Smad-independent Manner Research Articles

Activation of TGF-β - SMAD2 signaling by IL-6 drives neuroendocrine differentiation of prostate cancer through p38MAPK

Neuroendocrine prostate cancer (NEPC) is an aggressive, androgen independent PCa and it is detected in patients undergoing androgen deprivation therapy (ADT). Interleukin-6 (IL-6) is a pleiotropic cytokine elevated in PCa patients promotes neuroendocrine differentiation (NED). In this study, PCa cells were differentiated with IL-6 in in-vitro to identify novel targets or signaling pathways associated with emergence of NEPC on deprivation of androgens. From the results, we observed an activation of TGF-β signaling pathway is altered through multiple proteins in differentiated LNCaP cells. Hence, we investigated the role of TGF-β axis in PCa cells differentiation. LNCaP cells treated with IL-6 in androgens deprived media release excess TGF-β ligand and this as conditioned media added to cells stimulated NED of PCa cells. TGF-β released by IL-6 stimulated cells activate p38MAPK through SMAD2 thereby promote NED. Inhibition of TGF-βRI and TGF-βRII signaling activation in LNCaP cells treated with IL-6 did not reversed the NED of cells, possibly due to the reason that the inhibition of TGF-β axis is further activating p38MAPK through SMAD independent manner in PCa cells. However, siRNA mediated knock down or inhibition p38MAPK inactivated TGF-β – SMAD axis in differentiating cells and attenuated NED of LNCaP cells. This result suggests that p38MAPK is the central node for receiving IL-6 signals and promotes NED of LNCaP cells in androgens free media. Remarkably, downregulation or inhibition of p38MAPK in NCI-H660 reversed NED characteristics as well as markers along with inactivation of SMAD2 whereas no effect observed in WPMY-1 normal prostate cells. Taken together these findings unveil that p38MAPK and its upstream regulators are potential targets to overcome the progression of NED of PCa and develop novel therapeutic measures along ADT for effective treatment of PCa.

Periostin gene expression in neu-positive breast cancer cells is regulated by a FGFR signaling cross talk with TGF\u03b2/PI3K/AKT pathways

BackgroundBreast cancer is a highly heterogeneous disease with multiple drivers and complex regulatory networks. Periostin (Postn) is a matricellular protein involved in a plethora of cancer types and other diseases. Postn has been shown to be involved in various processes of tumor development, such as angiogenesis, invasion, cell survival and metastasis. The expression of Postn in breast cancer cells has been correlated with a more aggressive phenotype. Despite extensive research, it remains unclear how epithelial cancer cells regulate Postn expression.MethodsUsing murine tumor models and human TMAs, we have assessed the proportion of tumor samples that have acquired Postn expression in tumor cells. Using biochemical approaches and tumor cell lines derived from Neu+ murine primary tumors, we have identified major regulators of Postn gene expression in breast cancer cell lines.ResultsHere, we show that, while the stromal compartment typically always expresses Postn, about 50% of breast tumors acquire Postn expression in the epithelial tumor cells. Furthermore, using an in vitro model, we show a cross-regulation between FGFR, TGFβ and PI3K/AKT pathways to regulate Postn expression. In HER2-positive murine breast cancer cells, we found that basic FGF can repress Postn expression through a PKC-dependent pathway, while TGFβ can induce Postn expression in a SMAD-independent manner. Postn induction following the removal of the FGF-suppressive signal is dependent on PI3K/AKT signaling.ConclusionOverall, these results reveal a novel regulatory mechanism and shed light on how breast tumor cells acquire Postn expression. This complex regulation is likely to be cell type and cancer specific as well as have important therapeutic implications.

Modulation of EndMT by Hydrogen Sulfide in the Prevention of Cardiovascular Fibrosis.

