In distinction, mobile designs that Determine six. Myocardin-connected transcription issue signaling controls cell condition-dependent induction of SMA by TGF1. (A) Immunofluorescence staining for nuclei and MRTF-A in TGF1-taken care of NMuMG cells cultured on 400 m2 and 2500 m2 squares. (B) Quantification of the percentage of cells with nuclear (N), pan-mobile (N/C), and cytoplasmic (C) MRTF-A as a function of cell distribute region. (C) Quantification of the share of cells expressing SMA following simultaneous therapy with TGF1 and ethanol motor vehicle or CCG-1423. p < 0.01 p < 0.001 compared to ethanol control. Erioglaucine disodium saltOverexpression of Flag-tagged MRTF-A and Flag-tagged MRTF-A-N increase the expression of SMA. (D) Quantification of the percentage of cells expressing SMA for YFP, MRTF-A, MRTF-A-C, and MRTF-A-N transfected NMuMG cells treated with TGF1 or control vehicle. p < 0.001 compared to YFP. Scale bars: 25 m.prevented actin polymerization blocked TGF1-mediated upregulation of myofibroblast markers. Our findings suggest a model whereby cell adhesion and shape modulate the relative levels of monomeric and filamentous actin within the cell thereby controlling the nuclear localization of MRTF-A, as shown in Figure 7. Once in the nucleus, MRTF-A cooperates with other TGF1-induced signaling cascades to regulate the expression of cytoskeletal associated proteins. Changes in cell morphology have previously been reported to control a variety of cell behaviors including cell division [51], proliferation and apoptosis [52], migration [53], and differentiation [32,40,54,55]. Our results show that culture of epithelial cells on small islands blocks TGF1-induced increases in the expression of SMA while culture on large islands that permit cell spreading promotes the myofibroblast phenotype. Decreasing cytoskeletal tension in cells that were permitted to spread blocked the upregulation of SMA by TGF1. These results are consistent with previous studies that have demonstrated a role for RhoA/ROCK signaling and Figure 7. Model proposing how cell shape, MRTF-A, and TGF1 signaling regulate the development of myofibroblasts from epithelial cells cytoskeletal tension in the induction of SMA expression and EMT and in the control of myofibroblast properties [34,42]. Given the roles of cell shape and Rho/ROCK signaling in regulating a variety of other cell behaviors including cell cycle progression [56] and the differentiation of mesenchymal stem cells [40,54], it will be interesting to determine whether SRF/ MRTF-A signaling acts in concert with cell shape to control these cell processes. In addition to changes in cell spread area, cell elongation is also known to modulate cell fate and function including stem cell differentiation[32], macrophage phenotype[57], and contractile force[58]. We find that increased cell elongation for a fixed cell spread area promotes increased expression of SMA and cytoskeletal associated proteins in TGF1 treated cells. Previous studies have demonstrated that cell elongation induces stress fiber alignment in endothelial cells[59] and correlates with increased focal adhesion size and increased traction forces in fibroblasts[60]. Activation of focal adhesion kinase (FAK), a component of focal adhesions, is required for TGF1-induced EMT[61]. Furthermore, FAK cooperates with gelsolin to mediate force-induced nuclear localization of MRTFA and SMA expression in fibroblasts[29]. Thus, it is possible that cell elongation promotes increased expression levels of SMA in TGF1 treated epithelial cells through increased nuclear localization of MRTF-A or increased focal adhesion signaling. Loss of cell-cell contacts is an early event that occurs during EMT. Previous studies have proposed a two-hit model whereby disruption of intercellular contacts in combination with TGF enhances MRTF-A signaling and myofibroblast marker expression by promoting prolonged nuclear localization of MRTF-A [8,62]. Our results suggest that a lack of cell-cell contact is not sufficient to induce a myogenic program in epithelial cells in the presence of TGF1. We find that in addition to these factors, cell adhesion and cell shape control the development of myofibroblasts from epithelial cells. Recent studies have demonstrated a link between cell adhesion and SRF signaling, with increased cell spreading enhancing SRF promoter activity [48,63]. Our findings are consistent with these results, as we show that MRTF-A nuclear localization and the expression levels of SRF/MRTF-A target genes are enhanced by cell spreading. However, our observation that significant increases in the expression of SMA are not observed in control cells that are permitted to spread (even though MRTF-A localizes to the nucleus in a fraction of these cells) suggests that other factors are involved in regulating the transcriptional activity of MRTFs in the context of TGF1-induced EMT. Our data suggest that translocation of MRTF-A to the cell nucleus in spread epithelial