins affect EMT. EMT may lead to myofibroblast differentiation. Myofibroblasts are required for wound healing, but also form fibrotic lesions and are present in peritumoral areas as cancer associated fibroblasts (CAFs).
Given that several LIM-domain proteins are localized to cell–cell junctions, including abLIM3 [61], LIMD1 [62], zyxin [63], Ajuba [64] and LMO7 [65], it is not unexpected that some of them, including FHL2 and Ajuba regulate the dissolution of cell–cell junctions and the expression of E-cadherin during EMT. FHL2 was found highly expressed in primary and in metastatic colon cancer as compared to normal samples and to be required for TGFβ-induced EMT, migration and adhesion [66]. Furthermore, FHL2 was also identified as a direct negative regulator of E-cadherin, which suppressed the transcription of E-cadherin via interaction with Snail, a transcription factor that promotes EMT [67]. Ajuba interacts with Snail and acts as a corepressor to suppress the expression of E-cadherin and is required for Snail induced neural crest development [68] (Fig. 3). 2.4. Expression of LIM-domain proteins in CAFs Interestingly, several LIM-domain proteins, including FHL2, PINCH and CRP1 are expressed in CAFs surrounding the tumor tissue (Fig. 3). FHL2 is co-expressed with αSMA in myofibroblasts of the tumor invasive front in sporadic colon and in hereditary non-polyposis colorectal cancers. FHL2 expression is regulated by TGFβ-signaling pathway in fibroblasts, and is required for TGFβ-induced migration. This indicates that tumor-derived TGFβ via FHL2 may induce the migration of the peritumoral fibroblasts [69]. These findings support close signaling between tumor cells and myofibroblasts. The expression of PINCH is increased in stroma during progression from normal mucosa to colorectal adenocarcinoma and metastasis [70]. Further expression of PINCH is higher in invasive front than in intratumoral stroma [70]. Expression of PINCH is primarily observed in fibroblasts, myofibroblasts, in some endothelial cells and correlates with worse prognosis, implicating that PINCH may affect the tumorstroma association and promote tumor progression [70]. Furthermore, CRP1 is also expressed in peritumoral fibroblasts positive for αSMA in cutaneous squamous cell carcinoma [71]. Interestingly, LIM-only proteins are well represented in the group of LIM-domain proteins associated with CAFs. (Fig. 4). Whether these LIM-domain proteins share a structural or functional similarity that leads to their capacity to transform normal fibroblasts into CAFs or
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Fig. 3. LIM-domain proteins involved in EMT and TGFβ regulation. LIM-domain proteins that are regulated by TGFβ, and which affect EMT are grouped based on their protein structure and family (LIM group). Their expression in myofibroblasts (MF), CAFs, fibrosis and tumors (cancer/met) has been indicated.
respond to specific cytokine cues remain to be determined. Likewise, whether CRP1 has a role in tumor progression, and whether there are more related LIM-domain proteins involved in CAF-promoted transformation, are relevant aspects that should be resolved in the future. 2.5. LIM-domain proteins in fibrotic diseases Injury in alveolar epithelial cells in conjunction with TGFβ activation may cause the accumulation of myofibroblasts in the lung and promote the formation of fibrosis. While several LIM-domain proteins are associated with mesenchymal cells and actin cytoskeleton, they are also expressed in myofibroblasts, known for their rigid actin cytoskeleton and contractile phenotype. The expression of LIM-domain proteins CRP1 [42], CRP2 [72], PINCH1 and PINCH2 [73] has been detected in fibrotic myofibroblasts. CRP1 was not detected in normal alveolar epithelial cells, whereas it co-localized with αSMA in fibroblastic foci. Significant difference in expression of CRP1 was detected in control and IPF lung samples [42]. Given that CRP1 expression is increased in IPF, and that it is regulated by TGFβ via Smad2/3 and p38 dependent pathways [42], it is possible that CRP1 is elevated in IPF due to the increased production of TGFβ. Both canonical and non-canonical TGFβ-induced pathways are involved in EMT in pulmonary epithelial cells [74]. Furthermore, MAP kinase pathways, including p38, are activated in IPF patient