Endothelial mesenchymal transition (EndMT) has been described as a fundamental process during embryogenesis; however, it can occur also in adult age, underlying pathological events, including fibrosis. Indeed, during EndMT, the endothelial cells lose their specific markers, such as vascular endothelial cadherin (VE-cadherin), and acquire a mesenchymal phenotype, expressing specific products, such as α-smooth muscle actin (α-SMA) and type I collagen; moreover, the integrity of the endothelium is disrupted, and cells show a migratory, invasive and proliferative phenotype. Several stimuli can trigger this transition, but transforming growth factor (TGF-β1) is considered the most relevant. EndMT can proceed in a canonical smad-dependent or non-canonical smad-independent manner and ultimately regulate gene expression of pro-fibrotic machinery. These events lead to endothelial dysfunction and atherosclerosis at the vascular level as well as myocardial hypertrophy and fibrosis. Indeed, EndMT is the mechanism which promotes the progression of cardiovascular disorders following hypertension, diabetes, heart failure and also ageing. In this scenario, hydrogen sulfide (H2S) has been widely described for its preventive properties, but its role in EndMT is poorly investigated. This review is focused on the evaluation of the putative role of H2S in the EndMT process.

Noncanonical TGF-β signaling leads to FBXO3-mediated degradation of ΔNp63α promoting breast cancer metastasis and poor clinical prognosis.

Transforming growth factor-β (TGF-β) signaling plays a critical role in promoting epithelial-to-mesenchymal transition (EMT), cell migration, invasion, and tumor metastasis. ΔNp63α, the major isoform of p63 protein expressed in epithelial cells, is a key transcriptional regulator of cell adhesion program and functions as a critical metastasis suppressor. It has been documented that the expression of ΔNp63α is tightly controlled by oncogenic signaling and is frequently reduced in advanced cancers. However, whether TGF-β signaling regulates ΔNp63α expression in promoting metastasis is largely unclear. In this study, we demonstrate that activation of TGF-β signaling leads to stabilization of E3 ubiquitin ligase FBXO3, which, in turn, targets ΔNp63α for proteasomal degradation in a Smad-independent but Erk-dependent manner. Knockdown of FBXO3 or restoration of ΔNp63α expression effectively rescues TGF-β-induced EMT, cell motility, and tumor metastasis in vitro and in vivo. Furthermore, clinical analyses reveal a significant correlation among TGF-β receptor I (TβRI), FBXO3, and p63 protein expression and that high expression of TβRI/FBXO3 and low expression of p63 are associated with poor recurrence-free survival (RFS). Together, these results demonstrate that FBXO3 facilitates ΔNp63α degradation to empower TGF-β signaling in promoting tumor metastasis and that the TβRI-FBXO3-ΔNp63α axis is critically important in breast cancer development and clinical prognosis. This study suggests that FBXO3 may be a potential therapeutic target for advanced breast cancer treatment.

Curcumin attenuates IL-17A mediated pulmonary SMAD dependent and non-dependent mechanism during acute lung injury in vivo.

Acute lung injury (ALI) is a pathologic condition responsible for incurable human chronic respiratory diseases. Recent studies have shown the involvement of the glycoprotein, IL17A secreted by IL-17 producing cells in chronic inflammation. The current investigation was carried out to study the IL-17A mediated activation of SMAD and non- SMAD signaling in alveolar epithelial cells and to assess the putative modulatory role of curcumin. C57BL/6 mice were exposed to IL-17A and curcumin was administered as an intervention to modulate the IL-17A-induced alveolar damage. Techniques like Immunofluorescence and real-time PCR were used. We found elevated expression of IL-17A and IL-17A-associated signaling pathways to be activated in mice lung tissues. Curcumin intervention in vivo promoted the resolution of IL-17A-induced ALI and attenuated pulmonary damage. Increase phosphorylation of non- SMAD proteins like P-EGFR, P-STAT-1, STAT-3, P-JAK-1/2, P-JNK, and also SMAD proteins like P- SMAD 2/3 and TGF-β1 was encountered upon IL-17A exposure, while curcumin intervention reversed the protein expression levels. Curcumin was found to block mRNA expressions of non- SMAD genes EGFR, JNK-1, JAK1, JAK2, STAT-1, STAT-3, MAPK14, also of TGF-β1 and SMAD genes like SMAD 2, SMAD 3. However, mRNA expressions of SMAD 6 and SMAD 7 were increased upon curcumin intervention. Our study indicates that IL-17A participates in the development of ALI in both SMAD dependent and independent manner and the IL-17A signaling components were effectively controlled by curcumin, suggesting probable anti-inflammatory use of curcumin during ALI.

