Smad Transcriptional Activity Research Articles

Emodin suppresses activation of hepatic stellate cells through p38 mitogen-activated protein kinase and Smad signaling pathways in vitro.

The aim of this study was to evaluate the hypothesis that emodin inhibits extracellular matrix (ECM)-related gene expression in activated hepatic stellate cells (HSCs) by blocking canonical or/and noncanonical components of transforming growth factor β1 (TGFβ1) intracellular signaling. Here, we demonstrate that emodin suppressed the gene expression of HSCs activation markers type I collagen, fibronectin, and α-smooth muscle actin, as well as HSCs proliferation. Mechanistically, emodin suppresses TGFβ1, TGFβ receptor II, TGFβ receptor I, and Smad4 gene expression, as well as Smad luciferase activity. Simultaneously, emodin reduced p38 mitogen-activated protein kinase (p38MAPK ) activity but not c-Jun N-terminal kinases and extracellular signal-regulated kinases 1 and 2 phosphorylation in HSC-T6 cells. Interestingly, deprivation of TGFβ using a neutralizing antibody abolished emodin-mediated inhibitions of the both Smad transcriptional activity and p38MAPK phosphorylation. Furthermore, emodin-mediated inhibition of HSCs activation could be partially blocked by PD98059 inhibition of p38MAPK or short hairpin RNA-imposed knockdown of Smad4. Conversely, simultaneous inhibition of Smad4 and p38MAPK pathways completely reverses the effects of emodin, suggesting that Smad and p38MAPK locate downstream of TGFβ1 and regulate collagen genes expression in HSCs. Collectively, these data suggest that emodin is a promising candidate for the treatment of hepatic fibrosis.

Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System.

Transforming Growth Factor β (TGF-β) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-β is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-β/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-β displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-β signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-β signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway <em>In Vitro</em> and <em>In Vivo</em> Using an Adenovirus Reporter System

Transforming Growth Factor β (TGF-β) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-β is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-β/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-β displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-β signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-β signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Mediator kinase CDK8/CDK19 drives YAP1-dependent BMP4-induced EMT in cancer.

CDK8 is a transcription-regulating kinase that controls TGF-β/BMP-responsive SMAD transcriptional activation and turnover through YAP1 recruitment. However, how the CDK8/YAP1 pathway influences SMAD1 response in cancer remains unclear. Here we report that SMAD1-driven epithelial-to-mesenchymal transition (EMT) is critically dependent on matrix rigidity and YAP1 in a wide spectrum of cancer models. We find that both genetic and pharmacological inhibition of CDK8 and its homologous twin kinase CDK19 leads to abrogation of BMP-induced EMT. Notably, selectively blocking CDK8/19 specifically abrogates tumor cell invasion, changes in EMT-associated transcription factors, E-cadherin expression and YAP nuclear localization both in vitro and in vivo in a murine syngeneic EMT model. Furthermore, RNA-seq meta-analysis reveals a direct correlation between CDK8 and EMT-associated transcription factors in patients. Our findings demonstrate that CDK8, an emerging therapeutic target, coordinates growth factor and mechanical cues during EMT and invasion.

YAP/TAZ regulates TGF-\u03b2/Smad3 signaling by induction of Smad7 via AP-1 in human skin dermal fibroblasts

BackgroundTranscription factors YAP and TAZ function as the primary mediators of the Hippo pathway. Yet, crosstalk of YAP and TAZ with other signaling pathways remains relatively unexplored. We have explored the impact of YAP and TAZ levels on the TGF-β/Smad signaling pathway in human skin dermal fibroblasts.MethodsYAP and TAZ levels in dermal fibroblasts were reduced in dermal fibroblasts by siRNA-mediated knockdown. The effects of YAP and TAZ reduction on TGF-β/Smad signaling were examined by quantitative real-time PCR, Western analysis, and immunostaining. Luciferase reporter assays and electrophoretic mobility shift assays were conducted to investigate the transcription factor DNA-binding and transcriptional activities.ResultsKnockdown of both YAP and TAZ (YAP/TAZ), but not either separately, impaired TGF-β1-induced Smad3 phosphorylation and Smad3 transcriptional activity, thereby inhibiting the expression of TGF-β target genes. This reduction by reduced levels of YAP/TAZ results from induction of inhibitory Smad7, which inhibits Smad3 phosphorylation and activity by TGF-β1. Conversely, prevention of Smad7 induction restores Smad3 phosphorylation and Smad3 transcriptional activity in fibroblasts that have reduced YAP/TAZ. In agreement with these findings, inhibition of YAP/TAZ transcriptional activity, similar to the reduction of YAP/TAZ levels, also significantly induced Smad7 and impaired TGF-β/Smad signaling. Further investigations revealed that reduced levels of YAP/TAZ led to induction of activator protein-1 (AP-1) activity, Activated AP-1 bound to DNA sequences in the Smad7 gene promoter, and deletion of these AP-1 binding sequences substantially reduced Smad7 promoter reporter activity.ConclusionYAP/TAZ functions in concert with transcription factor AP-1 and Smad7 to regulate TGF-β signaling, in human dermal fibroblasts. Reduction of YAP/TAZ levels leads to activation of AP-1 activity, which induces Smad7. Smad7 suppresses the TGF-β pathway.