lung samples [75]. MK2, a p38 kinase substrate, is involved in TGFβ induced expression of αSMA and in myofibroblast differentiation [76], suggesting that CRP1 may be increased in IPF via p38, possibly also involving type III receptor activated signaling. Interestingly, p38 kinase inhibitor (Esbriet) was recently approved for treatment of IPF in Europe [77]. CRP2 has been linked to formation of liver fibrosis and is highly expressed in hepatic stellate cells [72]. Transdifferentiation of hepatic stellate cells is thought to be important in formation of liver fibrosis. However, the function of CRP2 in formation of liver fibrosis remains unresolved. PINCH 1 and 2 double knock-out mice exhibit severe dilated cardiomyopathy and die of heart failure within 4 weeks. Cardiomyocytes from the knock-out mice are significantly altered and suffer from abnormal adhesion, cell growth, and cell death. Ventricules are thinner and fibrotic causing heart failure [73], suggesting that PINCH proteins may have a special relevance in fibrotic processes of the heart. Again, LIM-only proteins are well represented in the group of LIMdomain proteins associated with fibrotic disease. (Fig. 4). Whether the structural and functional similarity of these proteins is relevant for formation of fibrosis, or share similar upstream regulatory pathways remain to be determined. Furthermore, given that FHL proteins, being the third member of the LIM-only group, mediate TGFβinduced migration and adhesion of the peritumoral fibroblasts, it
Fig. 4. LIM-only proteins are expressed in cancer-associated fibroblasts (CAFs) and in fibrotic myofibroblasts. CAFs secrete growth factors, proteases, cytokines and enzymes and promote tumor progression by increasing proliferation, invasion, angiogenesis and metastases. Fibrotic myofibroblasts secrete components of the extracellular matrix and proteases and these together with contractility affect the formation of fibrosis.
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would be highly interesting to explore whether these proteins are also involved in fibrotic diseases. 3. Conclusions LIM-domain proteins are largely unknown and diverse group of proteins. While the knowledge of these proteins is increasing, it is coming evident that they share strikingly similar functions with an impact in several human diseases. The expression of several LIM-domain proteins is modulated by TGFβ-signaling pathway. Given that many LIM-domain proteins are associated with actin cytoskeleton and localize to cell–cell adhesions, they also regulate EMT triggered alterations including actin stress fiber formation, migration and dissolution of cell–cell junctions. LIMdomain proteins are also expressed in myofibroblasts found in fibrotic lesions and in peritumoral area as cancer associated fibroblasts. Further studies will be needed to explore whether there are further LIM-domain proteins involved in TGFβ-induced EMT, and to study the function and mechanisms of action of LIM-domain proteins in myofibroblasts in fibrosis and in tumor stroma. As EMT and myofibroblasts have crucial roles in human diseases, finding novel targets to inhibit transdifferentiation, cell motility and invasiveness could potentially provide therapeutic value. Acknowledgments We thank all the co-authors who contributed to the original work Järvinen et al. 2011. The original work was supported by Academy of Finland (grant no. 213485, 108828), Biocentrum Helsinki, and the Finnish Cancer Organization. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
[19] [20]
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Contents lists available at SciVerse ScienceDirect
Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
Review
LIM-domain proteins in transforming growth factor β-induced epithelial-to-mesenchymal transition and myofibroblast differentiation Päivi M. Järvinen a, Marikki Laiho a, b,⁎ a b
Molecular Cancer Biology Program and Haartman Institute, University of Helsinki, Helsinki, Finland Department of Radiation Oncology and Molecular Radiation Sciences and Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
a r t i c l e
i n f o
Article history: Received 14 October 2011 Received in revised form 15 November 2011 Accepted 4 December 2011 Available online 11 December 2011 Keywords: Transforming growth factor β Myofibroblast Epithelial to mesenchymal transition LIM-domain protein Cancer associated fibroblast Fibrosis
