Cardiovascular disease is a leading cause of death and a major economic burden in the developed and developing world. Many heart diseases, including post-myocardial infarction, include a fibrotic component with remodeling of the extracellular matrix in the myocardium. Cardiac myofibroblasts are non-myocyte cells derived from relatively quiescent fibroblasts and are the main mediators of collagen remodeling in disease states. TGF-β is recognized as an important contributor to adverse cardiac remodeling in heart disease. In this study we have investigated the role of c-Ski, which is an endogenous TGF-β inhibitor, in controlling/regulating myofibroblast function and phenotype. We have developed an adenoviral overexpression system to study these endpoints using Western blot, immunofluorescence, MTT assay, flow cytometry, procollagen type I amino terminal peptide secretion and qPCR analysis. We observed that the 95 kDa c-Ski form is overexpressed upon virus infection with adenovirus encoding c-Ski and this form of c-Ski is localized to the nucleus. c-Ski expression inhibited cardiac myofibroblast collagen synthesis and secretion as well as contractility. Phosphorylation and translocation of Smad2 into the nucleus was not affected in the presence of c-Ski overexpression. We found that c-Ski overexpression was associated with diminution of the myofibroblastic phenotype with reduced α-smooth muscle actin and extra domain-A fibronectin expression (but not non-muscle myosin heavy chain B expression). c-Ski may reduce cell viability via the induction of apoptosis. Finally, we have elucidated a putative mechanism for c-Ski-mediated reduction of myofibroblast phenotype through the upregulation of the homeodomain protein Meox2. Adenoviral overexpression of Meox2 was associated with a significant reduction of α-smooth muscle actin and extra domain-A fibronectin expression in a similar manner to that of c-Ski overexpression. Thus we have identified c-Ski as being an antifibrotic protein as well as a novel mechanism for modulation of cardiac myofibroblast phenotype, possibly through the induction of Meox2 expression.

Cardiovascular disease is a leading cause of death and a major economic burden in the developed and developing world. Many heart diseases, including post-myocardial infarction, include a fibrotic component with remodeling of the extracellular matrix in the myocardium. Cardiac myofibroblasts are non-myocyte cells derived from relatively quiescent fibroblasts and are the main mediators of collagen remodeling in disease states. TGF-β is recognized as an important contributor to adverse cardiac remodeling in heart disease. In this study we have investigated the role of c-Ski, which is an endogenous TGF-β inhibitor, in controlling/regulating myofibroblast function and phenotype. We have developed an adenoviral overexpression system to study these endpoints using Western blot, immunofluorescence, MTT assay, flow cytometry, procollagen type I amino terminal peptide secretion and qPCR analysis. We observed that the 95 kDa c-Ski form is overexpressed upon virus infection with adenovirus encoding c-Ski and this form of c-Ski is localized to the nucleus. c-Ski expression inhibited cardiac myofibroblast collagen synthesis and secretion as well as contractility. Phosphorylation and translocation of Smad2 into the nucleus was not affected in the presence of c-Ski overexpression. We found that c-Ski overexpression was associated with diminution of the myofibroblastic phenotype with reduced α-smooth muscle actin and extra domain-A fibronectin expression (but not non-muscle myosin heavy chain B expression). c-Ski may reduce cell viability via the induction of apoptosis. Finally, we have elucidated a putative mechanism for c-Ski-mediated reduction of myofibroblast phenotype through the upregulation of the homeodomain protein Meox2. Adenoviral overexpression of Meox2 was associated with a significant reduction of α-smooth muscle actin and extra domain-A fibronectin expression in a similar manner to that of c-Ski overexpression. Thus we have identified c-Ski as being an antifibrotic protein as well as a novel mechanism for modulation of cardiac myofibroblast phenotype, possibly through the induction of Meox2 expression.