The non-neuronal cyclin-dependent kinase 5 is a fibrotic mediator potentially implicated in systemic sclerosis and a novel therapeutic target.

The mechanisms underlying persistent fibroblast activation and myofibroblast phenoconversion in underlying multi-organ fibrosis in systemic sclerosis (SSc) remain incompletely understood, hindering effective therapies to slow or reverse the process. Cyclin-dependent kinase 5 (CDK5) is a pleiotropic member of the CDK family originally identified in neuronal cells. In contrast to other CDKs, CDK5 activity depends on its CDK5R1 subunit p35. Here we demonstrate that expression of p35 and CDK5 activity are induced by TGF-ß in fibroblasts and adipocytic cell types. Levels of p35 are markedly elevated in both SSc skin biopsies and explanted SSc fibroblasts, as well as in fibrotic skin in mice. Ectopic p35 and CDK5 suppressed adipogenic markers while stimulating collagen production and myofibroblast markers, whereas RNAi-mediated CDK5 knockdown abrogated TGF-β fibrotic responses in a Smad-independent manner. Pharmacological inhibitors of CDK5 likewise prevented and reversed TGF-β responses in fibroblast monolayers and in ex vivo human skin organ cultures, ameliorated collagen overproduction in SSc fibroblasts, and prevented and reversed skin fibrosis in two distinct mouse models of SSc. Together, these results reveal a previously unrecognized key function for p35/CDK5 as a mediator of mesenchymal cell fibrotic responses. The results suggest a potential pathogenic role for elevated p35 expression and CDK5 activity in SSc, and raise the possibility that their selective pharmacological targeting might represent a novel treatment approach in fibrosis.

TGF-β triggers rapid fibrillogenesis via a novel TβRII-dependent fibronectin-trafficking mechanism.

Fibronectin (FN) is a critical regulator of extracellular matrix (ECM) remodeling through its availability and stepwise polymerization for fibrillogenesis. Availability of FN is regulated by its synthesis and turnover, and fibrillogenesis is a multistep, integrin-dependent process essential for cell migration, proliferation, and tissue function. Transforming growth factor β (TGF-β) is an established regulator of ECM remodeling via transcriptional control of ECM proteins. Here we show that TGF-β, through increased FN trafficking in a transcription- and SMAD-independent manner, is a direct and rapid inducer of the fibrillogenesis required for TGF-β-induced cell migration. Whereas TGF-β signaling is dispensable for rapid fibrillogenesis, stable interactions between the cytoplasmic domain of the type II TGF-β receptor (TβRII) and the FN receptor (α5β1 integrin) are required. We find that, in response to TGF-β, cell surface-internalized FN is not degraded by the lysosome but instead undergoes recycling and incorporation into fibrils, a process dependent on TβRII. These findings are the first to show direct use of trafficked and recycled FN for fibrillogenesis, with a striking role for TGF-β in this process. Given the significant physiological consequences associated with FN availability and polymerization, our findings provide new insights into the regulation of fibrillogenesis for cellular homeostasis.