a b s t r a c t Epithelial to mesenchymal transition (EMT) is a process during which junctions of the cell–cell contacts are dissolved, actin cytoskeleton is deformed, apical–basolateral cell polarity is lost and cell motility is increased. EMT is needed during normal embryonal development and wound healing, but may also lead to pathogenic transformation and formation of myofibroblasts. Transforming growth factor β (TGFβ) is a multifunctional cytokine promoting EMT and myofibroblast differentiation, and its dysregulation is involved in pathological disorders like cancer and fibrosis. Lin11, Isl-1 and Mec-3 (LIM) domain proteins are associated with actin cytoskeleton and linked to regulation of cell growth, damage signaling, cell fate determination and signal transduction. LIM-domain proteins generally do not bind DNA, but are more likely to function via protein–protein interactions. Despite being a disparate group of proteins, similarities in their functions are observed. In this review we will discuss the role of LIM-domain proteins in TGFβ-signaling pathway and in EMT-driven processes. LIM-domain proteins regulate TGFβ-induced actin cytoskeleton reorganization, motility and adhesion, but also dissolution of cell–cell junctions during EMT. Finally, the role of LIM-domain proteins in myofibroblasts found in fibrotic foci and tumor stroma will be discussed. © 2012 Elsevier Inc. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. TGFβ induced EMT . . . . . . . . . . . . . . . . . . . 1.2. TGFβ induced myofibroblast differentiation . . . . . . . 1.3. TGFβ at the juncture of wound healing and fibrotic diseases 1.4. TGFβ in formation of idiopathic pulmonary fibrosis (IPF) . 1.5. TGFβ and cancer associated fibroblast (CAFs) . . . . . . 2. LIM-domain proteins . . . . . . . . . . . . . . . . . . . . . 2.1. TGFβ regulates expression of several LIM-domain proteins 2.2. LIM-domain proteins regulate TGFβ signaling . . . . . . 2.3. LIM-domain proteins in EMT and migration . . . . . . . 2.4. Expression of LIM-domain proteins in CAFs . . . . . . . 2.5. LIM-domain proteins in fibrotic diseases . . . . . . . . . 3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: CAF, cancer associated fibroblast; CRP, cysteine rich protein; ECM, extracellular matrix; EMT, epithelial to mesenchymal transition; ERK, extracellular signalregulated kinases; FHL, four and half LIM protein; HIC-5, hydrogen peroxide-inducible gene 5; IPF, idiopathic pulmonary fibrosis; JNK, jun N-terminal protein kinase; LIM, Lin11, Isl-1 & Mec-3; LMO, LIM only protein; MAPK, mitogen activated protein kinase; NOX-4, NAPDH/oxidase 4; PINCH, particularly interesting new cysteine-histidine rich protein; αSMA, α smooth muscle actin; TGFβ, transforming growth factor β. ⁎ Corresponding author at: Molecular Cancer Biology Program, University of Helsinki, PO Box 63, FIN-00014 Helsinki, Finland. Tel.: + 358 9 19125542; fax: + 358 9 19125554. E-mail address: [email protected] (M. Laiho). 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.12.004
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1. Introduction
1.1. TGFβ induced EMT
TGFβ signaling pathway regulates growth, apoptosis, differentiation, adhesion, invasion and extracellular matrix production [1,2]. TGFβ is a pleiotropic cytokine ubiquitously expressed by most cells and tissues in the body, essential for embryogenesis and maintenance of tissue homeostasis. The effect of TGFβ is context dependent; broadly, in epithelial cells it acts as a growth inhibitor, whereas in fibroblasts it stimulates proliferation. Dysregulation of TGFβ signaling pathway is involved in pathological disorders like cancer and fibrosis [3]. TGFβ ligands recognize and bind TGFβ type II receptor. Binding of the ligand causes the activation of intracellular serine/threonine kinase domain of the type II receptor, which phosphorylates the cytoplasmic tail of type I receptor and activates its respective serine/ threonine kinase domain. The activated heterotetrameric receptor complex regulates downstream Smad and non-Smad pathways [4]. Receptor activation will ultimately lead to the phosphorylation of receptor-regulated Smads 2 and 3, and heteromeric complex formation with Smad4, nuclear translocation, and activation of transcription [4]. Besides the canonical Smad-pathway, the functional receptor complex also activates so-called non-Smad mediated pathways (Fig. 1). These include MAP kinases (ERK, JNK, p38), Rho-like GTPase signaling and phosphatidylinositol-3-kinase/AKT pathways, which are especially relevant in EMT [5].