Metformin suppresses melanoma progression by inhibiting KAT5-mediated SMAD3 acetylation, transcriptional activity and TRIB3 expression.

Metformin has beneficial effects of preventing and treating cancers on type 2 diabetic patients. However, the role of metformin in non-diabetic cancer patients and the precise molecular mechanisms against cancer have not yet been sufficiently elucidated. We recently reported that the pseudokinase protein TRIB3 acts as a stress sensor linking metabolic stressors to cancer promotion by inhibiting autophagy and ubiquitin-proteasomal degradation systems; genetically abrogating of TRIB3 expression reduces tumourigenesis and cancer progression. Thus, TRIB3 is a potential therapeutic target for diverse cancers. In this study, we found that metformin attenuates melanoma growth and metastasis by reducing TRIB3 expression in non-diabetic C57BL/6 mice and diabetic KK-Ay mice; overexpression of TRIB3 protects metformin from the activation of autophagic flux, the clearance of accumulated tumour-promoting factors and the attenuation of tumour progression. We further elucidated that TRIB3 acts as an adaptor to recruit lysine acetyltransferase 5 (KAT5) to SMAD3 and induce a phosphorylation-dependent K333 acetylation of SMAD3, which sustains transcriptional activity of SMAD3 and subsequently enhances TRIB3 transcription. Metformin suppresses SMAD3 phosphorylation and decreases the KAT5/SMAD3 interaction, to attenuate the KAT5-mediated K333 acetylation of SMAD3, reduce the SMAD3 transcriptional activity and subsequent TRIB3 expression, thereby antagonizes melanoma progression. Together, our study not only defines a molecular mechanism by which metformin protects against melanoma progression by disturbing the KAT5/TRIB3/SMAD3 positive feedback loop in diabetes and non-diabetes mice, but also suggests a candidate diverse utility of metformin in tumour prevention and therapy because of suppressing stress protein TRIB3 expression.

The methyltransferase SET9 regulates TGFB1 activation of renal fibroblasts via interaction with SMAD3.

Chronic kidney disease (CKD) is a global socioeconomic problem. It is characterised by the presence of differentiated myofibroblasts, which cause tissue fibrosis in response to TGFB1, leading to renal failure. Here, we define a novel interaction between the SET9 lysine methyltransferase (also known as SETD7) and SMAD3, the principal mediator of TGFB1 signalling in myofibroblasts. We show that SET9-deficient fibroblasts exhibit globally altered gene expression profiles in response to TGFB1, whilst overexpression of SET9 enhances SMAD3 transcriptional activity. We also show that SET9 facilitates nuclear import of SMAD3 and controls SMAD3 protein degradation via ubiquitylation. On a cellular level, we demonstrate that SET9 is broadly required for the effects of TGFB1 in diseased primary renal fibroblasts; SET9 promotes fibroblast migration into wounds, expression of extracellular matrix proteins, collagen contractility and myofibroblast differentiation. Finally, we demonstrate that SET9 is recruited to the α-smooth muscle actin gene in response to TGFB1, providing a mechanism by which SET9 regulates myofibroblast contractility and differentiation. Together with previous studies, we make the case for SET9 inhibition in the treatment of progressive CKD.