Transforming Growth Factor-β (TGF-β) Directly Activates the JAK1-STAT3 Axis to Induce Hepatic Fibrosis in Coordination with the SMAD Pathway

Transforming growth factor-β (TGF-β) signals through both SMAD and non-SMAD pathways to elicit a wide array of biological effects. Existing data have shown the association and coordination between STATs and SMADs in mediating TGF-β functions in hepatic cells, but it is not clear how STATs are activated under these circumstances. Here, we report that JAK1 is a constitutive TGFβRI binding protein and is absolutely required for phosphorylation of STATs in a SMAD-independent manner within minutes of TGF-β stimulation. Following the activation of SMADs, TGF-β also induces a second phase of STAT phosphorylation that requires SMADs, de novo protein synthesis, and contribution from JAK1. Our global gene expression profiling indicates that the non-SMAD JAK1/STAT pathway is essential for the expression of a subset of TGF-β target genes in hepatic stellate cells, and the cooperation between the JAK1-STAT3 and SMAD pathways is critical to the roles of TGF-β in liver fibrosis.

Role of Cation Channel TRPV4 in Mechanosensing, Myofibroblast Differentiation, and Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with marginally effective medical treatment. Myofibroblasts are critical to the fibrogenic lung repair process; however, the mechanism whereby the mechanical signal that leads to myofibroblast generation is sensed and transmitted remains to be determined. As transient receptor potential vanilloid 4 (TRPV4) ion channels are activated by plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 plays a role in myofibroblast differentiation and/or in vivo lung fibrosis. TRPV4 inhibition by small-molecule inhibitors almost completely abrogated both the calcium influx response to TPRV4 agonist in a dose-dependent manner and the myofibroblast differentiation response to transforming growth factor-β. Similar findings were noted on TRPV4 deletion with small interfering RNA or in TRPV4 KO murine lung fibroblasts. Further, TRPV4 exhibited its mechanosensing and myofibroblast-differentiating effect under conditions of matrix stiffness in the pathophysiological range (1–25 kPa). Mechanistically, TRPV4 activity modulates transforming growth factor β actions in a Smad-independent manner, in part through enhanced actomyosin remodeling with resultant nuclear translocation of the α–smooth muscle actin transcription coactivator (myocardin-related transcription factor-A). The myofibroblast differentiation response to actual fibrotic lung tissue was also completely inhibited on TRPV4 blockade. Furthermore, TRPV4 deficiency protects mice from fibrosis-inducing effects of bleomycin (1 or 4 U/kg dose) at the biochemical (75% less hydroxyproline; P < 0.05), histologic, and physiologic levels (57% less impairment of compliance; P < 0.05). These data identify TRPV4 as a long-elusive mechanosensor/transducer that mediates myofibroblast differentiation and in vivo pulmonary fibrogenesis. Successful manipulation of TRPV4 channel activity may be a novel therapeutic approach for fibrotic diseases of the lung and other organs.

TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-β; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-β1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases.

ZEB2–Sp1 cooperation induces invasion by upregulating cadherin-11 and integrin α5 expression

Epithelial-mesenchymal transition (EMT) is a process implicated in invasion and metastasis. EMT is characterized by repression of epithelial markers and induction of mesenchymal markers. ZEB2 is a transcriptional repressor of E-cadherin, leading to EMT. Previously, we have shown that ZEB2 directly upregulates integrin α5 transcription by cooperating with the transcription factor Sp1. In this study, we investigated the precise mechanism by which ZEB2 modulates invasion and EMT events and the role of Sp1 in ZEB2-induced invasion. We found that ZEB2 directly induced cadherin-11 transcription in an Sp1-dependent, but Smad- and E-box-independent, manner and repressed E-cadherin expression in an Sp1- and Smad-independent manner, leading to cadherin switch. Furthermore, ZEB2 upregulated Sp1 by enhancing Sp1 protein stability, and Sp1 was found to be critical for ZEB2-induced cancer cell invasion, mainly through induction of cadherin-11 and integrin α5. Expression levels of cadherin-11 and integrin α5 were interdependent and both modulated c-Jun N-terminal kinase-signaling activity and invasion. Immunofluorescence analysis showed that nuclear expression of ZEB2 was positively correlated with Sp1 expression in human colorectal cancers. Together, these findings demonstrate a previously unrecognized interplay between ZEB2, Sp1, cadherin-11 and integrin α5 that is, probably, significant in tumor progression and metastasis.