Fibrotic diseases are a leading cause of morbidity and mortality in the developed world. Despite this, the lack of therapies for fibrotic pathological disease states is severe. A large part of the reason for this lack of viable therapies is due to an incomplete understanding of the early processes driving tissue fibrosis, as well as the dismal results of pharmacologic monotherapies at the clinical trial stage in humans thus far. Therefore, better understanding of the upstream mechanisms driving tissue fibrosis is imperative. One of the common mechanisms underlying all fibroses is the presence and activity of the myofibroblast, a contractile mesenchymal cell that deposits high levels of extracellular matrix. Overpersistence of myofibroblasts in the wound site lead to deposition of an acellular, nonfunctional, mechanically aberrant scar that can result in loss of tissue function and, in severe cases, eventual organ failure. Here we investigate the mechanisms under which fibroblast growth factor 2 (FGF2), one member of the mammalian fibroblast growth factor family, antagonizes activation of fibroblasts to myofibroblasts. We identify a gene and protein expression signature induced by FGF2 that is antagonistic to activated myofibroblasts, and we demonstrate that induction of this antifibrotic gene expression paradigm is antagonized by inhibition of the mitogen-activated protein kinase pathways ERK and JNK, each of which lies canonically downstream of FGF2/FGFR signaling, suggesting that the antifibrotic effects of FGF2 as an antagonist to fibroblast activation are likely dependent at least in part upon activation of these cellular signaling pathways. We further demonstrated that, independent of exogenous FGF2 stimulation, inhibition of ERK or JNK signaling in proliferating human dermal fibroblasts was sufficient to induce fibroblast activation, accompanied by a pro-fibrotic extracellular matrix gene expression paradigm. Inhibition of these pathways also resulted in distinct changes in transforming growth factor beta (TGF-β) gene expression paradigms, modulating the expression of both ligands and receptors involved in this pathway, and we verified that activation of fibroblasts via MAPK inhibition was dependent at least in part on activation of TGF-βR signaling. In contrast, inhibition of p38 MAPK was sufficient to antagonize fibroblast activation and subsequent fibrosis-associated extracellular matrix deposition, both in the presence and absence of exogenous TGF-β, via changes in gene expression antagonistic to pro-fibrotic TGF-β/TGF-βR signaling. Broadly, these data suggest that activation of ERK and JNK pathways broadly antagonize fibroblast activation and fibrosis, while activation of p38 drives fibroblast activation and pro-fibrotic fibroblast phenotypes. It is our hope that this information will lead to a better understanding of the way that cellular signaling pathways interact in order to drive fibroblast activation, and better inform the potential effects of kinase inhibitors or related therapeutics for use as anti-fibrotic therapies.

Cardiac fibrosis is the excess deposition of myocardial extracellular matrix components, which increases tissue stiffness and heterogeneity, causing impaired diastolic/systolic function and arrhythmias, and eventually leading to heart failure and death. There are no available treatments for cardiac fibrosis. Myofibroblasts mediate fibrosis, and are characterized by hypersynthesis of collagens, decreased migration, and increased α-smooth muscle actin, which is incorporated into stress fibers, imparting contractility. Scleraxis is a transcriptional regulator of collagen-rich tissues, increased in response to the same stimuli that drive the myofibroblast phenotype, such as cyclic stretch. We show that Scleraxis mediates the conversion of cardiac fibroblasts to myofibroblasts, by increasing myofibroblast marker expression and contraction, and decreasing migration. Additionally, a proximal 1500 bp human SCLERAXIS promoter is activated by stretch and is responsive to transforming growth factor-β1. Thus, Scleraxis is a specific mechanoresponsive regulator of the myofibroblast, representing a novel target for the treatment of cardiac fibrosis.