Epithelial cells are immobile and polar cells, and are characterized by strong adhesions between neighboring cells. These cell-cell contacts include tight and adherens junctions and desmosomes, which are connected to the circumferential actin belt. E-cadherin is a key component of the adherens junctions and is vital for initiating and maintaining epithelial architecture in vitro and in vivo [6]. During EMT, epithelial junctions are disintegrated and cells become more mobile and apolar fibroblast-cell like. EMT is a normal process during embryonal development and wound healing, but occurs also during pathological conditions like carcinogenesis and in fibrotic diseases. Molecular characteristics of EMT are loss of epithelial markers such as E-cadherin, zona occludens-1, and an increase in mesenchymal markers like N-cadherin, α smooth muscle actin (αSMA), matrix metalloproteinases and extracellular matrix (ECM) components including collagen and fibronectin [2]. Especially as about 90% of the cancers are of epithelial origin, EMT is of high relevance during cancer progression [7]. TGFβ is a key regulator of EMT and drives it both by Smaddependent and independent manner, and also by regulating the expression of well known EMT-inducer transcription factor families Snail, ZEB and bHLH [8]. 1.2. TGFβ induced myofibroblast differentiation Myofibroblasts are cells with features of both fibroblasts and smooth muscle cells. They may differentiate from several origins; mesenchymal cells such as fibroblasts, hepatic stellate or smooth muscle cells, epithelial or endothelial cells through EMT, bone marrow cells and from fibrocytes [9]. Myofibroblasts are identified by the expression of αSMA, contractility and production of specific ECM proteins, and are needed for normal wound repair. TGFβ drives the transdifferentiation of fibroblasts to myofibroblasts in vitro and in vivo [10,11]. TGFβ induces the myofibroblast differentiation of hepatic stellate cells [12], and causes myofibroblast differentiation through EMT [13]. Furthermore, myofibroblast marker αSMA is a direct target of TGFβ [14]. 1.3. TGFβ at the juncture of wound healing and fibrotic diseases
Fig. 1. Several LIM-domain proteins are activated by TGFβ either via Smad or nonSmad pathways. TGFβ is required to maintain normal tissue homeostasis, but may also promote tumorigenesis or fibrotic processes.
During wound healing, damaged epithelial or endothelial cells produce inflammatory mediators that activate inflammatory cells and fibroblasts, which start the excess deposition of ECM. Myofibroblasts, epithelial and endothelial cells produce matrix metalloproteinases that degrade the basement membrane and thus enhance the migration of inflammatory cells to the site of the injury. Macrophages and neutrophils engulf the tissue debris, dead cells and pathogens and secrete cytokines and chemokines, which invite endothelial cells to surround the injured site and to form new blood vessels. Furthermore, lymphocytes and other cells become activated and start to secrete profibrotic cytokines and growth factors such as TGFβ, IL-13 and platelet derived growth factor, which further activate macrophages and fibroblasts. Activated myofibroblasts help the wound closure through contraction [15]. The wound healing process is completed by multiplication of epithelial and endothelial cells, which migrate over the basal layer to replace the injured tissue. While myofibroblasts disappear after successful wound healing, in fibrotic lesions these cells persist, and are responsible for scarring of tissue and excessive production of ECM altering the tissue architecture, and in severe cases, lead to the organ failure [9]. Fibrosis is a pathological condition that occurs in various organs, such as lung, liver, kidney and the cardiovascular system, and is characterized by overgrowth, stiffening, scarring and excess production of ECM. Fibrosis is also caused by chronic inflammatory reactions induced by various stimuli including persistent infection, autoimmune
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reaction, allergic response, chemical insult, radiation, and tissue injury [15]. TGFβ has been implicated as a major inducer of fibrosis in many tissues, including the lung [11]. 1.4. TGFβ in formation of idiopathic pulmonary fibrosis (IPF) IPF is an interstitial lung disease and an example of TGFβ-induced fibrotic diseases. Chronic inflammation and accumulation of myofibroblasts in fibroblastic foci is observed in the lung leading to decreased alveolar gas exchange and pulmonary volume restriction [16]. IPF has a poor prognosis and the 5-year survival is only 20% [17]. Although the etiology of IPF is largely unknown, persistent lung injury, inflammation and inefficient wound repair contribute to the disease. Furthermore, acquired or hereditary genetic alterations, including telomerase mutations, predispose to IPF [18]. A key process of repairing lung injury is the activation of TGFβ, and its uncontrolled activity contributes to IPF. An emerging concept is that IPF is a disease of deregulated EMT crosstalk [19]. IPF can be triggered by alveolar injury that leads to the activation of TGFβ and alveolar basement membrane disruption. Subsequently, TGFβ may promote enhanced epithelial apoptosis and EMT, as well as fibroblast and fibrocyte differentiation into myofibroblasts. Deposition of excess extracellular matrix by the myofibroblasts disposes to the development of IPF [19]. Elevated levels of TGFβ have been detected in IPF lung specimens as compared to the controls [20,21], and the presence of TGFβ1 in the lung epithelium indicates chronic injury [22]. Polymorphism in the TGFB1 gene has been found in codons 10 and 25, and while they do not predispose to the development of IPF, they may affect disease progression [23]. Moreover, specific inhibitors of the type I receptor reduce myofibroblast transformation and collagen gel contraction in a rat bleomycin-induced lung fibrosis model [24]. Several genes are reported to mediate TGFβ-induced effects in IPF. NAPDH/oxidase 4 (NOX-4) belongs to a group of enzymes that catalyze the formation of reactive oxygen species and is required for TGFβinduced myofibroblast differentiation, production and contractility [25]. Furthermore, silencing or pharmacological targeting of NOX-4 abrogated the formation of fibrosis in two murine models with lung injury [25]. Peroxisome proliferator-activated receptor-γ has been shown to repress TGFβ-induced myofibroblast differentiation via Smadindependent manner by affecting two TGFβ-dependent pro-survival pathways involved in myofibroblast differentiation [26].