A novel role of BMP4 in adult hematopoietic stem and progenitor cell homing via Smad independent regulation of integrin-α4 expression

AbstractAlthough it is well established that BMP4 plays an important role in the development of hematopoietic system, it is less well understood whether BMP4 affects adult hematopoiesis and how. Here, we describe a novel mechanism by which BMP4 regulates homing of murine as well as human hematopoietic stem/progenitor cells (HSPCs). BMP4 treatment of murine BM derived c-kit+Lin−Sca-1+ (KLS) and CD150+CD48−KLS cells for up to 5 days in vitro prevented the culture-induced loss of Integrin-α4 (ITGA4) expression as well as homing. The effect on ITGA4 expression in response to BMP4 is mediated via SMAD-independent phosphorylation of p38 MAPK, which activates microphthalmia-associated transcription factor (MITF), known to induce ITGA4 expression. Elevated ITGA4 expression significantly enhanced HSPC attachment to bone marrow stromal cells, homing and long-term engraftment of the BMP4 treated cells compared with the cells cultured without BMP4. BMP4 also induced expression of ITGA4 on human BM derived Lin−CD34+ cells in culture, which was associated with improved homingpotential. Thus, BMP4 prevents culture-induced loss of ITGA4 expression on HSPCs in a SMAD-independent manner, resulting in improved homing of cultured HSPCs and subsequent hematopoietic reconstitution.

Abstract 3252: Divergence of activin and TGFβ-induced SMAD-independent signaling in colon cancer

Abstract Introduction: Activin and TGFβ are members of the TGFβ superfamily with growth suppressive and pro-migratory properties. While growth suppressive SMAD 2/3/4 signaling is shared, we have evidence of diverging SMAD-independent migratory signaling involving the CDK inhibitor p21. Here, we dissect the participation of respective mitogenic pathways in activin and TGFβ-specific SMAD-independent signaling in colon cancer. Methods: Colon cancer cell lines with and without SMAD4 were assessed for SMAD, PI3K and MEK pathway influences on activin or TGFβ signaling and associated effects using pharmacologic inhibition, knock-down, co-immunoprecipitation, Western blotting, and functional assays of migration. Results: Irrespective of SMAD4 status, activin-induced migration was PI3K-dependent and associated with PI3K-induced p21 downregulation, while TGFβ-induced migration was MEK1/2-dependent. Activin but not TGFβ lead to colocalization of ACVR1, ACVR2's primary binding partner, with p85, the regulatory subunit of PI3K, in an ACVR1-dependent SMAD-independent manner. Further, activin-induced p21 downregulation was PI3K dependent and associated with proteasomal degradation, while TGFβ stabilized p21 via MEK/ERK. Inhibition of proteasomal degradation of p21 lead to a decrease in migration with activin implicating p21 downregulation in activin-induced migration but not in TGFβ-induced migration. Conclusion: Activin and TGFβ diverge in their pro-migratory SMAD-independent signaling in colon cancer affecting distinct targets to include p21 and employing diverse mitogenic signals. Dissecting ligand-specific functions in colon cancer may be exploited for risk stratification or therapeutic intervention in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3252. doi:1538-7445.AM2012-3252

Abstract 5266: TGF-β1 modulates the homeostasis between MMPs and MMPs inhibitors through p38 MAPK and ERK½ in highly invasive human breast cancer cells

