TGF-ß/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells.

The TGF-ß and Hippo pathways are critical for liver size control, regeneration, and cancer progression. The transcriptional cofactor TAZ, also named WWTR1, is a downstream effector of Hippo pathway and plays a key role in the maintenance of liver physiological functions. However, the up-regulation of TAZ expression has been associated with liver cancer progression. Recent evidence shows crosstalk of TGF-ß and Hippo pathways, since TGF-ß modulates TAZ expression through different mechanisms in a cellular context-dependent manner but supposedly independent of SMADs. Here, we evaluate the molecular interplay between TGF-ß pathway and TAZ expression and observe that TGF-ß induces TAZ expression through SMAD canonical pathway in liver cancer HepG2 cells. Therefore, TAZ cofactor is a primary target of TGF-ß/SMAD-signaling, one of the pathways altered in liver cancer.

Quantitative Expression of Key Cancer Markers in the AS-30D Hepatocarcinoma Model.

Hepatocellular carcinoma is one of the cancers with the highest mortality rate worldwide. HCC is often diagnosed when the disease is already in an advanced stage, making the discovery and implementation of biomarkers for the disease a critical aim in cancer research. In this study, we aim to quantify the transcript levels of key signaling molecules relevant to different pathways known to participate in tumorigenesis, with special emphasis on those related to cancer hallmarks and epithelial-mesenchymal transition, using as a model the murine transplantable hepatocarcinoma AS-30D. Using qPCR to quantify the mRNA levels of genes involved in tumorigenesis, we found elevated levels for Tgfb1 and Spp1, two master regulators of EMT. A mesenchymal signature profile for AS-30D cells is also supported by the overexpression of genes encoding for molecules known to be associated to aggressiveness and metastatic phenotypes such as Foxm1, C-met, and Inppl1. This study supports the use of the AS-30D cells as an efficient and cost-effective model to study gene expression changes in HCC, especially those associated with the EMT process.

Activation of ALDH1A1 by omeprazole reduces cell oxidative stress damage.

Under physiological conditions, cells produce low basal levels of reactive oxygen species (ROS); however, in pathologic conditions ROS production increases dramatically, generating high concentrations of toxic unsaturated aldehydes. Aldehyde dehydrogenases (ALDHs) are responsible for detoxification of these aldehydes protecting the cell. Due to the physiological relevance of these enzymes, it is important to design strategies to modulate their activity. It was previously reported that omeprazole activation of ALDH1A1 protected Escherichia coli cells overexpressing this enzyme, from oxidative stress generated by H2 O2 . In this work, omeprazole cell protection potential was evaluated in eukaryotic cells. AS-30D cell or hepatocyte suspensions were subjected to a treatment with omeprazole and exposure to light (that is required to activate omeprazole in the active site of ALDH) and then exposed to H2 O2 . Cells showed viability similar to control cells, total activity of ALDH was preserved, while cell levels of lipid aldehydes and oxidative stress markers were maintained low. Cell protection by omeprazole was avoided by inhibition of ALDHs with disulfiram, revealing the key role of these enzymes in the protection. Additionally, omeprazole also preserved ALDH2 (mitochondrial isoform) activity, diminishing lipid aldehyde levels and oxidative stress in this organelle, protecting mitochondrial respiration and transmembrane potential formation capacity, from the stress generated by H2 O2 . These results highlight the important role of ALDHs as part of the antioxidant system of the cell, since if the activity of these enzymes decreases under stress conditions, the viability of the cell is compromised.

Kinetic modeling of glucose central metabolism in hepatocytes and hepatoma cells.

BACKGROUND: Kinetic modeling and control analysis of a metabolic pathway may identify the steps with the highest control in tumor cells, and low control in normal cells, which can be proposed as the best therapeutic targets. METHODS: Enzyme kinetic characterization, pathway kinetic modeling and control analysis of the glucose central metabolism were carried out in rat (hepatoma AS-30D) and human (cervix HeLa) cancer cells and normal rat hepatocytes. RESULTS: The glycogen metabolism enzymes in AS-30D, HeLa cells and hepatocytes showed similar kinetic properties, except for higher AS-30D glycogen phosphorylase (GP) sensitivity to AMP. Pathway modeling indicated that fluxes of glycogen degradation and PPP were mainly controlled by GP and NADPH consumption, respectively, in both hepatocytes and cancer cells. Likewise, hexose-6-phosphate isomerase (HPI) and phosphoglucomutase (PGM) exerted significant control on glycolysis and glycogen synthesis fluxes in cancer cells but not in hepatocytes. Modeling also indicated that glycolytic and glycogen synthesis fluxes could be strongly decreased when HPI and PGM were simultaneously inhibited in AS-30D cells but not in hepatocytes. Experimental assessment of these predictions showed that both the glycolytic and glycogen synthesis fluxes of AS-30D cells, but not of hepatocytes, were inhibited by oxamate, by inducing increased Fru1,6BP levels, a competitive inhibitor of HPI and PGM. CONCLUSION: HPI and PGM seem suitable targets for decreasing glycolytic and glycogen synthesis fluxes in AS-30D cells but not in hepatocytes. GENERAL SIGNIFICANCE: The present study identified new therapeutic targets within glucose central metabolism in the analyzed cancer cells, with no effects on non-cancer cells.