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proteins have been suggested in development, gene expression, cytoarchitecture, cell adhesion and motility, signal transduction, cell fate determination and tumor formation [32,33]. These activities are mediated by protein–protein interactions, and depending on their interacting partners, a LIM-domain protein can have several different functions [33]. These include their actions as adaptors, competitors, autoinhibitors or localizers of other proteins. LIM-domain proteins have been categorized into four main families based on their functional domains and localization [33]. Nterminal tandem LIM-domain proteins such as LHX and nuclear LIM only protein (LMO) proteins are found in the nucleus and act as a transcription factors or cofactors. LIM-only proteins are detected both in the nucleus and cytoplasm, and include cysteine rich protein (CRP), four and half LIM (FHL) and particularly interesting new cysteine–histidine rich protein (PINCH) protein families. LIMdomain proteins that have additional protein–protein interaction motifs (PDZ, leucine-aspartate repeats, or actin-target domains) are represented by zyxin, EPLIN, and ABLIM protein families. Lastly, LIM-domain proteins LIMK and MICAL contain additional functional domains such as mono-oxygenase or kinase catalytic motifs [33]. Most LIM-domain proteins are associated with actin cytoskeleton in the cytoplasm. Several shuttle between cytoplasm and nucleus. For example, translocation of zyxin is triggered by extracellular stimulation by UV [34]. Furthermore, zyxin responds to cell stretching, and translocates from focal adhesions into nucleus to regulate gene expression [35]. Thus, LIM-domain proteins are involved in mediating signals between the nucleus and cytoplasm [32]. CRP family consists of three members: CRP1, CRP2 and CRP3/MLP [36]. CRPs are small proteins, 22 kDa of size, and comprise of two functional LIM-domains. Three-dimensional structure analyses of CRPs predict that these proteins may act as molecular adapters [37]. CRPs exhibit a differential expression pattern in chicken; CRP1 expression is detected in most tissues, and is especially enriched in smooth muscle cells, whereas CRP2 is expressed in smooth muscle cells especially in vasculature and in fibroblasts, and CRP3/mlp in striated muscle [38]. CRP1 exhibits dual localization pattern in cell; it is found both in the nucleus and in cytoplasm. In nucleus, CRP1 functions as a transcriptional co-regulator [39]. It also directly interacts with actin. CRP1 may play a role in actin cytoskeleton remodeling by bundling the stress fibers by crossing actin filaments and by stabilizing the interactions of α-actinin with actin filament bundles [40,41].