Interleukin 1 β-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFβ signaling in primary human mesenchymal stem cells

BackgroundChondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications. ResultsCo-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA12-luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2–4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1β-induced SMAD2 linker modifications and CAGA12-luciferase activity. ConclusionsTGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1β-induced TGFβ signal modulation.

ENMD-1068 inhibits liver fibrosis through attenuation of TGF-\u03b21/Smad2/3 signaling in mice

Protease-activated receptor 2 (PAR-2) plays an important role in the pathogenesis of liver fibrosis. We studied the effect of N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068), a PAR-2 antagonist, on the development of CCl4-induced liver fibrosis in mice and activation of hepatic stellate cells (HSCs) isolated from the mice. Before CCl4 injection, the mice were injected intraperitoneally with either 25 mg/kg or 50 mg/kg ENMD-1068 or with 200 μL of the vehicle control twice per week for 4 weeks. The isolated HSCs were stimulated by TGF-β1 with or without ENMD-1068 to evaluate the role of PAR-2 in TGF-β1 induced HSCs activation and collagen production. We showed that the levels of ALT/AST, collagen content, and α-smooth muscle actin (α-SMA) were significantly reduced by treatment with ENMD-1068 in CCl4-induced fibrotic mice. Interestingly, we found TGF-β1 signaling-related expression levels of α-SMA, type I and III collagen, and C-terminal phosphorylation of Smad2/3 were significantly decreased in the ENMD-1068 treated HSCs. Moreover, we showed ENMD-1068 treatment inhibited trypsin or SLIGRL-NH2 stimulated calcium release and TGF-β1 induced Smad transcriptional activity in HSCs. We demonstrated that ENMD-1068 reduces HSCs activation and collagen expression through the inhibiton of TGF-β1/Smad signal transduction.

A GEF activity-independent function for nuclear Net1 in Nodal signal transduction and mesendoderm formation.

Net1 is a well-characterized oncoprotein with RhoA-specific GEF activity. Oncogenic Net1, which lacks the first 145 amino acids, is present in the cytosol and contributes to the efficient activation of RhoA and the formation of actin stress fibers in a number of tumor cell types. Meanwhile, wild-type Net1 is predominantly localized in the nucleus at steady state due to its N-terminal nuclear localization sequences, where the function of nuclear Net1 has not been fully determined. Here, we find that zebrafish net1 is expressed specifically in mesendoderm precursors during gastrulation. Endogenous Net1 is located in the nucleus during early embryonic development. Gain- and loss-of-function experiments in zebrafish embryos and mammalian cells demonstrate that, regardless of its GEF activity, nuclear Net1 is critical for zebrafish mesendoderm formation and Nodal signal transduction. Detailed analyses of protein interactions reveal that Net1 associates with Smad2 in the nucleus in a GEF-independent manner, and then promotes Smad2 activation by enhancing recruitment of p300 (also known as EP300) to the transcriptional complex. These findings describe a novel genetic mechanism by which nuclear Net1 facilitates Smad2 transcriptional activity to guide mesendoderm development.

Structural Basis of Intracellular TGF-β Signaling: Receptors and Smads.

Stimulation of the transforming growth factor β (TGF-β) family receptors activates an intracellular phosphorylation-dependent signaling cascade that culminates in Smad transcriptional activation and turnover. Structural studies have identified a number of allosteric mechanisms that control the localization, conformation, and oligomeric state of the receptors and Smads. Such mechanisms dictate the ordered binding of substrate and adaptor proteins that determine the directionality of the signaling process. Activation of the pathway has been illustrated by the various structures of the receptor-activated Smads (R-Smads) with SARA, Smad4, and YAP, respectively, whereas mechanisms of down-regulation have been elucidated by the structural complexes of FKBP12, Ski, and Smurf1. Interesting parallels have emerged between the R-Smads and the Forkhead-associated (FHA) and interferon regulatory factor (IRF)-associated domains, as well as the Hippo pathway. However, important questions remain as to the mechanism of Smad-independent signaling.

Tumor suppressor bromodomain-containing protein 7 cooperates with Smads to promote transforming growth factor-β responses.