Abstract Matrix metalloproteinases (MMPs) and their specific inhibitors, known as tissue inhibitors of MMPs (TIMPs) and the membrane-associated MMP inhibitor (RECK), are involved in several steps of the metastatic process. Previous results from our laboratory showed a positive correlation between high mRNA expression levels of MMPs and their inhibitors upon breast cancer progression, both in cellular models and in tumor tissue samples. However, the molecular mechanisms underlying this coordinate regulation of MMPs, TIMPs and RECK expression remain unknown. Since transforming growth factor beta 1 (TGF-β1) has been related to mechanisms which regulate the expression of some MMPs and MMPs inhibitors in different models, we investigated whether this multifunctional cytokine could have a role as a common regulator of MMPs, TIMPs and RECK in the human breast cancer model. The mRNA expression levels of TGF-β isoforms and their receptors were examined, by qRT-PCR, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. We demonstrated that TGF-β1, TGF-β2 and TβRII are significantly overexpressed in more aggressive mammary cell lines. Furthermore, using the Spearman correlation test, we showed a significantly positive correlation between the mRNA expression levels of TGF-β1 and MMP-2, MMP-14, TIMP-1, TIMP-3 and RECK. In addition, upon treatment with TGF-β1, the MDA-MB-231 breast cancer cell line presented significantly increased mRNA expression levels of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 was also able to induce the protein expression levels of MMP-2, MMP-9, MMP-14 and TIMP-2. On the other hand, RECK protein expression was significantly downregulated in TGF-β1-treated-MDA-MB-231 cells. In addition to the classical TGF-β-induced signal transduction by Smads, it is well known that this cytokine also signals in a Smad-independent manner, by induction of other pathways. Here, we analyzed the involvement of p38 MAPK and ERK½, two well established Smad-independent pathways, in the proposed mechanism of coordinate regulation of MMPs, TIMPs and RECK by TGF-β1 in the breast cancer model. Firstly, we confirmed that TGF-β1 is able to induce phosphorylation of p38MAPK and ERK½ proteins in the MDA-MB-231 cell line. Inhibition of ERK½ pathway led to increased levels of the MMPs inhibitors (TIMP-2 and RECK) mRNA in MDA-MB-231 cells treated (or not) with TGF-β1. In contrast, cells treated with a pharmacological p38MAPK inhibitor showed blockage of TGF-β1-induced mRNA expression of MMPs (MMP-2, MMP-9 and MMP-14) and MMPs inhibitors (TIMP-2 and RECK). Altogether, our results suggest that the balance between p38MAPK and ERK½ activities mediates the role of TGF-β1 as an important homeostasis regulator between MMPs and MMPs inhibitors in the MDA-MB231 breast cancer cell line. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5266. doi:10.1158/1538-7445.AM2011-5266

Rap1-GTP Augments Activation of Smad and p38 Mediated Signaling Downstream of TGF-β Receptor In T Lymphocytes