1.5. TGFβ and cancer associated fibroblast (CAFs) 2.1. TGFβ regulates expression of several LIM-domain proteins Tumor microenvironment has become a subject of intensive research. During tumor development, tissue homeostasis between different cell types is disturbed. The invasive nature of tumor cells is an end result of interaction between tumor stroma and the epithelial cells. The tumor stroma includes cancer associated fibroblasts (CAFs), immune cells, vasculature and extracellular matrix [27]. CAFs are often present in carcinomas, and they play a role in tumorigenesis by secreting paracrine growth factors and in this way promote tumor growth, angiogenesis and invasion [28]. TGFβ significantly increases the percentage of myofibroblasts in CAF population [29]. Several studies show that CAFs produce TGFβ at higher level than normal fibroblasts. TGFβ and SDF1 are released in cell autonomous fashion during tumorigenesis by stromal fibroblasts leading to CAF differentiation in the breast [30]. Similarly, CAFs isolated from prostate show increased TGFβ immunoreactivity as compared to normal human prostate fibroblasts [31]. 2. LIM-domain proteins LIM-domains consist of two zinc fingers and have been found in a wide variety of eukaryotes. So far, 135 LIM-domains have been identified within 58 human genes [32]. Diverse roles for LIM-domain
LIM-domain proteins act both as downstream targets and modulators of TGFβ signaling. TGFβ positively regulates the expression of several LIM-domain proteins: CRP1 [42], CRP2 [43,44], LMO1 [45], zyxin [46] and hydrogen peroxide-inducible 5 (HIC-5) [47] (Fig. 1). In addition, the expression of paxillin is modulated by TGFβpathway, increasing paxillin phosphorylation on Tyr31 and Tyr118. Conversely, paxillin delta lacking these phosphorylation sites is downregulated during TGFβ-induced EMT [48]. Regulation of the LIM-domain proteins by TGFβ is mediated through the Smad-pathway leading to transcriptional activation of the respective targets, with one exception. Interestingly, the increase in CRP1 by TGFβ is independent of TGFβ-induced transcriptional regulation [42]. This is in contrast to the close relative, CRP2 [44], even though the kinetics of elevated protein expression of CRP1 and CRP2 post TGFβ-treatment are similar. Furthermore, the regulation of CRP1 by TGFβ appeared biphasic. TGFβ-treatment led to a rapid increase of CRP1 within one hour followed by a decrease. A sustained increase of CRP1 by TGFβ then ensued a few hours later and was stable for few days [42]. Although the sustained increase of CRP1 appeared dependent on type I receptor and activated Smadpathway, the rapid induction was independent of both [42]. However,
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both the rapid and sustained increases in CRP1 were dependent on p38 signaling pathway. Interestingly, p38/MAPKs have been reported to mediate signaling from TGFβ receptor III independently of Smads [49]. In addition, p38 MAPK has been shown to mediate TGFβ regulated apoptosis and EMT independently of Smads [50], and p38 MAPK pathway is activated by TGFβ type III receptor (betaglycan) [51,52]. Given that the type I receptor inhibitor used here was without effect on the rapid response, it is possible that the response is mediated via type III receptor and p38 MAPK signaling [42]. 2.2. LIM-domain proteins regulate TGFβ signaling Certain LIM-domain proteins modulate TGFβ signaling pathway. HIC-5 is a coactivator of androgen receptor and has four LIMdomains. HIC-5 modulates TGF-β-signaling by interaction with Smad3, Smad4 [53] and Smad7 [54], but does not interact with Smad2 [53]. HIC-5 suppresses Smad7 by physically interacting with Smad7 through its third LIM-domain causing loss of Smad7 [54]. Loss of Smad7, the negative regulator of Smads 2 and 3, leads to enhanced TGFβ signaling [54]. The third LIM-domain is also needed for interaction between HIC-5 and Smad3 [53]. The suppressive activity of HIC-5 on Smad3 and Smad7 enhances Smad2 activity thus suggesting that HIC-5 could be a factor involved in switch in TGFβ from being a tumor suppressor to being an oncogene during tumor progression [54]. Furthermore, FHL proteins (FHL1, FHL2, FHL3) modulate TGFβsignaling pathway via interaction with Smads (Smad2, Smad3, Smad4). FHL proteins are positive regulators of TGFβ-responsive transcription. They enhance phosphorylation of Smad2/3, increase interaction between Smad2/3 and Smad4 and their nuclear accumulation via casein kinase 1δ independently of TGFβ-receptor signaling. Interestingly, FHL proteins are often downregulated in hepatocellular carcinoma, and the levels correlate with decreased TGFβ-like responses in the clinical samples [55]. These findings suggest that LIM-domain proteins act both by activating and repressing TGFβ signaling, and contribute to both TGFβ physiological and pathophysiological regulation. 