cells is necessary but not sufficient to induce the expression of all SRF/MRTF target genes. In light of this, we posit that increases in cell adhesion and spreading predispose epithelial cells to enhanced MRTF transcriptional activity by promoting increases in cytosolic actin polymerization which then drives MRTF to the cell nucleus. Once in the nucleus, the activity of MRTF may be determined by a variety of factors including the relative levels of negative regulators such as intranuclear G-actin [64] or Smad3 [8]. Indeed, activation of nuclear mDia can induce polymerization of nuclear actin and SRF activity [65]. Recent studies have also shown that the level of Smad3, a protein which binds to MRTFs [26] and subsequently inhibits the transcriptional activity of MRTFs [8], is reduced in epithelial cells following treatment with TGF1 [8,62]. Further studies are necessary to identify the key signaling factors involved in regulating the cell shape-dependent transcriptional activity of MRTF-A during TGF1-induced EMT. Although our results suggest an important role for MRTF-A signaling in the regulation of TGF1-induced gene expression by cell shape, we cannot entirely dismiss the possibility that other factors may also contribute to cell shape effects on TGF1 mediated development of myofibroblasts from epithelial cells. For example, it is possible that cell shape may impact the transcription and translation of EMT associated genes. Cell shape regulates nuclear morphology[59,63,66] and cell rounding leads to histone deacetylation in mammary epithelial cells[67] while cell elongation promotes histone acetylation in mesenchymal stem cells[68]. Additionally, the subcellular localization of mRNAs can regulate gene expression[69,70]. Interestingly, mRNAs have been observed to associate with actin filaments and it has been suggested that this may facilitate translation[713]. Thus, it is possible that differences in the cytoskeletal architecture within rounded and spread cells may lead to differences in mRNA transport and translation. A recent study demonstrated that mammary epithelial cells express high levels of cytokeratins when cultured on both small and large ECM islands and TGF1 treatment induces downregulation of cytokeratins and upregulation of vimentin across a range of cell spread areas[17]. Therefore, although cell shape may affect the transcriptional and translational activity of some genes, these results suggest that restricting cell spreading does not globally downregulate the expression of all proteins associated with EMT. Overall, our findings demonstrate that the combined effects of cell shape and TGF1 signaling are critical for MRTF activity and increased expression of myofibroblast markers during EMT. In vivo, EMT occurs during normal morphogenic processes of the embryo and contributes to pathological conditions including fibrosis and cancer. Whether cell shape and MRTF signaling regulate aspects of EMT in all of these biological settings is yet to be determined. Analysis of the precise interplay between cell adhesion and MRTF signaling will permit a clearer view of how biochemical cues and mechanical forces act in concert to influence gene expression.Humans are constantly exposed to factors causing oxidative stress including pollutants, radiation and oxidized food [1]. Oxidative stress, defined as a loss of balance between the cellular concentration of reactive oxygen species and the cell's antioxidant capacity, is implicated in the onset of various diseases [2]. The human body has several endogenous systems [3] with which it can protect itself against oxidative stress, but antioxidant factors acquired from food also play a key role. Indeed, certain micronutrients obtained from food have potent antioxidant properties and may play an important role in maintaining the oxidative/antioxidative balance, especially if the diet is rich in these constituents [4]. In recent years, a variety of vegetables that contain antioxidants potentially capable of preventing oxidative stress reactions, such as those mediated by the formation of free radical species have been studied. Tomatoes, spinach, green peppers and cabbage are important sources of vitamin C. In vivo, this vitamin acts as scavenger of oxygen radicals and also as competitive inhibitor of nitrosamine synthesis from nitrite and amines in vivo [5]. Isothiocyanates are a family of molecules which are abundant in cruciferous vegetables such as broccoli, watercress and cauliflower. Sulforaphane, the best known isothiocyanate, induces drug metabolizing enzymes such as glutathione Stransferase A1/2 isoforms and NAD(P)H:quinone oxidoreductase (NQO1) in primary hepatocytes [6]. Whole grains are good source of B group vitamins, vitamin E, some minerals (zinc, magnesium and phosphorous), and they contain a variety of phytochemicals such as phytoestrogens, phytate, proteins, polysaccharides, phenols and lignans that are able to minimize oxydive damage [7]. All these components may act synergically [8]. By contrast, refined grains have a reduced nutrient content as the milling process results in the loss of dietary fibre, vitamins, minerals, lignans, phytoestrogens, phenolic compounds and phytic acid [9]. Many wheat proteins contain reduced sulfhydryl groups, which can have some free radical scavenging activity. Phytic acid can protect tissues against oxidative reactions by sequestering and inactivating pro-oxidative transition metals [3]. In epidemiological studies, whole grain consumption is associated with improvements in body mass index (BMI) [10] and insulin sensitivity [11] as well as with lower incidences of type 2 diabetes [12], cardiovascular diseases [13], and colorectal cancer [14]. Little is known about how cereals effect cells and to our knowledge, no research has yet been done on the antioxidant properties of whole grain products in primary hepatocytes. Several studies have shown that some phytochemicals can modulate antioxidant and phase II enzymes through the activation of nuclear factor E2-related protein (Nrf2) [15]. Nrf2 is a basicleucine zipper transcription factor that under basal conditions, is present in an inactive form in the cytoplasm, bound to the Kelchlike ECH- associated protein 1 (Keap1) [16]. Various agents including Antioxidant Response Element (ROS) and weak electrophiles (e.g. isothiocyanates) can alter the Keap1-Nrf2 protein complex and free Nrf2 through phosphorilation or alkylation of one or more of the 27 cysteine residues in Keap1 [17]. When this occurs, Nrf2 translocates into the nucleus. Upon activation, Nrf2 dimerizes with a small Maf protein then binds to antioxidant responsive element (ARE) sites in the promoter regions of antioxidant and phase II genes, thereby inducing their transcription [18]. In recent years, many authors have suggested the existence of cross-talk between Nrf2/ARE and the nuclear factor-kappa B (NF-kB) signaling pathways in response to inflammation [191]. The Nrf2 and NF-kB signaling pathways interface at several points to control the transcription or function of downstream target proteins [22]. In addition, ROS now appear to act as second messengers in numerous signaling pathways [234]. One signaling pathway that engages in cross-talk with ROS involves NF-kB family transcription factors [257]. It had already been shown twenty years ago by Schreck and coworkers [28] that oxidative stresses, such as addition of extracellular hydrogen peroxide, can induced NF-kB nuclear translocation in several cell lines. The NF-kB family is made up of NF-kB1 (p50), NFkB2 (p52), RelA (p65), c-Rel and RelB. In the absence of stimuli, NF-kB, is associated with the inhibitor protein, IkBa, and sequestered in the cytosol. Upon stimulation with a NF-kB inducers, IkBa is rapidly phosphorylated on two serine residues (S32 and S36), which targets the inhibitor for ubiquitination and degradation by proteosome. Lisosan G is a powder obtained from Triticum Sativum (wheat) and it is registered with the Italian Ministry of Health as a nutritional supplement. In the production process, the wholegrain is first ground to a rough powder. From this intermediary product, the bran and germ are separated and collected for further treatment which consists in the following: water is added to moisten the mix, then selected microbic starting cultures are inoculated to initiate fermentation. The starting cultures typically consist of a mix of lacto-bacillus and natural yeast strains. Once the product is sufficiently fermented, it is dried. The resulting dry powder is now Lisosan G, which is widely used in food production thanks to its rich nutritional content. It contains vitamins, minerals and polyunsatured fatty acids as well as having significant antioxidant activity [29]. 5320621In vivo, Lisosan G protects against cisplatin induced toxicity [30], and a recent paper showed that Lisosan G helps prevent microcirculatory dysfunction [31]. The authors of these works suggested that the protective effect of Lisosan G could be associated with the attenuation of oxidative stress and the preservation of antioxidant enzymes. To date, no studies have attempted to identify the molecular mechanism that determines antioxidant properties of Lisosan G. For this reason, in the present study, we investigated the effects of Lisosan G on the antioxidant and drug-metabolising enzymes at transcriptional, catalytic and protein levels using cultures of primary rat hepatocytes oxygenase-1 (sc-10789), NFkB (sc-7178), b-actin (sc-130657), PARP-1 (sc-25780) and goat anti-rabbit (1:2000 or 1:5000) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Collagen (type I) was prepared by the method of Beken et al. (1998).Hepatocytes were isolated from 20000 g Wistar male rats with free access to drinking water and food and on a 12 h light/ dark cycle. The research with the use of animals was approved by the Italian Ministry of Health in compliance with European Community law n. 116/92. The approved protocol number is 10/ 09.