Smad proteins are central mediators in the canonical transforming growth factor-β (TGF-β) signaling pathway in mammalian cells. We report here that bromodomain-containing protein 7 (BRD7) functions as a novel transcription coactivator for Smads in TGF-β signaling. BRD7 forms a TGF-β inducible complex with Smad3/4 through its N-terminal Smad-binding domain. BRD7 simultaneously binds to acetylated histones to promote Smad-chromatin association, and associates with histone acetyltransferase p300 to enhance Smad transcriptional activity. Ectopic expression of BRD7, but not its mutants defective in Smad binding, enhances TGF-β transcriptional, tumor suppressing and epithelial-mesenchymal transition (EMT) responses. Conversely, depletion of BRD7 inhibits TGF-β responses. Thus, our study provides compelling evidence for a new function of BRD7 in fine-tuning TGF-β physiological responses.

Abstract P5-04-13: Pin1 negatively impacts Smad3 tumor suppression in triple negative breast cancer cell lines

Abstract Introduction: Triple negative breast cancer (TNBC) is an aggressive subtype associated with poor outcomes. Accordingly, there is an urgent need to develop novel and targeted therapeutics for patients with this disease subtype. Cyclins D and E and the corresponding activation of CDK4/2 represent promising therapeutic targets for the treatment of TNBC. CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, to promote the transition from tumor suppressive to oncogenic TGFβ activity in cyclin-overexpressing breast cancers. We identified a Smad3 interaction with Pin1, a cis-trans isomerase also overexpressed in aggressive breast cancers and associated with CDK-mediated Smad3 phosphorylation. Smad3 interaction with Pin1 can influence protein function and fidelity through recruitment of Smurf2 and subsequent proteasomal degradation. Based on these findings, we hypothesized that inhibition of the CDK-mediated Smad3-Pin1 interaction would stabilize Smad3 protein expression and restore tumor-suppressive Smad3 activity. Methods: Pin1 expression was knocked-down (KD) in MDA-MB-231 TNBC cells by transfecting with Pin1-targeting siRNA (siPin1) or control non-specific siRNA (siNS). KD efficiency was confirmed by immunoblotting. To assay Smad3 transcriptional activity with Pin1 KD, luciferase reporter studies were performed. Also, following Pin1 KD, immunoblotting was used to determine expression of Smad3 and associated protein targets. MTS assays were utilized to determine cellular proliferation after Pin1 KD. Transwell migration assays were used to assay the effect of Pin1 KD or CDK2 inhibitor treatment, which blocked non-canonical Smad3 Thr179 phosphorylation, on TNBC cell migration. Results: KD of Pin1 expression in TNBC cell lines resulted in an increase in Smad3 transcriptional activity compared to control cells, and correlated with an increase in expression of cdki p15 and a decrease in c-myc, Smad3-target genes and cell cycle regulators. Additionally, Pin1 KD resulted in a significant decrease in TNBC cell proliferation compared to siNS control TNBC cells. Smad3 protein levels increased following Pin1 KD, suggesting Pin1 action may negatively impact Smad3 stability. We also found that KD of Pin1 or treatment with a CDK2 inhibitor, which blocked Smad3 noncanonical Thr179 phosphorylation, resulted in significantly reduced TNBC cell migration. Conclusions: Inhibiting the Smad3-Pin1 interaction by knock-down of Pin1 expression in TNBC cells restored Smad3 transcriptional activity, which correlated to an increase in expression of the Smad3 associated protein cdki p15, decrease in c-myc, and a decrease in cellular proliferation. Additionally, Pin1 KD enhanced Smad3 protein levels, suggesting a role of Pin1 in mediating Smad3 stability. Inhibiting the Smad3-Pin1 interaction with Pin1 KD or CDK2 inhibitor treatment also reduced TNBC cell migration. Collectively, these data suggest that the Smad3-Pin1 interaction, facilitated by noncanonical CDK-mediated Smad3 phosphorylation, is associated with pro-tumorigenic and pro-migratory TGFβ signaling, and inhibition of this interaction may provide an important therapeutic option for TNBC patients. Citation Format: Thomas AL, Hamdan R, Hong A, Rosenthal E, Thomas AJ, Jeruss JS. Pin1 negatively impacts Smad3 tumor suppression in triple negative breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-13.

Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome.