Fibroblasts are abundant mesenchymal cells present in all tissues in a quiescent state, which contribute to wound healing when activated. Cytokine transforming growth factor-β1 (TGF-β1) stimulates fibroblast-myofibroblast differentiation, which induces extracellular matrix secretion, tissue contraction and promotes cancer cell migration. Hence, chronic activity of stromal myofibroblasts correlates with a poor prognosis for cancer and organ fibrosis patients. Therefore, modulating myofibroblast activity may reduce the severity of these diseases. Previous research suggests blockade of transmembrane integrin receptors expressed by fibroblasts prevents TGF-β1- induced differentiation, indicating integrins are attractive therapeutic targets. However, fibroblasts derived from different organs exhibit heterogeneity, although their integrin expression and integrin-regulated differentiation has not been directly compared. The aim of my research was 1) to understand and compare how integrins regulate TGF-β1-induced activation of fibroblasts derived from normal skin, lung and breast tissue; 2) to examine the global gene expression of TGF-β1-treated lung fibroblasts; 3) to identify novel therapeutic targets that modulate TGF-β1-induced activation of lung fibroblasts using a drug library. qPCR showed skin, lung and breast fibroblasts differentially expressed TGF-β1- induced activation markers, including ACTA2, FN1, TIMP3, CTGF and SERPINE1, in addition to integrin genes for α1, α4, α11 and β3. Small-molecule inhibitors of αv integrins only reduced the invasion of TGF-β1-exposed skin fibroblasts, but not lung or breast fibroblasts. siRNA against α11, β3 and β5 decreased TGF-β1-induced collagen contraction and activation marker expression in skin and lung fibroblasts, while α1 siRNA prevented collagen contraction by breast fibroblasts only. RNA sequencing of TGF-β1-treated lung fibroblasts revealed pro-inflammatory and profibrotic pathways were significantly enriched, while screening TGF-β1-treated lung fibroblasts with a FDA-approved drug library identified 46 hits that significantly reduced α-smooth muscle actin and fibronectin expression. Overall, genes are differentially expressed in TGF-β1-treated skin, lung and breast fibroblasts, while different integrins in each fibroblast appear to regulate invasion, TGF-β1-induced collagen contraction and gene expression. RNA sequencing revealed TGF-β1 promotes the expression of a pro-tumour signature in lung fibroblasts and several novel therapeutic targets that modulate the activation of lung fibroblasts have been identified. Understanding these integrin-dependent and independent mechanisms will facilitate the generation of myofibroblast-targeted treatments for cancer and organ fibrosis.

Die im Rahmen dieser Arbeit behandelten Fragestellungen vermitteln neue Kenntnisse über die Pathogenese der Lungenfibrose auf zellulärer Ebene. Bei der Lungenfibrose handelt es sich um eine chronische Erkrankung, die durch eine initiale Inflammation und das Auftreten von Myofibroblasten gekennzeichnet ist. Die Myofibroblasten führen zu einer vermehrten Produktion von EZM, was in einer Zerstörung der Lungenarchitektur, Narbenbildung und folglich einem verminderten Gasaustausch resultiert. Eine modulatorische Rolle von Stickstoffmonoxid (NO) bei der Entwicklung der Lungenfibrose wird vermutet, dennoch sind die Effektorzellen in der Lunge noch nicht bekannt. Daher wurde im ersten Teil dieser Arbeit die Lokalisation des NO-Rezeptors, der NO-sensitiven Guanylyl-Cyclase (NO-GC), in der Lunge untersucht. Dazu wurden Knockout-Mäuse generiert, bei denen die NO-GC global (GCKO) oder Perizyten-spezifisch (PDGFRβ-GCKO, SMMHC-GCKO, NG2-GCKO und SMMHC/NG2-GCKO) deletiert ist. Zudem wurden tdTomato-Reportermäuse verwendet, die das Fluoreszenzprotein unter Kontrolle eines spezifischen Reporters exprimieren (PDGFRβ/tomato, SMMHC/tomato, NG2/tomato, FoxD1/tomato und Tie2/tomato). In der Lunge sind Perizyten der NO-GC-exprimierende Zelltyp. Durch Immunhistochemie konnten zudem zwei verschiedene Subpopulationen von NO-GC-exprimierenden Perizyten identifiziert werden: Eine große Population an SMMHC/PDGFRβ-positiven Perizyten und eine kleine Population an NG2/PDGFRβ-positiven Perizyten. Im zweiten Teil dieser Arbeit wurde die Funktion der NO-GC während der Bleomycin-induzierten Lungenfibrose untersucht. Bleomycin führt zu einer fibrotischen Antwort in allen Genotypen, was durch ein erhöhtes Lungengewicht und einen erhöhten Kollagengehalt deutlich wird. Der Schweregrad der Lungenverletzung ist in NO-GC-defizienten Mäusen größer als in Anwesenheit der NO-GC. Dies deutet auf eine Rolle der NO-GC bei der Bleomycin-induzierten Lungenfibrose hin. Während der Entstehung der Lungenfibrose kommt es zur Bildung von Myofibroblasten, die als die Schlüsselzellen der Wundheilung und fibrotischer Prozesse bezeichnet werden. Diese Zellen kommen unter physiologischen