2.3. LIM-domain proteins in EMT and migration Given that LIM-domain proteins classically have been linked to actin cytoskeleton, their levels may be elevated simply due to actin cytoskeleton reorganization during TGFβ-induced EMT. Although we did not find evidence that CRP1 is involved in regulation of EMT [42], several LIM-domain proteins, like zyxin [56], HIC-5 [57], LMO2 [58] and LIMK [59], are required for TGFβ-driven stress fiber reorganization, cell motility and adhesion (Fig. 2). Zyxin is a target of TGFβ signaling cascade and positive regulator of EMT. It is required for stress fiber reorganization and cell migration during EMT in mouse mammary gland epithelial cells and in endocardial cells [56]. Furthermore, depletion of Hic-5 in TGFβ treated cells leads to decreased cell migration, actin stress fiber formation, retains the epithelial organization and thus inhibits EMT [57]. Conversely, expression of LMO2 protein in prostate cancer cells downregulates Ecadherin and promotes EMT by enhancing cell motility and invasiveness [58]. Using similar silencing approach towards LIMK, it was shown that while LIMK is not required for TGFβ-induced 2D motility, but it is needed for TGFβ-induced actin stress fiber reorganization and for cell invasion in matrix [59]. Although the expression of LIM-domain proteins in many cases is associated with mesenchymal cells, also LIM-domain proteins negatively regulating EMT exist. EPLIN (epithelial protein lost in neoplasm) is downregulated during EMT, and its depletion leads to the marked increase of mesenchymal markers. Downregulation of EPLIN was further demonstrated in lymph node metastases of human solid tumors suggesting that it may be a frequent event during tumor formation [60].
Fig. 2. LIM-domain proteins affect EMT. EMT may lead to myofibroblast differentiation. Myofibroblasts are required for wound healing, but also form fibrotic lesions and are present in peritumoral areas as cancer associated fibroblasts (CAFs).
Given that several LIM-domain proteins are localized to cell–cell junctions, including abLIM3 [61], LIMD1 [62], zyxin [63], Ajuba [64] and LMO7 [65], it is not unexpected that some of them, including FHL2 and Ajuba regulate the dissolution of cell–cell junctions and the expression of E-cadherin during EMT. FHL2 was found highly expressed in primary and in metastatic colon cancer as compared to normal samples and to be required for TGFβ-induced EMT, migration and adhesion [66]. Furthermore, FHL2 was also identified as a direct negative regulator of E-cadherin, which suppressed the transcription of E-cadherin via interaction with Snail, a transcription factor that promotes EMT [67]. Ajuba interacts with Snail and acts as a corepressor to suppress the expression of E-cadherin and is required for Snail induced neural crest development [68] (Fig. 3). 2.4. Expression of LIM-domain proteins in CAFs Interestingly, several LIM-domain proteins, including FHL2, PINCH and CRP1 are expressed in CAFs surrounding the tumor tissue (Fig. 3). FHL2 is co-expressed with αSMA in myofibroblasts of the tumor invasive front in sporadic colon and in hereditary non-polyposis colorectal cancers. FHL2 expression is regulated by TGFβ-signaling pathway in fibroblasts, and is required for TGFβ-induced migration. This indicates that tumor-derived TGFβ via FHL2 may induce the migration of the peritumoral fibroblasts [69]. These findings support close signaling between tumor cells and myofibroblasts. The expression of PINCH is increased in stroma during progression from normal mucosa to colorectal adenocarcinoma and metastasis [70]. Further expression of PINCH is higher in invasive front than in intratumoral stroma [70]. Expression of PINCH is primarily observed in fibroblasts, myofibroblasts, in some endothelial cells and correlates with worse prognosis, implicating that PINCH may affect the tumorstroma association and promote tumor progression [70]. Furthermore, CRP1 is also expressed in peritumoral fibroblasts positive for αSMA in cutaneous squamous cell carcinoma [71]. Interestingly, LIM-only proteins are well represented in the group of LIM-domain proteins associated with CAFs. (Fig. 4). Whether these LIM-domain proteins share a structural or functional similarity that leads to their capacity to transform normal fibroblasts into CAFs or
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Fig. 3. LIM-domain proteins involved in EMT and TGFβ regulation. LIM-domain proteins that are regulated by TGFβ, and which affect EMT are grouped based on their protein structure and family (LIM group). Their expression in myofibroblasts (MF), CAFs, fibrosis and tumors (cancer/met) has been indicated.