AbstractAbstract 956Rap1A, a member of the Ras superfamily, was discovered as a gene product that reverted K-Ras-induced transformation. Although it was initially thought that Rap1A (thereafter referred to as Rap1) opposes Ras-induced transformation by competing for common downstream effector(s), it has now become apparent that Ras and Rap1 proteins operate in different signaling networks and mediate distinct functions. In lymphocytes Rap1 is activated by BCR and TCR mediated signals and is involved in inside-out activation of integrins. To understand the role of Rap1 in T cell responses we generated transgenic (Tg) mice that express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg and littermate control mice had no statistically significant difference in absolute thymocyte numbers and differentiation profiles of double positive and single positive thymocytes. In contrast to thymocyte numbers, Rap1E63-Tg mice had reduced T cell numbers in peripheral lymphoid organs. In peripheral lymph nodes and spleen, the total T cell numbers were decreased to approximately one third to one forth of those in littermate controls. Strikingly, Rap1E63-Tg mice had a 4-fold increase in the CD4+CD103+ T cell fraction in spleens and lymph nodes compared to their littermate controls. CD103 defines a subset of peripherally generated Treg with potent suppressive function. CD4+CD103+ Treg cells in RapE63-Tg mice expressed CD45RBlow, CD44high, CD54high, CD62Llow and LFA-1high, findings consistent with an effector memory phenotype. TGF-β is the strongest stimulus for induction of CD103 expression. To examine whether Rap1 affected TGF-β-mediated signaling in T cells, we used stable Jurkat T cell lines expressing Rap1E63, Jurkat T cell lines, in which endogenous Rap1 was depleted by shRNA (Rap1-KD), and primary T cells from Rap1E63-Tg mice and Rap1-KO mice. TGF-β signaling involves the interaction of two membrane-bound receptors, TGF-βRI and II. TGF-β binds the exracellular domain of TGF-βRII, which then recruits TGF-βRI resulting in transphosphorylation. TGF-βRI activation propagates downstream signaling via the Smad family proteins Smad2 and Smad3 and also via p38 MAPK, in a Smad-independent manner. Incubation of Rap1E63 Jurkat T cells with TGF-β resulted in enhanced and sustained phosphorylation of Smad2 and Smad3, which was observed with very low concentrations of TGF-β that were incapable of inducing detectable phosphorylation of Smad2 or Smad3 in control cells. In contrast, diminished level and duration of Smad2 and Smad3 phosphorylation was observed in Rap1-KD Jurkat cells. Activation of p38 downstream of TGF-βRI displayed a dramatically augmented and sustained induction, whereas the opposite effect was observed in Rap1-KD cells. Similar patterns of responses to those observed in Rap1E63 Jurkat T cells and in Rap1-KD cells were observed in primary mouse T cells isolated from Rap1E63-Tg mice and Rap1 deficient mice, respectively. Assessment of TGF-β binding on TGF-β surface receptors by using biotinylated TGF-β revealed that Rap1E63 expressing cells displayed significantly enhanced TGF-β binding compared to their relevant controls, whereas reduced TGF-β binding was observed in Rap1 deficient cells. These results indicate that active Rap1 modulates TGF-β receptor binding and enhances TGF-β mediated signaling, gene transcription, and functional outcome. Our data reveal a novel and unexpected mechanism by which Rap1 regulates T cell responses that may have important implications on TGF-β-mediated T cell homeostasis and maintenance of immune quiescence. Disclosures:No relevant conflicts of interest to declare.

HSP27 modulates epithelial to mesenchymal transition of lung cancer cells in a Smad-independent manner

Epithelial to mesenchymal transition (EMT) is induced by transforming growth factor-β1 (TGF-β1) and is a crucial event for cancer cells to acquire invasive and metastatic phenotypes. However, the signals that induce EMT in cancer cells have yet to be adequately defined. In this study, a proteomic investigation was performed to understand the signaling pathway of the EMT of lung cancer using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. The protein expression profiles of A549 were compared to those of A549 cells treated with TGF-β1. Of more than 2,000 protein spots shown by 2D-DIGE, 53 were found to be up- or down-regulated upon induction with TGF-β1. In the 53 protein spots, the protein level of heat shock protein (HSP) 27 was found to increase significantly. HSP27 protein was higher in two different lung cancer cell lines, demonstrating the EMT phenomenon with TGF-β1. Notably, the silencing of HSP27 enhanced spindle integration, resulting in an additive effect with TGF-β1-induced EMT. Furthermore, the TGF-β1-induced HSP27 increase was not affected by the suppression of Smad2 and Smad3 in A549 cells. These results suggest that HSP27 was involved in TGF-β1-induced EMT in a Smad-independent manner in lung cancer cells and may provide an effective clinical strategy in lung cancer patients whose tumors are dependent on TGF-β1-induced EMT.