Bedingungen kaum vor und ihre Herkunft ist nach wie vor nicht eindeutig geklärt. Da Perizyten als mögliche Vorläuferzellen betrachtet werden, wurde Lineage Tracing von Perizyten durchgeführt. Erstmals wurden zwei verschiedene Myofibroblasten-Subtypen durch die Expression von NO-GC unterschieden: (1) NO-GC-positive Myofibroblasten, die in der Alveolarwand lokalisiert sind und von Perizyten abstammen und (2) NO-GC-negative Myofibroblasten, die sich innerhalb der Alveolen befinden, deren Ursprung jedoch nicht Perizyten sind. Diese Myofibroblasten zeigen jedoch eine de novo-Synthese von PDGFRβ. Durch Lineage Tracing-Versuche sowie immunhistochemische Analysen können Perizyten, Endothelzellen und Fibrozyten als Vorläuferzellen ausgeschlossen werden. Die Ursprungszelle der intra-alveolären Myofibroblasten ist somit bislang nicht identifiziert. Im letzten Teil der Arbeit wurde die Rolle der an der Lungenfibrose beteiligten Zelltypen näher untersucht. Dazu wurde die Auflösung der reversiblen Bleomycin-induzierten Lungenschäden betrachtet. Der Verlust der beiden Myofibroblasten-Subtypen weist darauf hin, dass sie zwar die Effektorzellen der Wundheilungsreaktion, jedoch nicht an der Entstehung der chronisch manifesten Fibrose beteiligt sind. Perizyten proliferieren in Folge der Gabe von Bleomycin und sind vermehrt im Lungenparenchym auch nach Auflösung der Bleomycin-induzierten Lungenverletzung vorzufinden. Diese Ergebnisse führen zu der Annahme, dass es sich hierbei um die Effektorzellen der chronisch manifesten Lungenfibrose handelt, die durch eine Verdickung der Alveolarwand gekennzeichnet ist. Um die zellulären Mechanismen der Lungenfibrose umfassend aufzuklären, müssen weitere Untersuchungen an irreversiblen Fibrosemodellen folgen, die auch die chronischen Charakteristiken der Erkrankung berücksichtigen. / This project provides new insights into the pathogenesis of pulmonary fibrosis on the cellular level. Pulmonary fibrosis is a chronic disease characterized by signs of inflammation and the appearance of myofibroblasts that are responsible for excessive production of extracellular matrix (ECM). This leads to destroyed lung architecture, scar formation and reduced gas exchange. A modulatory role of nitric oxide (NO) in the development of pulmonary fibrosis has been proposed. However, the effector cells in the lung are remain elusive. The first part of the thesis focused on the localization of NO-sensitive guanylyl cyclase (NO-GC) in lung. Pericytes are the major NO-GC-expressing cell type in lung. Knock-out mice were generated lacking NO-GC globally (GCKO) as well as pericyte-specific GCKO mice (PDGFRβ-GCKO, SMMHC-GCKO, NG2-GCKO und SMMHC/NG2-GCKO). In addition, reporter mice were used that express tdTomato following cre-mediated recombination (PDGFRβ/tomato, SMMHC/tomato, NG2/tomato, FoxD1/tomato und Tie2/tomato). Immunohistochemical analysis shows the existence of two subpopulations of pericytes expressing NO-GC in lung: SMMHC/PDGFRβ-positive pericytes and a smaller subpopulation of NG2/PDGFRβ-positive pericytes. In the second part of the thesis, the role of NO-GC during bleomycin-induced lung injury was investigated. Bleomycin led to a fibrotic response in all genotypes as seen by an increase of lung weight and collagen content. Severity of lung injury in NO-GC-deficient mice was greater compared to wild type (WT) mice following instillation of bleomycin. These results indicate a possible role of NO-GC during bleomycin-induced lung injury. The development of pulmonary fibrosis is characterized by the formation of myofibroblasts that are known to be key players of wound healing and fibrotic processes. These cells do not occur under physiological conditions and their origin is still under debate. Lineage tracing of pericytes showed that NO-GC-expression allows to differentiate interstitial from intra-alveolar myofibroblasts: (1) NO-GC-positive, pericyte-derived myofibroblasts located in the alveolar wall and (2) NO-GC-negative, intra-alveolar myofibroblasts that are not derived from pericytes but, surprisingly, show de novo-expression of PDGFRβ after injury. The precursor cell type of intra-alveolar myofibroblasts is not identified yet. Pericytes, endothelial cells and fibrocytes do not transdifferentiate into myofibroblasts. Investigation of different cell types during resolution of lung fibrosis showed the disappearance of both types of myofibroblast. NO-GC-expressing pericytes that proliferate following administration of bleomycin are still present in an increased number. These results implicate a major role of myofibroblasts during wound healing responses but pericytes could be the effectors of chronic and manifest pulmonary fibrosis that is characterized by thickening of the alveolar wall. For a further understanding of the cellular mechanisms during pulmonary fibrosis investigations on irreversible models of fibrosis need to be performed.