Nucleoporins (NUPs) are essential components of the nuclear pore complex (NPC).1 Only few diseases have been attributed to NPC dysfunction.2-4 Steroid resistant nephrotic syndrome (SRNS), a frequent cause of chronic kidney disease, is caused by dysfunction of glomerular podocytes.5 Here we identify in 8 families with SRNS mutations of NUP93, its interaction partner NUP205, or exportin5 (XPO5) as a hitherto unrecognized monogenic cause of SRNS. NUP93 mutations caused disrupted NPC assembly. NUP93 knockdown reduced the presence of NUP205 in the NPC and, reciprocally, a NUP205 mutation abrogated NUP93 interaction. We demonstrate that NUP93 and XPO5 interact with the signaling protein SMAD4, and that NUP93 mutations abrogated interaction with SMAD4. Significantly, NUP93 mutations interfered with BMP7-induced SMAD transcriptional reporter activity. We hereby demonstrate that mutations of NUPs cause a distinct renal disease, and reveal SMAD signaling as a novel disease mechanism of SRNS, opening a potential new avenue for treatment.

Epigenetic regulators, including SETD7, as new targets for the treatment of chronic kidney disease

BackgroundFibrosis is common to all forms of chronic kidney disease and leads to kidney failure. 1·3% of the National Health Service budget and 7% of the US Medicare budget is spent on managing complications of the disease, yet there is no available treatment for the underlying fibrosis. We studied specific epigenetic regulators, including SETD7, to provide a paradigm for these enzymes as future targets in the treatment of chronic kidney disease. MethodsHuman renal fibroblasts (obtained after ethics approval), mesangial cells, and mouse embryonic fibroblasts were used to study myofibroblast transformation, expression of extracellular matrix proteins by immunofluorescence, wound healing, and global gene expression with whole-genome microarrays (Illumina, San Diego, CA, USA). Tubular epithelial cells were used to study the SETD7 lysine methyltransferase interaction with SMAD3 by immunoprecipitation, SMAD3 stability, and SMAD3 nuclear import. FindingsSETD7-deficient cells failed to undergo myofibroblast transformation in response to profibrotic transforming growth factor β1 (TGF β1). They also expressed lower concentrations of fibrotic collagen and fibronectin-1 than SETD7-proficient cells and showed impaired wound healing. In response to TGFβ1, SMAD3 interacted with SETD7 resulting in enhanced SMAD3 stability in cells. Additionally, SMAD3 nuclear import was dependent on SETD7, as determined by immunofluorescence and subcellular fractionation. SETD7 overexpression enhanced SMAD3 transcriptional activity on natural (plasminogen activator inhibitor-1) and synthetic (CAGA) cis-regulatory elements, whereas SETD7 depletion reduced SMAD3 transcriptional activity, as demonstrated in reporter gene assays. Gene Ontology analysis indicated that SETD7 regulated expression of profibrotic genes. InterpretationWe conclude that SETD7 is crucial in the development of the fibrotic phenotype that characterises chronic kidney disease and other fibrotic disorders. Pharmacological inhibition of this epigenetic regulator might therefore be of clinical use in this context. Our development of in-vitro models to study fibrosis, including human primary kidney fibroblasts, should overcome translational difficulties with animal models, thereby allowing further assessment of epigenetic enzymes as targets in the treatment of chronic kidney disease. FundingNorthern Counties Kidney Research Fund, Academy of Medical Sciences.

ELL Protein-associated Factor 2 (EAF2) Inhibits Transforming Growth Factor β Signaling through a Direct Interaction with Smad3

A series of in vitro and in vivo studies has shown that EAF2 can affect multiple signaling pathways involved in cellular processes. However, the molecular mechanisms underlying its effects have remained elusive. Here we report the discovery of a new functional link between EAF2 and TGF-β signaling. Promoter reporter assays indicated that EAF2 suppresses Smad3 transcriptional activity, resulting in inhibition of TGF-β signaling. Coimmunoprecipitation assays showed that EAF2 specifically interacts with Smad3 in vitro and in vivo but not with other Smad proteins. In addition, we observed that EAF2 binding does not alter Smad3 phosphorylation but causes Smad3 cytoplasmic retention, competes with Smad4 for binding to Smad3, and prevents p300-Smad3 complex formation. Furthermore, we demonstrated that EAF2 suppresses both TGF-β-induced G1 cell cycle arrest and TGF-β-induced cell migration. This study identifies and characterizes a novel repressor of TGF-β signaling.