TGF-β receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation

Skeletal muscle differentiation is strongly inhibited by transforming growth factor type β (TGF-β), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-β-receptors (TGF-β-Rs) during skeletal muscle differentiation. We found a decrease of TGF-β signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-β. No change in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-β-R type I (TGF-β-RI) and type II (TGF-β-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-β-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-β-RII lacking the cytoplasmic domain. The TGF-β-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-β-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-β receptors independent of Smad proteins are essential for skeletal muscle differentiation.

Four-and-a-half LIM protein 2 promotes invasive potential and epithelial-mesenchymal transition in colon cancer

Cancer invasion and metastasis may associate with the phenotype transition called epithelial-mesenchymal transition (EMT). We aim to evaluate the impact of four-and-a-half LIM protein 2 (FHL2) on EMT and invasion of colon cancer. The functional role of FHL2 in EMT was determined by overexpression or small interfering RNA-mediated depletion of FHL2. Mechanisms of FHL2 on expression or activity of E-cadherin and beta-catenin were assessed. FHL2 was highly expressed in primary and metastatic colon cancer but not in normal tissues. FHL2 was critical for cancer cell adhesion to extracellular matrix, migration and invasion. FHL2 expression was stimulated by transforming growth factor (TGF)-beta1. Moreover, FHL2 acted as a potent EMT inducer by stimulating vimentin and matrix metalloproteinase-9 expressions and causing a loss of E-cadherin, whereas those alterations of EMT markers were not affected by silencing of Smad molecules (typical TGF-beta signal mediators) in FHL2 stable transfectant cells. Therefore, FHL2 induced EMT in a TGF-beta-dependent and Smad-independent manner. FHL2 downregulated E-cadherin expression and inhibited the formation of membrane-associated E-cadherin-beta-catenin complex. FHL2 also stabilized nuclear beta-catenin, resulting in enforcement of beta-catenin transactivation activity. FHL2 is a potent EMT inducer and might be an important mediator for invasion and/or metastasis of colon cancer.

Activin A induces neuronal differentiation and survival via ALK4 in a SMAD-independent manner in a subpopulation of human neuroblastomas

Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-β superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine β-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-β target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-β target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.

Expression and Activity of Phosphodiesterase Isoforms during Epithelial Mesenchymal Transition: The Role of Phosphodiesterase 4

Epithelial-mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer and is typically induced by the multifunctional cytokine transforming growth factor (TGF)-beta1. The present study was undertaken to evaluate the potential role of phosphodiesterases (PDEs) in TGF-beta1-induced EMT in the human alveolar epithelial type II cell line A549. Stimulation of A549 with TGF-beta1 induced EMT by morphological alterations and by expression changes of the epithelial phenotype markers E-cadherin, cytokeratin-18, zona occludens-1, and the mesenchymal phenotype markers, collagen I, fibronectin, and alpha-smooth muscle actin. Interestingly, TGF-beta1 stimulation caused twofold increase in total cAMP-PDE activity, contributed mostly by PDE4. Furthermore, mRNA and protein expression demonstrated up-regulation of PDE4A and PDE4D isoforms in TGF-beta1-stimulated cells. Most importantly, treatment of TGF-beta1 stimulated epithelial cells with the PDE4-selective inhibitor rolipram or PDE4 small interfering RNA potently inhibited EMT changes in a Smad-independent manner by decreasing reactive oxygen species, p38, and extracellular signal-regulated kinase phosphorylation. In contrast, the ectopic overexpression of PDE4A and/or PDE4D resulted in a significant loss of epithelial marker E-cadherin but did not result in changes of mesenchymal markers. In addition, Rho kinase signaling activated by TGF-beta1 during EMT demonstrated to be a positive regulator of PDE4. Collectively, the findings presented herein suggest that TGF-beta1 mediated up-regulation of PDE4 promotes EMT in alveolar epithelial cells. Thus, targeting PDE4 isoforms may be a novel approach to attenuate EMT-associated lung diseases such as pulmonary fibrosis and lung cancer.