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The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated α-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated α-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-β-induced EMT. We found that TGF-β increased the activity of HDAC6, a major deacetylase of α-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-β type I receptor inhibitor SB431542 restored the level of acetylated α-tubulin and consequently blocked EMT. Our results demonstrate that acetylated α-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.

TGF-β controls a variety of cellular functions during development. Abnormal TGF-β responses are commonly found in human diseases such as cancer, suggesting that TGF-β signaling must be tightly regulated. Here, we report that protein tyrosine phosphatase non-receptor 3 (PTPN3) profoundly potentiates TGF-β signaling independent of its phosphatase activity. PTPN3 stabilizes TGF-β type I receptor (TβRI) through attenuating the interaction between Smurf2 and TβRI. Consequently, PTPN3 facilitates TGF-β-induced R-Smad phosphorylation, transcriptional responses, and subsequent physiological responses. Importantly, the leucine-to-arginine substitution at amino acid residue 232 (L232R) of PTPN3, a frequent mutation found in intrahepatic cholangiocarcinoma (ICC), disables its role in enhancing TGF-β signaling and abolishes its tumor-suppressive function. Our findings have revealed a vital role of PTPN3 in regulating TGF-β signaling during normal physiology and pathogenesis.

Loss of TGF-β tumour suppressive response is a hallmark of human cancers. As a central player in TGF-β signal transduction, SMAD4 (also known as DPC4) is frequently mutated or deleted in gastrointestinal and pancreatic cancer. However, such genetic alterations are rare in most cancer types and the underlying mechanism for TGF-β resistance is not understood. Here we describe a mechanism of TGF-β resistance in ALK-positive tumours, including lymphoma, lung cancer and neuroblastoma. We demonstrate that, in ALK-positive tumours, ALK directly phosphorylates SMAD4 at Tyr 95. Phosphorylated SMAD4 is unable to bind to DNA and fails to elicit TGF-β gene responses and tumour suppressing responses. Chemical or genetic interference of the oncogenic ALK restores TGF-β responses in ALK-positive tumour cells. These findings reveal that SMAD4 is tyrosine-phosphorylated by an oncogenic tyrosine kinase during tumorigenesis. This suggests a mechanism by which SMAD4 is inactivated in cancers and provides guidance for targeted therapies in ALK-positive cancers.

Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation.

© 2014 Elsevier Inc. TGF-β signaling is essential in many processes, including immune surveillance, and its dysregulation controls various diseases, including cancer, fibrosis, and inflammation. Studying the innate host defense, which functions in most cell types, we found that RLR signaling represses TGF-β responses. This regulation is mediated by activated IRF3, using a dual mechanism of IRF3-directed suppression. Activated IRF3 interacts with Smad3, thus inhibiting TGF-β-induced Smad3 activation and, in the nucleus, disrupts functional Smad3 transcription complexes by competing with coregulators. Consequently, IRF3 activation by innate antiviral signaling represses TGF-β-induced growth inhibition, gene regulation and epithelial-mesenchymal transition, and the generation of Treg effector lymphocytes from naive CD4+lymphocytes. Conversely, silencing IRF3 expression enhances epithelial-mesenchymal transition, TGF-β-induced Treg cell differentiation upon virus infection, and Treg cell generation invivo. Wepresent a mechanism of regulation of TGF-β signaling by the antiviral defense, with evidence for its role in immune tolerance and cancer cell behavior.

Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation.

Cytosolic nucleic acid sensing elicits interferon production for primary antiviral defense through cascades controlled by protein ubiquitination and Ser/Thr phosphorylation. Here we show that TBK1, a core kinase of antiviral pathways, is inhibited by tyrosine phosphorylation. The Src family kinases (SFKs) Lck, Hck, and Fgr directly phosphorylate TBK1 at Tyr354/394, to prevent TBK1 dimerization and activation. Accordingly, antiviral sensing and resistance were substantially enhanced in Lck/Hck/Fgr triple knockout cells and ectopic expression of Lck/Hck/Fgr dampened the antiviral defense in cells and zebrafish. Small-molecule inhibitors of SFKs, which are conventional anti-tumor therapeutics, enhanced antiviral responses and protected zebrafish and mice from viral attack. Viral infection induced the expression of Lck/Hck/Fgr through TBK1-mediated mobilization of IRF3, thus constituting a negative feedback loop. These findings unveil the negative regulation of TBK1 via tyrosine phosphorylation and the functional integration of SFKs into innate antiviral immunity.