Interplay between interferon-stimulated gene 15/ISGylation and interferon gamma signaling in breast cancer cells.

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that conjugates to its target proteins to modify them through ISGylation, but the relevance of ISG15 expression and its effects have been not completely defined. Herein, we examined the interplay between ISG15/ISGylation and the interferon-gamma (IFN-γ) signaling pathway in mammary tumors and compared it with that in normal mammary tissues. Our results indicated that mammary tumors had higher levels of ISG15 mRNA and ISG15 protein than the adjacent normal mammary tissue. Furthermore, the expression of IFN-γ signaling components was altered in breast cancer. Interestingly, IFN-γ treatment induced morphological changes in MCF-7 and MDA-MB-231 breast cancer cell lines due to cytoskeletal reorganization. This cellular process seems to be related to the increase in ISGylation of cytoplasmic IQ Motif Containing GTPase Activating Protein 1 (IQGAP1). Interactome analysis also indicated that IFN-γ signaling and the ISGylation system are associated with several proteins implicated in cytoskeletal remodeling, including IQGAP1. Thus, ISG15 may present a potential biomarker for breast cancer, and IFN-γ signaling and protein ISGylation may participate in the regulation of the cytoskeleton in breast cancer cells.

Fabrication of low-cost micropatterned polydimethyl-siloxane scaffolds to organise cells in a variety of two-dimensioanl biomimetic arrangements for lab-on-chip culture platforms.

We present the rapid-prototyping of type I collagen micropatterns on poly-dimethylsiloxane substrates for the biomimetic confinement of cells using the combination of a surface oxidation treatment and 3-aminopropyl triethoxysilane silanisation followed by glutaraldehyde crosslinking. The aim of surface treatment is to stabilise microcontact printing transfer of this natural extracellular matrix protein that usually wears out easily from poly-dimethylsiloxane, which is not suitable for biomimetic cell culture platforms and lab-on-chip applications. A low-cost CD-DVD laser was used to etch biomimetic micropatterns into acrylic sheets that were in turn replicated to poly-dimethylsiloxane slabs with the desired features. These stamps were finally inked with type I collagen for microcontact printing transfer on the culture substrates in a simple manner. Human hepatoma cells (HepG2) and rat primary hepatocytes, which do not adhere to bare poly-dimethylsiloxane, were successfully seeded and showed optimal adhesion and survival on simple protein micropatterns with a hepatic cord geometry in order to validate our technique. HepG2 cells also proliferated on the stamps. Soft and stiff poly-dimethylsiloxane layers were also tested to demonstrate that our cost-effective process is compatible with biomimetic organ-on-chip technology integrating tunable stiffness with a potential application to drug testing probes development where such cells are commonly used.

Actin-cytoskeleton polymerization differentially controls the stability of Ski and SnoN co-repressors in normal but not in transformed hepatocytes.

BACKGROUND: Ski and SnoN proteins function as transcriptional co-repressors in the TGF-ß pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-ß signalling, malignant transformation, and alterations in cell proliferation. METHODS: We carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-ß, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-ß/Smad-driven gene transcription. RESULTS: Ski and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-ß-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-ß-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-ß-target genes. CONCLUSION: Normal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells. GENERAL SIGNIFICANCE: This work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.

Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

TGF-ß-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-ß signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-ß and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-ß/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-ß and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l(-1) insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Calcium-sensing receptor inhibits TGF-ß-signaling by decreasing Smad2 phosphorylation.

Calcium-sensing receptor (CaSR) contributes to maintain homeostatic levels of extracellular calcium. In addition, CaSR controls other cellular activities such as proliferation and migration, particularly in cells not related to extracellular calcium homeostasis, potentially by cross-talking with parallel signaling pathways. Here we report that CaSR attenuates transforming growth factor-ß (TGF-ß)-signaling in hepatic C9 cells and in transfected HEK293 cells. Wild type CaSR interferes with TGF-ß-dependent Smad2 phosphorylation and induces its proteasomal degradation, resulting in a decrease of TGF-ß-dependent transcriptional activity, whereas an inactivating CaSR mutant does not transduce an inhibitory effect of extracellular calcium on TGF-ß signaling. Attenuation of TGF-ß signaling in response to extracellular calcium is linked to Rab11-dependent CaSR-trafficking with the intervention of CaSR carboxyl-terminal tail. Our data suggest that CaSR might regulate TGF-ß-dependent cellular responses mediated by TGF-ß signaling inhibition.

Downregulation of SnoN oncoprotein induced by antibiotics anisomycin and puromycin positively regulates transforming growth factor-ß signals.