Cardiac fibroblast to myofibroblast phenoconversion is a critical step during the development of cardiac fibrosis. Myofibroblasts chronically remodel extracellular matrix that results in myocardial stiffening, cardiac dysfunction and eventually heart failure. Previously we showed that Meox2, a homeobox transcription factor, can inhibit myofibroblast phenoconversion. Here we show that Zeb2, a repressor of Meox2, plays a crucial role during this phenoconversion process. Zeb2 overexpression significantly upregulates the expression of three key myofibroblast markers: α-SMA, SMemb and ED-A fibronectin in primary rat cardiac myofibroblast. We show that Zeb2 is highly expressed in myofibroblast nuclei whereas it is minimally expressed in fibroblast nuclei. Zeb2 overexpression in myofibroblasts results in a less migratory and more contractile mature myofibroblast phenotype. Moreover, Zeb2 overexpression represses Meox2 expression in endothelial cells. Thus, the current study enhances our understanding of the mechanism behind myofibroblast phenoconversion and provides a basis for developing Zeb2-based novel anti-fibrotic drug in the future. / February 2016

The trans-differentiation of injury-activated fibroblasts to myofibroblasts is a process that provides contractile strength for wound closure. Persistent myofibroblast differentiation, however, is associated with fibrotic pathologies such as organ fibrosis, vascular remodeling, and atherosclerotic plaque formation. Myofibroblasts acquire a contractile phenotype with biochemical properties characteristic of both smooth muscle cells and stromal fibroblasts. The cyto-contractile protein, smooth muscle α-actin (SMαA) is a biomarker of myofibroblast differentiation. Expression of the SMαA gene, ACTA2, is regulated by cis-acting elements and transcription factors that activate or repress the ACTA2 promoter. Purine-rich element binding proteins A (Purα) and B (Purβ) are sequence-specific, single-stranded DNA (ssDNA)/RNA-binding proteins that act as transcriptional repressors of ACTA2 expression. Both Pur proteins interact with the purine-rich strand of a cryptic muscle-CAT (MCAT) enhancer motif in 5'-flanking region of the ACTA2 promoter. Despite significant sequence homology with Purα, Purβ was identified as the dominant repressor of ACTA2 expression in mouse embryonic fibroblasts and vascular smooth muscle cells by virtue of gain-of function and loss-of-function analyses in cultured cells. Biophysical studies indicated that Purβ reversibly self-associates in solution to form a homodimer. Quantitative DNA-binding assays revealed that Purβ interacts with the purine-rich strand of the ACTA2 MCAT motif via a cooperative, multisite binding mechanism to form a high-affinity 2:1 Purβ-ssDNA complex. In this dissertation, a combination of computational, biochemical, and cell-based approaches were employed to elucidate the molecular basis of Purβ repressor interaction with the ACTA2 gene. Limited proteolysis of recombinant mouse Purβ in the presence and absence of the purine-rich strand of the ACTA2 MCAT element led to the identification of a core ssDNA-binding region that retains the ability to dimerize in solution. Knockdown of endogenous Purβ in mouse embryonic fibroblasts via RNA interference induced SMαA expression and conversion to a myofibroblast-like phenotype. To map the specific structural domains in the core region of Purβ that account for its unique ACTA2 repressor and ssDNA-binding functions, computational homology models of the Purβ monomer and dimer were generated based on the x-ray crystal structure of an intramolecular subdomain of Drosophila melanogaster Purα. Empirical biochemical and cell-based analyses of rationally-designed Purβ truncation proteins revealed that the assembled Purβ homodimer is composed of three separate purine-rich ssDNA-binding subdomains. Evaluation of the effects of anionic detergent and high-salt on the binding of Purβ to ssDNA implicated the involvement of hydrophobic and electrostatic interactions in mediating high-affinity nucleoprotein complex formation. This inference was validated by site-directed mutagenesis experiments, which identified several basic amino acid residues required for the ACTA2 repressor activity of Purβ. Collectively, the findings described herein establish the structural and chemical basis for the cooperative interaction of Purβ with the ACTA2 MCAT enhancer and for Purβ-dependent suppression of myofibroblast differentiation.