respond to specific cytokine cues remain to be determined. Likewise, whether CRP1 has a role in tumor progression, and whether there are more related LIM-domain proteins involved in CAF-promoted transformation, are relevant aspects that should be resolved in the future. 2.5. LIM-domain proteins in fibrotic diseases Injury in alveolar epithelial cells in conjunction with TGFβ activation may cause the accumulation of myofibroblasts in the lung and promote the formation of fibrosis. While several LIM-domain proteins are associated with mesenchymal cells and actin cytoskeleton, they are also expressed in myofibroblasts, known for their rigid actin cytoskeleton and contractile phenotype. The expression of LIM-domain proteins CRP1 [42], CRP2 [72], PINCH1 and PINCH2 [73] has been detected in fibrotic myofibroblasts. CRP1 was not detected in normal alveolar epithelial cells, whereas it co-localized with αSMA in fibroblastic foci. Significant difference in expression of CRP1 was detected in control and IPF lung samples [42]. Given that CRP1 expression is increased in IPF, and that it is regulated by TGFβ via Smad2/3 and p38 dependent pathways [42], it is possible that CRP1 is elevated in IPF due to the increased production of TGFβ. Both canonical and non-canonical TGFβ-induced pathways are involved in EMT in pulmonary epithelial cells [74]. Furthermore, MAP kinase pathways, including p38, are activated in IPF patient
lung samples [75]. MK2, a p38 kinase substrate, is involved in TGFβ induced expression of αSMA and in myofibroblast differentiation [76], suggesting that CRP1 may be increased in IPF via p38, possibly also involving type III receptor activated signaling. Interestingly, p38 kinase inhibitor (Esbriet) was recently approved for treatment of IPF in Europe [77]. CRP2 has been linked to formation of liver fibrosis and is highly expressed in hepatic stellate cells [72]. Transdifferentiation of hepatic stellate cells is thought to be important in formation of liver fibrosis. However, the function of CRP2 in formation of liver fibrosis remains unresolved. PINCH 1 and 2 double knock-out mice exhibit severe dilated cardiomyopathy and die of heart failure within 4 weeks. Cardiomyocytes from the knock-out mice are significantly altered and suffer from abnormal adhesion, cell growth, and cell death. Ventricules are thinner and fibrotic causing heart failure [73], suggesting that PINCH proteins may have a special relevance in fibrotic processes of the heart. Again, LIM-only proteins are well represented in the group of LIMdomain proteins associated with fibrotic disease. (Fig. 4). Whether the structural and functional similarity of these proteins is relevant for formation of fibrosis, or share similar upstream regulatory pathways remain to be determined. Furthermore, given that FHL proteins, being the third member of the LIM-only group, mediate TGFβinduced migration and adhesion of the peritumoral fibroblasts, it
Fig. 4. LIM-only proteins are expressed in cancer-associated fibroblasts (CAFs) and in fibrotic myofibroblasts. CAFs secrete growth factors, proteases, cytokines and enzymes and promote tumor progression by increasing proliferation, invasion, angiogenesis and metastases. Fibrotic myofibroblasts secrete components of the extracellular matrix and proteases and these together with contractility affect the formation of fibrosis.
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would be highly interesting to explore whether these proteins are also involved in fibrotic diseases. 3. Conclusions LIM-domain proteins are largely unknown and diverse group of proteins. While the knowledge of these proteins is increasing, it is coming evident that they share strikingly similar functions with an impact in several human diseases. The expression of several LIM-domain proteins is modulated by TGFβ-signaling pathway. Given that many LIM-domain proteins are associated with actin cytoskeleton and localize to cell–cell adhesions, they also regulate EMT triggered alterations including actin stress fiber formation, migration and dissolution of cell–cell junctions. LIMdomain proteins are also expressed in myofibroblasts found in fibrotic lesions and in peritumoral area as cancer associated fibroblasts. Further studies will be needed to explore whether there are further LIM-domain proteins involved in TGFβ-induced EMT, and to study the function and mechanisms of action of LIM-domain proteins in myofibroblasts in fibrosis and in tumor stroma. As EMT and myofibroblasts have crucial roles in human diseases, finding novel targets to inhibit transdifferentiation, cell motility and invasiveness could potentially provide therapeutic value. Acknowledgments We thank all the co-authors who contributed to the original work Järvinen et al. 2011. The original work was supported by Academy of Finland (grant no. 213485, 108828), Biocentrum Helsinki, and the Finnish Cancer Organization. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
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