Transforming growth factor β (TGF-β) family signaling dictates highly complex programs of gene expression responses, which are extensively regulated at multiple levels and vary depending on the physiological context. The formation, activation, and destruction of two major functional complexes in the TGF-β signaling pathway (i.e., the TGF-β receptor complexes and the Smad complexes that act as central mediators of TGF-β signaling) are direct targets for posttranslational regulation. Dysfunction of these complexes often leads or contributes to pathogenesis in cancer and fibrosis and in cardiovascular, and autoimmune diseases. Here we discuss recent insights into the roles of posttranslational modifications in the functions of the receptor-activated Smads in the common Smad4 and inhibitory Smads, and in the control of the physiological responses to TGF-β. It is now evident that these modifications act as decisive factors in defining the intensity and versatility of TGF-β responsiveness. Thus, the characterization of posttranslational modifications of Smads not only sheds light on how TGF-β controls physiological and pathological processes but may also guide us to manipulate the TGF-β responses for therapeutic benefits.

An enzyme safeguards antiviral recognition machinery to avoid leaking and excessive danger response., Cytosolic RNA sensing is a prerequisite for initiation of innate immune response against RNA viral pathogens. Signaling through RIG-I (retinoic acid–inducible gene I)–like receptors (RLRs) to TBK1 (Tank-binding kinase 1)/IKKε (IκB kinase ε) kinases is transduced by mitochondria-associated MAVS (mitochondrial antiviral signaling protein). However, the precise mechanism of how MAVS-mediated TBK1/IKKε activation is strictly controlled still remains obscure. We reported that protein phosphatase magnesium-dependent 1A (PPM1A; also known as PP2Cα), depending on its catalytic ability, dampened the RLR-IRF3 (interferon regulatory factor 3) axis to silence cytosolic RNA sensing signaling. We demonstrated that PPM1A was an inherent partner of the TBK1/IKKε complex, targeted both MAVS and TBK1/IKKε for dephosphorylation, and thus disrupted MAVS-driven formation of signaling complex. Conversely, a high level of MAVS can dissociate the TBK1/PPM1A complex to override PPM1A-mediated inhibition. Loss of PPM1A through gene ablation in human embryonic kidney 293 cells and mouse primary macrophages enabled robustly enhanced antiviral responses. Consequently, Ppm1a−/− mice resisted to RNA virus attack, and transgenic zebrafish expressing PPM1A displayed profoundly increased RNA virus vulnerability. These findings identify PPM1A as the first known phosphatase of MAVS and elucidate the physiological function of PPM1A in antiviral immunity on whole animals.

Cytosolic RNA/DNA sensing elicits primary defense against viral pathogens. Interferon regulatory factor 3 (IRF3), a key signal mediator/transcriptional factor of the antiviral-sensing pathway, is indispensible for interferon production and antiviral defense. However, how the status of IRF3 activation is controlled remains elusive. Through a functional screen of the human kinome, we found that mammalian sterile 20-like kinase 1 (Mst1), but not Mst2, profoundly inhibited cytosolic nucleic acid sensing. Mst1 associated with IRF3 and directly phosphorylated IRF3 at Thr75 and Thr253. This Mst1-mediated phosphorylation abolished activated IRF3 homodimerization, its occupancy on chromatin, and subsequent IRF3-mediated transcriptional responses. In addition, Mst1 also impeded virus-induced activation of TANK-binding kinase 1 (TBK1), further attenuating IRF3 activation. As a result, Mst1 depletion or ablation enabled an enhanced antiviral response and defense in cells and mice. Therefore, the identification of Mst1 as a novel physiological negative regulator of IRF3 activation provides mechanistic insights into innate antiviral defense and potential antiviral prevention strategies.

A new methodology, three-variable hypersurface splines, is presented for depth solution of geological surfaces in a certain exploration field based on the integration of elevation data, obtained from drilling, and travel time data, obtained from high-resolution seismic exploration. It is based on two-variable surface splines and could be used to generate a three-variable transformation function so that the travel time data could be globally inverted to the elevations of geological surfaces for a whole exploration field. In this procedure, no velocity parameters are used, so the selection of a suitable velocity background model is not necessary in the area with complex geological bodies. Another characteristic of this method is the ability to solve the discordance between travel time data and elevation data which always occurs while using conventional interpretation method and ensure the depths of geological surfaces transformed from seismic data full coinciding with those from original drilling data.

Ectodomain shedding mediated by tumor necrosis factor–α (TNF-α)–converting enzyme [TACE; also known as ADAM17 (a disintegrin and metalloproteinase 17)] provides an important switch in regulating cell proliferation, inflammation, and cancer progression. TACE-mediated ectodomain cleavage is activated by signaling of the mitogen-activated protein kinases (MAPKs) p38 and ERK (extracellular signal– regulated kinase). Here, we found that under basal conditions, TACE was predominantly present as dimers at the cell surface, which required its cytoplasmic domain and enabled efficient association with tissue inhibitor of metalloproteinase-3 (TIMP3) and silencing of TACE activity. Upon activation of the ERK or p38 MAPK pathway, the balance shifted from TACE dimers to monomers, and this shift was associated with increased cell surface presentation of TACE and decreased TIMP3 association, which relieved the inhibition of TACE by TIMP3 and increased TACE-mediated proteolysis of transforming growth factor–α. Thus, cell signaling altered the dimer-monomer equilibrium and inhibitor association to promote activation of TACE-mediated ectodomain shedding, a regulatory mechanism that may extend to other ADAM proteases.

Regulating TGF-beta receptor presentation provides an avenue to alter a cell's responsiveness to TGF-beta. We report that activation of the Erk MAP kinase pathway decreases the TGF-beta-induced Smad3 activation due to decreased cell surface levels of the type I receptor TbetaRI, but not the type II receptor. Inhibition of TACE activity or expression enhanced the cell surface TbetaRI levels and TGF-beta-induced Smad3 and Akt activation. Accordingly, silencing TACE expression in cancer cells enhanced the TbetaRI presentation and TGF-beta responsiveness, including the antiproliferative effect of TGF-beta, and epithelial-to-mesenchymal transition. These results establish a mechanism for downregulating TGF-beta signaling through TACE activation by the Erk MAP kinase pathway and a strategy for evasion of tumor suppression and modulation of epithelial-to-mesenchymal transition during cancer progression. The decreased growth inhibition by TGF-beta, due to elevated TACE activity, complements the growth stimulation resulting from increased release of TGF-alpha family ligands.

The liver receptor homolog 1 (LRH-1) belongs to the Fushi tarazu factor 1 nuclear receptor subfamily, and its biological functions are just being unveiled. The molecular mechanism for the transcriptional regulation by LRH-1 is not clear yet. In this report, we use mutagenesis and reporter gene assays to carry out a detailed analysis on the hinge region and the proximal ligand binding domain (LBD) of human (h) LRH-1 that possess important regulatory functions. Our results indicate that helix 1 of the LBD is essential for the activity of hLRH-1 and that the steroid receptor coactivator (SRC)-1 interacts directly with the LBD of hLRH-1 and significantly potentiates the transcriptional activity of hLRH-1. Cotransfection assays demonstrate that overexpressed SRC-1 potentiates hLRH-1 mediated activation of the cholesterol 7-alpha-hydroxylase promoter and increases the transcription of the endogenous cholesterol 7-alpha-hydroxylase in Huh7 cells. The interaction between SRC-1 and hLRH-1 assumes a unique pattern that involves primarily a region containing the glutamine-rich domain of SRC-1, and helix 1 and activation function-2 of hLRH-1 LBD. Mutagenesis and molecular modeling studies indicate that, similar to mouse LRH-1, the coactivator-binding cleft of hLRH-1 LBD is not optimized. An interaction between helix 1 of hLRH-1 LBD and a region containing the glutamine-rich domain of SRC-1 can provide an additional stabilizing force and enhances the recruitment of SRC-1. Similar interaction is observed between hLRH-1 and SRC-2/transcriptional intermediary factor 2 or SRC-3/acetyltransferase. Moreover, transcriptional intermediary factor 2 and acetyltransferase also potentiate the transcriptional activity of hLRH-1, suggesting a functional redundancy among SRC family members. These findings collectively demonstrate an important functional role of helix 1 in cofactor recruitment and reveal a novel molecular mechanism of transcriptional regulation and cofactor recruitment mediated by hLRH-1.

The human homologue of the Drosophila melanogaster orphan nuclear receptor fushi tarazu factor 1 (Ftz-F1), NR5A2 (hB1F), was initially identified as a regulatory factor that binds and activates enhancer II of hepatitis B virus. NR5A2 (hB1F) is expressed specifically in pancreas and liver, playing important roles in the regulation of several liver-specific genes. A detailed analysis on the genomic structure and promoter activity will greatly promote future studies on the function of the NR5A2 (hB1F) gene. In this report, a bacterial artificial chromosome clone and several phage clones covering the NR5A2 (hB1F) gene were isolated and the complete genomic sequence was obtained. Alignment of different cDNAs of the NR5A2 (hB1F) gene with the genomic sequence facilitated the delineation of its structural organization, which spans over 150 kb and consists of eight exons interrupted by seven introns. RT-PCR and 3'-RACE revealed that utilization of two polyadenylation signals results in the 3.8 and 5.2 kb transcripts that were observed previously. The transcription start site of the NR5A2 (hB1F) gene was mapped downstream of a canonical TATA box. An upstream fragment containing binding sites for several liver-specific and ubiquitous transcription factors exhibits hepatocyte-specific promoter activity. Transient transfections indicated that hepatocyte nuclear factors HNF1 and HNF3beta could activate NR5A2 (hB1F) promoter.

Autoimmunity is a direct consequence of a lack of immune homeostasis and unregulated destruction of self-tissues. The lack of regulation has been attributed to the reduced efficacy of a highly specialized regulatory T cell (Treg). Accumulating evidence suggests that defective regulation is due, at least in part, to the instability and loss of Tregs in the inflamed tissues. Treg instability and loss results from reduced IL-2 sensitivity and IL-2 signaling via STAT5 as instability is reversed, and immune regulation restored, with IL-2 therapy. In addition, recent data suggest that the inflammatory milieu can alter cytokine activities including TGFb. This presentation will focus on several aspects of Treg, IL-2 and TGFb biology including their function and stability in mouse and human autoimmune settings. This work is supported by NIH, JDRF and Pfizer Corp.

TGF-β family signaling through Smads is conceptually a simple and linear signaling pathway, driven by sequential phosphorylation, with type II receptors activating type I receptors, which in turn activate R-Smads. Nevertheless, TGF-β family proteins induce highly complex programs of gene expression responses that are extensively regulated, and depend on the physiological context of the cells. Regulation of TGF-β signaling occurs at multiple levels, including TGF-β activation, formation, activation and destruction of functional TGF-β receptor complexes, activation and degradation of Smads, and formation of Smad transcription complexes at regulatory gene sequences that cooperate with a diverse set of DNA binding transcription factors and coregulators. Here we discuss recent insights into the roles of post-translational modifications and molecular interaction networks in the functions of receptors and Smads in TGF-β signal responses. These layers of regulation demonstrate how a simple signaling system can be coopted to exert exquisitely regulated, complex responses.