"During heart valve remodeling, and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype which is characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (alpha-SMA)-containing stress fibers, the hallmark of activated myofibroblasts, are also observed when VICs are placed under tension due to altered mechanical loading in vivo or during in vitro culture on stiff substrates or under high mechanical loads and in the presence of transforming growth factor-beta 1 (TGF-beta 1). The work presented herein describes three distinct model systems for application of controlled mechanical environment to VICs cultured in vitro. The first system uses polyacrylamide (PA) gels of defined stiffness to evaluate the response of VICs over a large range of stiffness levels and TGF-beta 1 concentration. The second system controls the boundary stiffness of cell-populated gels using springs of defined stiffness. The third system cyclically stretches soft or stiff two-dimensional (2D) gels while cells are cultured on the gel surface as it is deformed. Through the use of these model systems, we have found that the level of 2D stiffness required to maintain the quiescent VIC phenotype is potentially too low for a material to both act as matrix to support cell growth in the non-activated state and also to withstand the mechanical loading that occurs during the cardiac cycle. Further, we found that increasing the boundary stiffness on a three-dimensional (3D) cell populated collagen gel resulted in increased cellular contractile forces, alpha-SMA expression, and collagen gel (material)stiffness. Finally, VIC morphology is significantly altered in response to stiffness and stretch. On soft 2D substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates. Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. These studies provide critical information for characterizing how VICs respond to mechanical stimuli. Characterization of these responses is important for the development of tissue engineered heart valves and contributes to the understanding of the role of mechanical cues on valve pathology and disease onset and progression. While this work is focused on valvular interstitial cells, the culture conditions and methods for applying mechanical stimulation could be applied to numerous other adherent cell types providing information on the response to mechanical stimuli relevant for optimizing cell culture, engineered tissues or fundamental research of disease states."

Epithelial mesenchymal/-myofibroblast transition (EMT/EMyT) has emerged as one of the central mechanisms in wound healing and tissue fibrosis. The main feature of EMyT is the activation of a myogenic program, leading to the induction of the α-smooth-muscle actin (SMA) gene in the transitioning epithelium. Recent research suggests that intercellular contacts are not merely passive targets, but are active contributors to EMT/EMyT. Indeed, our group showed previously that contact uncoupling or injury is necessary for TGFβ to induce EMyT (two-hit paradigm). Further, our previous work also revealed that Smad3, the main TGFβ-regulated transcription factor, binds to the Myocardin Related Transcription Factor (MRTF), the prime driver of SMA promoter, and inhibits MRTF’s transcriptional activity. During EMyT, Smad3 eventually degrades, which liberates the MRTF-driven myogenic program. However the mechanisms whereby cell contacts regulate the fate of Smad3 and MRTF during EMyT are poorly understood. Accordingly, the central aim of my studies was to explore the role of intercellular contacts, in particular that of Adherens Junction (AJs) in the induction of the myogenic reprogramming of the injured epithelium. This thesis describes two novel molecular mechanisms through which AJs impact EMyT. In the first part, we show β-catenin, an AJs component and transcriptional co-activator counteracts the inhibitory action of Smad3 on MRTF. Moreover we reveal that β-catenin is necessary to maintain MRTF stability via protecting MRTF from proteasomal degradation. Thus, β-catenin is an indispensable permissive factor for SMA expression. In the second part, we demonstrate that contact injury and TGFβ suppress the expression of the phosphatase PTEN. EMyT-related reduction or absence of PTEN potentiates Smad3 degradation. EMyT is associated with enhanced phosphorylation of the T179 residue in Smad3 linker region, and this event is necessary for Smad3 degradation. PTEN silencing increases the stimulatory effect of contact uncoupling and TGFβ on SMA promoter activity and SMA protein expression. Thus, the integrity of intercellular contacts regulates the level of PTEN, which in turn controls Smad3 stability through impacting on T179 phosphorylation. This new knowledge holds promises for targeted therapies and more effective prevention of the currently incurable fibroproliferative and fibrocontractile diseases.