the EMT process induced by TGF-β1 , and Nrf2 ac- tivation can restore the expression of klotho and then at- tenuates oxidative stress and inflammation in CKD . In our study, treatment with OA could restore the expres- sion of klotho in NRK-52E cells which is down-regulated by TGF-β1. The results showed that OA attenuates renal EMT induced by TGF-β1 in NRK-52E cells may be primar- ily involved the upregulation of Nrf2 and klotho expression. To further address the mechanism by which OA inhibits EMT in NRK-52E cells induced by TGF-β1, we focused on components downstream of TGF-β1 signaling. TGF-β1/ Smads pathway plays a critical role in TGF-β1-induced EMT in epithelial cells. It has been demonstrated that acti- vation of TGF-β1 signaling triggers a dramatic induction of Smad2/3 phosphorylation. Previous reports showed that Nrf2 is involved in the inhibition of smad activation path- way by TGF-β1. Moreover, many reports have shown that klotho suppresses TGF-β1-induced EMT responses in cul- tured cells, including decreased epithelial marker expres- sion, increased mesenchymal marker expression, and/or increased cell migration by inhibiting TGF-β1/Smads path- way . The results in this study suggests that OA attenu- ates TGF-β1-induced EMT in NRK-52E cells associated with the modulation of the TGF-β1/Smads pathway. Snail,
Abstract: Epithelial-mesenchymal transition (EMT) is considered as the first and key step for the migration and inva- sion of cancers including colon cancer. Ubiquitin-conjugating enzyme E2T (UBE2T), a member of the E2 family, plays an important role in tumorigenesis and progression. However, its role in colon cancer has not been investigated. In this study, we investigated the effect of UBE2T on transforming growth factor-β1 (TGF-β1)-mediated EMT in colon cancer cells and its underlying mechanisms. Our results showed that UBE2T was highly expressed in human colon cancer tissues and cell lines, and TGF-β1 treatment obviously induced the expression of UBE2Tin colon cancer cells. In addition, knockdown of UBE2T inhibited TGF-β1-induced EMT process, as well as migration and invasion in colon cancer cells. Furthermore, knockdown of UBE2T inhibits phosphorylation of PI3K and Akt in TGF-β1-stimulated colon cancer cells. In conclusion, we demonstrated that UBE2T silencing prevented TGF-β1-induced EMT in colon cancer cells by inhibiting the PI3k/Akt signaling pathway. Therefore, UBE2T may be a potential therapeutic target for the treatment of colon cancer.
cell surface to the nucleus, which contributes to the pro- liferation and differentiation of cells, such as prolifera- tion of lung fibroblasts and production of extracellular matrix (ECM). Researchers have proved that the ECM process may be achieved partly via the activation of TGF-β pathway, involving the TGF-β1-induced MAPK pathway [38, 39]. Taken together, the similarly upregula- tion of the protein level of pulmonary ERK1/2 in BLM- treated mice observed in this study may be explained by the above mechanism. However, there is no direct report about the relationship between A2aR and TGF-β1- induced ERK1/2 pathway in pulmonary fibrosis. Our data shown a significantly increased protein expression of TGF-β1 and phospho-ERK1/2 in KO model mice, which illustrated that A2aR could down-regulate the TGF-β1-induced ERK1/2 pathway in lung fibrosis for the first time. From another point of view, these results confirmed the protective effect of A2aR on pulmonary fibrosis. It is interesting to note that in the western blot graph of p-ERK1/2, the increase degree of p-ERK1 in WM group seems higher than that of p-ERK2, but no obvious difference was found in the rest of groups be- tween p-ERK1 and p-ERK2. We speculate that there are two probable reasons: the first reason is that it was caused by the differences between individual mice; the second reason is that the pathway of ERK1 was indeed more associated with IPF than ERK2. Though the under- lying mechanism is still unclear, we will perform further researches to clarify it.
of TGF-β1-induced Smad3 activation and growth inhibition was due to enhanced phosphorylation of Smad3 linker region (pSmad3L) through activation of BLT1- NAD(P)H oxidase (NOX)-reactive oxygen species (ROS)-epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3-K)-extracellular signal-activated kinase1/2 (ERK1/2)-linked signaling cascade. Furthermore, the LTB 4 /BLT1 signaling pathway leading to pSmad3L was constitutively activated in breast cancer cells and was correlated with TGF-β1-resistant growth of the cells in vitro and in vivo. In human breast cancer tissues, the expression level of pSmad3L (Thr179) had a positive correlation with BLT1 expression. Collectively, our data demonstrate for the first time that the induction of pSmad3L through BLT1-NOX-ROS-EGFR-PI3K-ERK1/2 signaling pathway is a key mechanism by which LTB 4 blocks the anti-proliferative responses of TGF-β1, providing a novel mechanistic insight into the connection between enhanced inflammatory signal and cancer cell growth.
Abstract: 3,3’-Diindolylmethane (DIM) is a natural component of cruciferous plants. Previous studies have shown that DIM has multiple physiological effects including anti-angiogenic, anti-inflammatory and anti-cancer effect. How- ever, little is known about the role of DIM on myofibroblast differentiation and extracellular matrix (ECM) production. This study investigated the effect of DIM on myofibroblast differentiation and ECM production in neonatal rat cardiac fibroblasts induced by transforming growth factor β1 (TGF-β1). We found that DIM blunted TGF-β1induced conver- sion of cardiac fibroblast into myofibroblast, and reduced the mRNA and protein expressions of α-smooth muscle actin (α-SMA). Furthermore, DIM also significantly decreased the mRNA expression of fibrosis markers (Collagen I, Collagen III, connective tissue growth factor (CTGF) in neonatal rat cardiac fibroblasts induced by TGF-β1. DIM atten- uated the phosphorylation AKT and glycogen synthase kinase-3β (GSK-3β) induced by TGF-β1. Our results showed that DIM was a potential drug to attenuate myofibroblast differentiation and excessive ECM production induced by TGF-β1 through down-regulated AKT/GSK-3β signaling pathways.
ASMC migration is not only essential for devel- opment of respiratory system but also impor- tant for airway remodeling in asthma [20, 21]. Previous studies showed that ASMCs migration toward the airway epithelium in response to inflammatory mediators such as TGF-β1 con- tributes to the airway remodeling [22-24]. It is interesting that apigenin inhibited human umbilical vein endothelial cells (HUVECs) migra- tion, compared with the control . Here, we found that TGF-β1 significantly increased ASMCs migration, whereas, apigenin significa- ntly suppressed TGF-β1-induced ASMCs migra- tion.
Despite their functional cooperation in PDAC development, little information is available on a possible crosstalk between the Rac1/Rac1b and the TGF-β signalling pathways in the control of oncogenic cellular responses. We have shown earlier that Rac1 antagonized TGF-β1-induced growth inhibition and promoted cell migration in PDAC cells . However, in studying Rac1-induced signalling and function one has to bear in mind that all Rac1 siRNA-mediated silencing approaches also result in co-depletion of Rac1b mRNA transcripts. Thus, most of these studies [8, 21] did not discriminate between the effects of Rac1 and Rac1b. In the light of our recent discovery of Rac1b expression in PDAC cells, a reinvestigation of the distinct roles played by Rac1 and Rac1b in the regulation of TGF-β1-induced cell motility appeared therefore mandatory. In contrast to Rac1, the role of Rac1b in these processes is unclear at present. Although it was reported that Rac1b promotes cell motility induced by MMP-3 , another study demonstrated that activated Rac1b was unable to stimulate lamellipodia formation . Here we have used Rac1b-specific RNA interference and ectopic overexpression of Rac1b in non- malignant and malignant pancreatic ductal epithelial cells. The non-malignant pancreatic ductal epithelial cell line H6c7 as well as the PDAC cell lines Panc-1 and Colo357 were particularly suitable because i) their TGF- β1-responsiveness is well characterized, ii) all cell lines respond to TGF-β1 with cell migration in vitro [21, 22] and iii) they were frequently employed in animal models for assessing the therapeutic activities of TGF-β inhibitors for suppressing pancreatic cancer growth and metastasis [23-25].
Transforming growth factor-β (TGF-β) plays an important role in several diseases that characteristically involve changes in tissue rigidity, such as cancer and tissue fibrosis. To determine whether matrix rigidity regulates the effects of TGF-β, we examined NMuMG and MDCK epithelial cells cultured on polyacrylamide gels with varying rigidity and treated with TGF-β1. Decreasing matrix rigidity reduced cell spreading and increased TGF-β1-induced apoptosis, while increasing matrix rigidity resulted in epithelial-mesenchymal transition (EMT). To more carefully control cell spreading, microcontact printing was used to restrict ECM area and revealed that reducing cell spreading also increased apoptosis. Apoptosis on compliant substrates was associated with decreased FAK expression, and FAK overexpression rescued cell survival but not EMT. Further investigation revealed manipulations of FAK activity, using pharmacological inhibitors or expression of FAK mutants, did not affect apoptosis or EMT, suggesting that FAK regulates apoptosis through expression but not activity. Additional investigation into the signaling pathways regulated by rigidity revealed a role for PI3K/Akt. We observed increased Akt activity with increasing rigidity, and that PI3K/Akt activity was necessary for cell survival and EMT on rigid substrates. These findings demonstrate that matrix rigidity regulates a switch in TGF-β-induced cell functions through rigidity-dependent regulation of FAK and PI3K, and suggest that changes in tissue mechanics during disease contribute to the cellular response to TGF-β.
The best combination to partially reverse TGF-β1- induced EMT to a more epithelial phenotype was SAHA (a HDAC inhibitor) associated with a bromodomain in- hibitor. With this treatment, NRP2, SEMA3C, and PD-L1 expression were reduced. For other classical EMT genes, the response was gene-specific. More experiments would be necessary with dose response for each compound alone or in combination, associated with functional tests for cell migration and invasion. In addition, this first screening would be improved by use of a 3D cell culture model. Several studies highlight the benefits of epigenetic-based therapeutic strategy in mouse models. Combining the bromodomain inhibitor JQ1 with the histone deacetylase inhibitor SAHA in pancreatic cancer inhibits both MYC activity and inflammatory signals as well as in an estab- lished adenocarcinoma lung cancer model with KRas G12D
Proliferative vitreoretinopathy (PVR) is a complication that can follow rhegmatogenous retinal detachments. Proliferative membrane contraction can result in re- duced vision or blindness . ECM deposition and cell proliferation are associated with PVR development, how- ever, the mechanism underlying the relationship between ECM deposition and cell proliferation and PVR devel- opment is unclear. The KKS is one of the main pressure-releasing systems in the human body, and BK is the primary determinant of KKS activity and thus plays a vital role in regulating inflammation, cell prolif- eration, and matrix hyperplasia. The present study attempted to elucidate the mechanism by which BK protects against PVR development. Multiple types of cells and cytokines are involved in the pathogenesis of PVR, and TGF-β1-induced cell proliferation is closely related to PVR development.
TGF-β1/Smad signaling pathway plays an im- portant role in regulating the proliferation, migration and ECM expression of ASMCs. To further explore the molecular mechanisms by which PCA inhibited TGF-β1-induced ASMCs proliferation and migration, we examined the effect of PCA on the activation of TGF-β1/Sm- ad pathway using western blot. As shown in Figure 4A, TGF-β1 treatment significantly in- creased the phosphorylation of Smad2/3 in ASMCs. However, pre-treatment of ASMCs wi- th PCA markedly inhibited TGF-β1-induced the phosphorylation of Smad2/3. Quantification analysis of p-Smad2 and p-Smad3 was per- formed using Image-Pro Plus 6.0 software (Fi- gure 4B).
Tumor metastasis is a complex and multistep process and its exact molecular mechanisms remain unclear. We attempted to find novel microRNAs (miRNAs) contributing to the migration and invasion of breast cancer cells. In this study, we found that the expression of miR-487a was higher in MDA-MB-231breast cancer cells with high metastasis ability than MCF-7 breast cancer cells with low metastasis ability and the treatment with transforming growth factor β1 (TGF-β1) significantly increased the expression of miR-487a in MCF-7 and MDA-MB-231 breast cancer cells. Subse- quently, we found that the transfection of miR-487a inhibitor significantly decreased the expres- sion of vimentin, a mesenchymal marker, while increased the expression of E-cadherin, an epi- thelial marker, in both MCF-7 cells and MDA-MB-231 cells. Also, the inactivation of miR-487a inhibited the migration and invasion of breast cancer cells. Furthermore, our findings demon- strated that miR-487a directly targeted the MAGI2 involved in the stability of PTEN. The down-regulation of miR-487a increased the expression of p-PTEN and PTEN, and reduced the expression of p-AKT in both cell lines. In addition, the results showed that NF-kappaB (p65) sig- nificantly increased the miR-487a promoter activity and expression, and TGF-β1induced the in- creased miR-487a promoter activity via p65 in MCF-7 cells and MDA-MB-231 cells. Moreover, we further confirmed the expression of miR-487a was positively correlated with the lymph nodes metastasis and negatively correlated with the expression of MAGI2 in human breast cancer tissues. Overall, our results suggested that miR-487a could promote the TGF-β1-induced EMT, the mi- gration and invasion of breast cancer cells by directly targeting MAGI2.
. The initiation of TGF- β signaling is triggered through the binding of TGF- β cytokines to their cognate receptors (TGF- β RI and RII). Upon ligand binding, the activated TGF- β RI recruits and phosphorylates Smad proteins, i.e. Smad 2/3 for TGF- β s, Activin, Nodals, and Smad 1/5/8 for BMPs . Many studies [36, 37] have reported that it is Smad 2/3 which play an important role in SMCs differentiation. However, there are also studies [13, 38] which have shown that BMP4 could me- diate SMCs differentiation via the Smad 1/5/8 pathway. Therefore in our study, we selected TGF- β 1 and BMP4 as two representative growth factors of the TGF- β super- family to induce SMCs differentiation. The results of this study conclusively demonstrated that BMP4 alone exerted negligible effects on the differentiation of SHED into SMCs. Furthermore, the effects of TGF- β 1 on SHED-SMCs differentiation could be weakened when combined with BMP4. We hypothesize that BMP4 might compete with TGF- β 1 for binding to TGF- β receptors (TGF- β RI and RII) during the process of SHED-SMCs differentiation. Although accumulated evidence shows that BMPs play crucial roles in the regulation of stem cell properties and lineage fate, their functions vary with different stem cell types. While the differentiation of SHED into SMCs could be mediated by TGF- β 1, no sig- nificant differences were observed between concentra- tions of 10 ng/ml to 30 ng/ml TGF- β 1. The results of our analysis of the Smad signaling pathway showed that upon interaction with TGF- β 1, the TGF receptors re- cruited and phosphorylated the downstream targets of Smad 2 and 3. Besides this classical signaling pathway, several other signaling pathways that affect SMC differ- entiation under TGF- β 1 stimulation have also been re- ported, such as the p38 , AKT , and RhoA  signaling pathways. In this study, we investigated the ef- fects of SB-431542, a specific inhibitor of ALK5 , on the differentiation of SHED into SMCs. In the presence of SB-431542, SHED could not be induced into SMCs under TGF-β1 stimulation, which thus confirmed the crucial role of the TGF-β1-ALK5 signaling pathway in modulating the differentiation of SHED into SMCs. This finding could explain why BMP4 had negligible ef- fects on the course of differentiation of SHED into SMCs. Unlike TGF-β1, BMP4 functions through Alk2, Alk3 and Alk6 that mediate phosphorylation of Smad1, Smad5, or Smad8, which in turn modulate target gene expression at the transcriptional level .
During the process of stimulating lung pericyte transdifferentiation by TGF-β1, the pericyte morphology transformed from a multi-star shape into a fusiform myofibroblast shape. The results demonstrated that the pericytes in the control group were multi-star or medusiform shaped with a plump endochylema and clear outline. Following stimulation with TGF-β1 for 24 h, some of the cells began to stretch and separate from peripheral cells to induce a dis- ordered arrangement. Following stimulation with TGF-β1 for 48 h, the shape of the pericytes changed significantly and formed into a strip. After a 20-nM concentration of FUT8siRNA was applied to the pericytes and the cells were incubated for 24 h, TGF-β1 was applied to stim- ulate pericytes for 24 h and 48 h; the majority of the pericytes under an optical microscope remained in a normal cell shape without obvi- ous stretching or changes in the strip shape. This indicates that inhibiting core fucosylation by FUT8siRNA could suppress the develop- ment of the pericyte shape caused by TGF-β1 stimulation.
Ovalbumin (OVA)‑induced murine asthma model. A total of 10 BALB/c male mice (6 weeks old; 18‑20 g), obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. Mice were housed in plastic boxes with a 12‑h light/dark cycle and constant temperature (19‑23˚C) and humidity (55±10%). Food and water were supplied ad libitum. Mice were random‑ ized into two groups: The control group and the OVA group, each group containing 5 mice. On day 0 and day 14, the mice of the OVA group were sensitized with 20 µg OVA (Sigma‑Aldrich; Merck KGaA) with 1 mg aluminum hydroxide (Thermo Fisher Scientific, Inc.) adsorbed in 200 µl PBS by intraperitoneal injection. On days 21‑23, the mice of the OVA group were challenged through the airway with 1% OVA (dissolved in PBS) for 30 min using an ultrasonic nebulizer (INQUA NEB Plus; PARI GmbH). The mice of the control group were treated with PBS. All the mice were euthanized by cervical dislocation on day 24. The present study was approved by the Nanjing Medical University Animal Experimental Ethics Committee (approval no. 2005020).
TGF-b1 has been identified as a potent stimulus for EMT, whereby epithelial cells acquire hyperplasticity and develop a mesenchymal-cell phenotype [5,13-15]. TGF-b1 treatment has been shown to alter the cell mor- phology of the human AECII derived A549 cell line from cobblestone-shaped to a fibroblastoid appearance [13,15]. Phenotypic markers associated with EMT included diminished expression of E-cadherin, a cell anchoring protein expressed specifically by epithelial cells, and elevated expression of N-cadherin, normally present at relatively higher levels in fibroblasts [8,10]. These alterations were accompanied by increased secre- tion of the gelatinase matrix metalloproteinase-2 (MMP- 2) [13,14], increased cell motility [15,16] and de novo synthesis of fibrillar collagen I and III . Given the established actions of TGF-b1 on fibroblast differentia- tion, EMT and collagen synthesis, studies have investi- gated potential therapeutic benefits of targeting profibrotic effects of TGF-b1.
Induction of HDM2 gene expression has been reported in response to stimuli other than genotoxic stress. Transcription fac- tors such as N-Myc, Ets family members, Sp1, and ERα can bind and induce HDM2 gene expression through the second promoter (P2) (32–34). After an in silico survey of the P2 promoter of HDM2 for possible transcription factor–binding elements that are acti- vated in response to TGF-β1, 2 possible binding elements in the promoter conformed to the GTCT Smad3 DNA–binding element (SBE). One binding element (SBE2) was located at nucleotide –245 in the P2 promoter region of HDM2 and a second site (SBE1) was located at nucleotide –585 in the P2 promoter region of HDM2. To explore the possibility of Smad3 inducing HDM2 gene expression, we determined whether Smad2 and Smad3 were active after TGF-β1 exposure at times when Hdm2 levels were elevated. Dimeriza- tion of TβRI/RII in response to TGF-β1 formed an active serine/ threonine kinase that phosphorylates and activates Smad2 and Smad3. We observed Smad3 activation using a phospho-antibody to Smad3 to probe a Western blot of cellular extracts from 293T, SKOV3, Vaco400:RII, and HCT116:3-6 cells treated with TGF-β1 for 48 hours. As expected, Smad3 activation was not observed in parental HCT116 cells treated with TGF-β1 (Figure 4A). Impor- tantly, Smad3 activation occurred and corresponded with induc- tion of the HDM2 gene. To further explore the involvement of Smads in the activation of HDM2 gene induction, we transiently transfected a dominant negative Smad4 (dnSmad4) in transient reporter assays. Smad4 is a binding partner of Smad3, and a dom- inant negative form would presumably impede the activation of the HDM2 promoter. Using the HDM2 promoter and SBE2X2 as a control, cells transiently transfected with the respective promot- ers were treated with or without TGF-β1. As predicted, dnSmad4 blocked induction of the synthetic Smad reporter SBE2X2 and also prevented the activation of HDM2 promoter (Figure 4B). Thus, these data support a role for the Smads in the induction of Hdm2 in response to TGF-β1 treatment.
The distribution of NF- κ B in the cytoplasm and the nucleus were further examined (Fig. 4c). The transloca- tion of NF-κB p65 from the cytoplasm to the nucleus was observed after EMT induction by either YY1 overex- pression (Fig. 4d, lane 2 vs lane 1) or TGF- β treatment (Fig. 4d, lane 3 vs lane 1) in A549. The translocation of the NF- κ B p65 protein was further validated by immuno- fluorescence staining (Fig. 4e). Quantitative analysis (Fig. 4f) indicated the significant upregulation of the nuclear NF- κ B p65 by both YY1 overexpression and TGF- β treatment. Since nuclear translocation of NF- κ B has been confirmed to associate with the pro-fibrotic phe- notypes, the current data suggests that cytoplasm to nucleus translocation of NF- κ B p65 promotes YY1 expression, which in turn activates NF-κB signaling, mediating TGF- β -induced EMT and fibrosis in the lung epithelial cells (Fig. 5).
tion of myofibroblasts can contribute to chang- es in the structure and tone of the vessel wall under pathophysiologic conditions [36, 37]. Collagen is a major component of ECM and accounts for more than fifty percent of vascular wall with stenosis . Among all the collagen subtypes, type I collagen is considered to be most relevant to arterial remodeling. Synthesis and accumulation of collagen are essential for vascular homeostasis, development, and wou- nd healing. However, excessive collagen expres- sion and deposition can lead to fibrotic disor- ders. AS an active component of the vessel wall, ECM plays an important role in the patho- physiology of vascular diseases. Considerable studies have demonstrated TGF-β1 could induce fibroblasts into myofibroblasts by stimu- lating α-SMA expression  and promoting the secretion of ECM proteins, which have been found to play a vital role in the process of path- ological vascular remodeling. In accordance with previous findings, the α-SMA mRNA and protein expression levels is obviously up-regu- lated in AFs stimulated by TGF-β1. Moreover, we observed that pretreatment of adventitial fibroblasts with CTRP3 markedly decreased the expression of α-SMA and type I collagen, which were induced by TGF-β1. These findings sug- gest that CTRP3 could blunt vascular remodel- ing by inhibiting the differentiation of adventi- tial fibroblasts and secretion of type I collagen. Figure 3. CTRP3 weakens TGF-β1-induced collagen synthesis. A: Collagen I protein levels were assessed by Western blot. GAPDH was a loading control. B: Level of quantification of Collagen I as a ratio of GAPDH in densitometric units was presented. C: Collagen I mRNA levels were assessed by quantitative real-time PCR.