BACKGROUND: SnoN and Ski proteins function as Smad transcriptional corepressors and are implicated in the regulation of diverse cellular processes such as proliferation, differentiation and transformation. Transforming growth factor-ß (TGF-ß) signaling causes SnoN and Ski protein degradation via proteasome with the participation of phosphorylated R-Smad proteins. Intriguingly, the antibiotics anisomycin (ANS) and puromycin (PURO) are also able to downregulate Ski and SnoN proteins via proteasome. METHODS: We explored the effects of ANS and PURO on SnoN protein downregulation when the activity of TGF-ß signaling was inhibited by using different pharmacological and non-pharmacological approaches, either by using specific TßRI inhibitors, overexpressing the inhibitory Smad7 protein, or knocking-down TßRI receptor or Smad2 by specific shRNAs. The outcome of SnoN and Ski downregulation induced by ANS or PURO on TGF-ß signaling was also studied. RESULTS: SnoN protein downregulation induced by ANS and PURO did not involve the induction of R-Smad phosphorylation but it was abrogated after TGF-ß signaling inhibition; this effect occurred in a cell type-specific manner and independently of protein synthesis inhibition or any other ribotoxic effect. Intriguingly, antibiotics seem to require components of the TGF-ß/Smad pathway to downregulate SnoN. In addition, SnoN protein downregulation induced by antibiotics favored gene transcription induced by TGF-ß signaling. CONCLUSIONS: ANS and PURO require TGF-ß/Smad pathway to induce SnoN and Ski protein downregulation independently of inducing R-Smad2 phosphorylation, which facilitates TGF-ß signaling. GENERAL SIGNIFICANCE: Antibiotic analogs lacking ribotoxic effects are useful as pharmacological tools to study TGF-ß signaling by controlling Ski and SnoN protein levels.

Transforming growth factor-ß/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-ß signaling. SNON protein levels are tightly regulated by the TGF-ß pathway: whereas a short stimulation with TGF-ß decreases SNON levels by its degradation via the proteasome, longer TGF-ß treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-ß response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-ß treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-ß signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-ß pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-ß target genes is modified.

Casiopeina II-gly and bromo-pyruvate inhibition of tumor hexokinase, glycolysis, and oxidative phosphorylation.

The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC50 = 0.74-6.7 µM) was more effective to inhibit 24-72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC50 = 45-100 µM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3-21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 µM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.

Inhibitory Smad7: emerging roles in health and disease.

Smad7 is an inhibitory Smad protein that blocks Transforming Growth Factor-beta (TGF-ß) signaling through a negative feedback loop, also capable of mediating the crosstalk between TGF-ß and other signaling pathways. Smad7 mRNA and protein levels are upregulated after TGF-ß signaling; subsequently, Smad7 protein binds TGF-ß type I receptor blocking R-Smad phosphorylation and eventually TGF-ß signaling. Because of this inhibitory function, Smad7 can antagonize diverse cellular processes regulated by TGF-ß such as cell proliferation, differentiation, apoptosis, adhesion and migration. Smad7 induction by different cytokines, besides TGF-ß, is also critical for crosstalk/integration of a variety of signaling pathways, and relevant in the pathology of some diseases. Thus, Smad7 plays a key role in the control of various physiological events, and even in some pathological processes including fibrosis and cancer. This review highlights the main known functions of Smad7 with a particular focus on the relevance that alterations of Smad7 function may have in homeostasis, also describing some Smad7 emerging roles in the development of several human diseases that identify this protein as a potential therapeutic target.

Plasma membrane calcium ATPase isoform 3 expression in single cells isolated from rat liver.

The plasma membrane Ca(2+)-ATPase (PMCA) located in the hepatocyte is a controversial molecule in itself since it displays different features to those regarded as canonical for P-type Ca(2+)-ATPases, and from which transcript expression as well as catalytic activity continues to be under active investigation. Our aim in this study was to explore at a first glance, pmca isoform distribution using isolated parenchymal and non-parenchymal cells from rat liver tissue. Expression of pmca transcripts was analyzed in fresh or cell-enriched culture preparations, confirming pmca1 and pmca4 as the housekeeping isoforms in all cell types studied (hepatocytes, Kupffer cells, and stellate cells). However, for the first time we show expression of pmca3 transcripts edited at two different sites in both hepatocytes and non-parenchymal cells. Interestingly, employing non-parenchymal cells we demonstrate the specific expression of pmca3e transcripts previously considered nearly exclusive of excitable tissues. Real-time PCR quantification shows a significant decrease of pmca3 transcripts in cultured Kupffer and hepatic stellate cells in comparison with fresh cells. The presence of pmca2 along with pmca3 in all liver cell types studied suggests that high affinity isoforms are relevant to the adequate management of calcium in liver tissue, particularly when hepatic cells become activated by diverse stimuli.

Angiotensin II increases mRNA levels of all TGF-beta isoforms in quiescent and activated rat hepatic stellate cells.

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF-beta (transforming growth factor-beta). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF-beta actions. Here, we studied the effect of AII on the mRNA levels of TGF-beta isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF-beta1, TGF-beta2 and TGF-beta3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF-beta isoforms, particularly TGF-beta2and TGF-beta3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF-beta protein after AII treatment. The mRNA expression of TGF-beta isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF-beta expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.

Determining and understanding the control of glycolysis in fast-growth tumor cells. Flux control by an over-expressed but strongly product-inhibited hexokinase.

Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.

SnoN co-repressor binds and represses smad7 gene promoter.

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.