Ionizing radiation induces a motile phenotype in human carcinoma cells in vitro through hyperactivation of the TGF-beta signaling pathway.

Radiotherapy, a major treatment modality against cancer, can lead to secondary malignancies but it is uncertain as to whether tumor cells that survive ionizing radiation (IR) treatment undergo epithelial-mesenchymal transition (EMT) and eventually become invasive or metastatic. Here, we have tested the hypothesis that the application of IR (10MeV photon beams, 2-20Gy) to lung and pancreatic carcinoma cells induces a migratory/invasive phenotype in these cells by hyperactivation of TGF-β and/or activin signaling. In accordance with this assumption, IR induced gene expression patterns and migratory responses consistent with an EMT phenotype. Moreover, in A549 cells, IR triggered the synthesis and secretion of both TGF-β1 and activin A as well as activation of intracellular TGF-β/activin signaling as evidenced by Smad phosphorylation and transcriptional activation of a TGF-β-responsive reporter gene. These responses were sensitive to SB431542, an inhibitor of type I receptors for TGF-β and activin. Likewise, specific antibody-mediated neutralization of soluble TGF-β, or dominant-negative inhibition of the TGF-β receptors, but not the activin type I receptor, alleviated IR-induced cell migration. Moreover, the TGF-β-specific approaches also blocked IR-dependent TGF-β1 secretion, Smad phosphorylation, and reporter gene activity, collectively indicating that autocrine production of TGF-β(s) and subsequent activation of TGF-β rather than activin signaling drives these changes. IR strongly sensitized cells to further increase their migration in response to recombinant TGF-β1 and this was accompanied by upregulation of TGF-β receptor expression. Our data raise the possibility that hyperactivation of TGF-β signaling during radiotherapy contributes to EMT-associated changes like metastasis, cancer stem cell formation and chemoresistance of tumor cells.

Ferulic acid suppresses activation of hepatic stellate cells through ERK1/2 and Smad signaling pathways in vitro

Hepatic stellate cells (HSCs) are the primary source of matrix components in hepatic fibrosis. Ferulic acid (FA) has antifibrotic potential in renal and cardiac disease. However, whether FA comprises inhibitive effects of HSCs activation remains to be clarified. This study aims at evaluating the hypothesis that FA inhibits extracellular matrix (ECM)-related gene expression by the interruption of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) or/and Smad signaling pathways in HSC-T6. Our results indicated that FA significantly inhibited both viability and activation of HSC-T6 cells in vitro. In addition, we demonstrated, for the first time, that FA dramatically inhibited the expression of α1(I) collagen (Col-I) and fibronectin at levels of transcription and translation. Moreover, FA treatment inhibited Smad transcriptional activity, as evaluated by transient transfection with a plasmid construction containing SMAD response element and the luciferase reporter gene. Furthermore, FA inhibition of HSCs activation involved in both focal adhesion kinase (FAK)-dependent ERK1/2 and Smad signaling pathways with independent manner. Blocking transforming growth factor-β by a neutralizing antibody caused a marked reduction in both ERK1/2 and Smad signaling. These results support FA as an effective therapeutic agent for the prevention and treatment of hepatic fibrosis.

SREBP-1 is a novel mediator of TGFβ1 signaling in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent studies showed that overexpression of the transcription factor SREBP-1 induces glomerulosclerosis. TGFβ1 is a key profibrotic mediator of glomerulosclerosis, but whether SREBP-1 regulates its effects is unknown. In kidney mesangial cells and in vivo, TGFβ1 activates SREBP-1. This requires SCAP, S1P, and PI3K/Akt signaling, but is independent of Smad3. Activation of the TGFβ1-responsive reporter plasmid p3TP-lux requires SREBP-1a, but not SREBP-1c, binding to an E-box adjacent to a Smad-binding element. SREBP-1a overexpression alone activates p3TP-lux. Smad3 is required for SREBP-1a transcriptional activation and TGFβ1 induces association between the two transcription factors. SREBP-1a K333 acetylation by the acetyltransferase CBP is required for Smad3 association and SREBP-1 transcriptional activity, and is also required for Smad3 transcriptional activity. Thus, both Smad3 and SREBP-1a activation cooperatively regulate TGFβ transcriptional responses. SREBP-1 inhibition provides a novel therapeutic strategy for diabetic kidney disease.

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development

TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left–right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate.