Sustainability of CD24 expression, cell proliferation and migration, cisplatin-resistance, and caspase-3 expression during mesenchymal-epithelial transition induced by the removal of TGF-β1 in A549 lung cancer cells.
ABSTRACT: Epithelial-mesenchymal transition (EMT) is a notable mechanism underlying cancer cell metastasis. Transforming growth factor β1 (TGF-β1) has been used to induce EMT; however, there is a lack of information regarding the role of TGF-β1 in mesenchymal-epithelial transition (MET). In the present study, EMT was induced in A549 lung cancer cells using TGF-β1 (TGF-β1-treated group) and MET was induced sequentially from the TGF-β1-treated group by removing the TGF-β1 (MET/return group). Untreated A549 lung cancer cells were used as a control. Characteristic features, including cancer stem cell markers [cluster of differentiation (CD)24, CD44 and CD133], cell proliferation and migration and diverse intracellular mechanisms, were observed in all groups. Using western blot analysis, the TGF-β1-treated group demonstrated increased vimentin and reduced E-cadherin expression, whereas the MET/return group demonstrated the opposite trend. Among cancer stem cell markers, the population of CD24low cells was reduced in the TGF-β1-treated group. Furthermore, the G2/M phase cell cycle population, cisplatin-sensitivity, and cell proliferation and migration ability were increased in the TGF-β1-treated group. These features were unaltered in the MET/return group when compared to the TGF-β1-treated group. Immunoblotting revealed an increase in the levels of SMAD3, phosphorylated SMAD3, phosphorylated extracellular signal-regulated kinase and caspase-3, and a decrease in active caspase-3 levels in the TGF-β1-treated group. Increased caspase-3 and reduced active caspase-3 levels were observed in the MET/return group, similar to those in the TGF-β1-treated group; however, levels of other signalling proteins were unchanged compared with the control group. EMT induced by TGF-β1 was not preserved; however, stemness-associated properties (CD24 expression, caspase-3 expression, cell proliferation and cisplatin-resistance) were sustained following removal of TGF-β1.
Project description:Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT.
Project description:Transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) contributes to the pathophysiological development of kidney fibrosis. Although it was reported that TGF-β1 enhances β(1) integrin levels in NMuMG cells, the detailed molecular mechanisms underlying TGF-β1-induced β(1) integrin gene expression and the role of β(1) integrin during EMT in the renal system are still unclear. In this study, we examined the role of β(1) integrin in TGF-β1-induced EMT both in vitro and in vivo. TGF-β1-induced augmentation of β(1) integrin expression was required for EMT in several epithelial cell lines, and knockdown of Smad3 inhibited TGF-β1-induced augmentation of β(1) integrin. TGF-β1 triggered β(1) integrin gene promoter activity as assessed by luciferase activity assay. Both knockdown of Smad3 and mutation of the Smad-binding element to block binding to the β(1) integrin promoter markedly reduced TGF-β1-induced β(1) integrin promoter activity. Chromatin immunoprecipitation assay showed that TGF-β1 enhanced Smad3 binding to the β(1) integrin promoter. Furthermore, induction of unilateral ureteral obstruction triggered increases of β(1) integrin in both renal epithelial and interstitial cells. In human kidney with chronic tubulointerstitial fibrosis, we also found a concomitant increase of β(1) integrin and α-smooth muscle actin in tubule epithelia. Blockade of β(1) integrin signaling dampened the progression of fibrosis. Taken together, β(1) integrin mediates EMT and subsequent tubulointerstitutial fibrosis, suggesting that inhibition of β(1) integrin is a possible therapeutic target for prevention of renal fibrosis.
Project description:IL-27 is a multifunctional cytokine that has both pro-inflammatory and anti-inflammatory functions. Although IL-27 has been shown to potently inhibit lung fibrosis, the detailed mechanism of IL-27 in this process is poorly understood. Epithelial-mesenchymal transition (EMT) is one of the key mechanisms involved in pulmonary fibrosis. We assessed the effects of IL-27 on TGF-β1-induced EMT in alveolar epithelial cells.A549 cells (a human AEC cell line) were incubated with TGF-β1, IL-27, or both TGF-β1 and IL-27, and changes in E-cadherin, β-catenin, vimentin and a-SMA levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT and TGF-β/Smad signalling pathways were examined by western blot.IL-27 increased the expression of epithelial phenotypic markers, including E-cadherin and β-catenin, and inhibited mesenchymal phenotypic markers, including vimentin and a-SMA in A549 cells. Moreover, TGF-β1-induced EMT was attenuated by IL-27. Furthermore, we found that TGF-β1 activated the phosphorylation of JAK1, STAT1, STAT3, STAT5, Smad1, Smad3 and Smad5, and IL-27 partially inhibited these changes in this process. When cells were treated with the STAT3 specific inhibitor wp1006 and the Smad3 specific inhibitor SIS3, the inhibition of EMT by IL-27 was significantly strengthened.Our results suggest that IL-27 attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the absence or presence of TGF-β1 through the JAK/STAT and TGF-β/Smad signalling pathways.
Project description:Epithelial-to-mesenchymal transition (EMT) is increasingly recognized as contributing to the pathogenesis of idiopathic pulmonary fibrosis. Therefore, novel plant-based natural, active compounds have been sought for the treatment of fibrotic EMT. The aim of the present study was to investigate the inhibitory effects of Astilbe rubra on TGF-β1-induced EMT in lung alveolar epithelial cells (A549). A. rubra was subjected to extraction using 70% ethanol (ARE), and ethanol extracts of the aerial part and that of the rhizome were further partitioned using various solvents. Protein expression and cell motility were investigated to evaluate the inhibitory effects of ARE on EMT. EMT occurred in A549 cells treated with TGF-β1, but was prevented by co-treatment with ARE. The dichloromethane fractions showed the strongest inhibitory effect on TGF-β1-induced EMT. β-Peltoboykinolic acid was isolated from the dichloromethane fractions of A. rubra by activity-oriented isolation. β-Peltoboykinolic acid not only attenuated TGF-β1-induced EMT, but also the overproduction of extracellular matrix components including type I collagen and fibronectin. The Smad pathway activated by TGF-β1 was inhibited by co-treatment with β-peltoboykinolic acid. Taken together, these results indicate that β-peltoboykinolic acid from A. rubra and dichloromethane fractions shows potential as an antifibrotic agent in A549 cells treated with TGF-β1.
Project description:Epithelial cancer cells can undergo an epithelial-mesenchymal transition (EMT), a complex genetic program that enables cells to break free from the primary tumor, breach the basement membrane, invade through the stroma and metastasize to distant organs. Myoferlin (MYOF), a protein involved in plasma membrane function and repair, is overexpressed in several invasive cancer cell lines. Depletion of myoferlin in the human breast cancer cell line MDA-MB-231 (MDA-231MYOFKD) reduced migration and invasion and caused the cells to revert to an epithelial phenotype. To test if this mesenchymal-epithelial transition was durable, MDA-231MYOFKD cells were treated with TGF-β1, a potent stimulus of EMT. After 48 hr with TGF-β1, MDA-231MYOFKD cells underwent an EMT. TGF-β1 treatment also decreased directional cell motility toward more random migration, similar to the highly invasive control cells. To probe the potential mechanism of MYOF function, we examined TGF-β1 receptor signaling. MDA-MB-231 growth and survival has been previously shown to be regulated by autocrine TGF-β1. We hypothesized that MYOF depletion may result in the dysregulation of TGF-β1 signaling, thwarting EMT. To investigate this hypothesis, we examined production of endogenous TGF-β1 and observed a decrease in TGF-β1 protein secretion and mRNA transcription. To determine if TGF-β1 was required to maintain the mesenchymal phenotype, TGF-β receptor signaling was inhibited with a small molecule inhibitor, resulting in decreased expression of several mesenchymal markers. These results identify a novel pathway in the regulation of autocrine TGF-β signaling and a mechanism by which MYOF regulates cellular phenotype and invasive capacity of human breast cancer cells.
Project description:Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-β1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-β1 on cell-cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-β1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-β1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-β1 enhanced gap junction formation and impacted cell-cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-β1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-β1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-β1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell-cell communication in osteocytes induced by TGF-β1.
Project description:Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and fetal morbidity and mortality with a prevalence of 6-8% of pregnancies. Although impaired trophoblast invasion in early pregnancy is known to be closely associated with preeclampsia, the underlying mechanisms remain elusive. Here we revealed that lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2) play a critical role in preeclampsia. Our results demonstrated that LOX and LOXL2 expression decreased in preeclamptic placentas. Moreover, knockdown of LOX or LOXL2 suppressed trophoblast cell migration and invasion. Mechanistically, collagen production was induced in LOX- or LOXL2-downregulated trophoblast cells through activation of the TGF-β1/Smad3 pathway. Notably, inhibition of the TGF-β1/Smad3 pathway could rescue the defects caused by LOX or LOXL2 knockdown, thereby underlining the significance of the TGF-β1/Smad3 pathway downstream of LOX and LOXL2 in trophoblast cells. Additionally, induced collagen production and activated TGF-β1/Smad3 were observed in clinical samples from preeclamptic placentas. Collectively, our study suggests that the downregulation of LOX and LOXL2 leading to reduced trophoblast cell migration and invasion through activation of the TGF-β1/Smad3/collagen pathway is relevant to preeclampsia. Thus, we proposed that LOX, LOXL2, and the TGF-β1/Smad3/collagen pathway can serve as potential markers and targets for clinical diagnosis and therapy for preeclampsia.
Project description:Epigenetic modifications, such as histone acetylation/deacetylation, have been shown to play a role in the pathogenesis of fibrotic disease. Peyronie's disease (PD) is a localized fibrotic process of the tunica albuginea, which leads to penile deformity. This study was undertaken to determine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of histone deacetylase 2 (HDAC2) in primary fibroblasts derived from human PD plaque. PD fibroblasts were pre-treated with HDAC2 siRNA and then stimulated with transforming growth factor-β1 (TGF-β1). Protein was extracted from treated fibroblasts for Western blotting and the membranes were probed with antibody to phospho-Smad2/Smad2, phospho-Smad3/Smad3, smooth muscle α-actin and extracellular matrix proteins, including plasminogen activator inhibitor-1, fibronectin, collagen I and collagen IV. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 in fibroblasts. Knockdown of HDAC2 in PD fibroblasts abrogated TGF-β1-induced extracellular matrix production by blocking TGF-β1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, and by inhibiting TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts. Decoding the individual function of the HDAC isoforms by use of siRNA technology, preferably siRNA for HDAC2, may lead to the development of specific and safe epigenetic therapies for PD.
Project description:Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor-β1 (TGF-β1)/Smad3-driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small-molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF-β1-induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin-1β and tumour necrosis factor-α) and reducing extracellular matrix deposition (α-smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF-κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down-regulated the expression of the fibrotic marker collagen I in TGF-β1-treated renal epithelial cells. Further, we found that petA dose-dependently suppressed Smad3-responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF-β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF-β/Smad3 signalling.
Project description:Transforming growth factor-β1 (TGF-β1) has an essential role in the development of kidney diseases. However, targeting TGF-β1 is not a good strategy for fibrotic diseases due to its multifunctional characteristic in physiology. A precise therapeutic target maybe identified by further resolving the underlying TGF-β1 driven mechanisms in renal inflammation and fibrosis. Smad signaling is uncovered as a key pathway of TGF-β1-mediated renal injury, where Smad3 is hyper-activated but Smad7 is suppressed. Mechanistic studies revealed that TGF-β1/Smad3 is capable of promoting renal inflammation and fibrosis via regulating non-coding RNAs. More importantly, involvement of disease- and tissue-specific TGF-β1-dependent long non-coding RNAs (lncRNA) have been recently recognized in a number of kidney diseases. In this review, current understanding of TGF-β1 driven lncRNAs in the pathogenesis of kidney injury, diabetic nephropathy and renal cell carcinoma will be intensively discussed.