TGF-β1 induces type I collagen deposition in granulosa cells via the AKT/GSK-3β signaling pathway-mediated MMP1 down-regulation

Type I collagen is the most abundant extracellular matrix (ECM) protein in the mammalian ovary, and comprises two COL1A1 subunits and one COL1A2 subunit. Matrix metalloproteinase 1 (MMP1) is a typical collagenase of type I collagen, that can be detected in ovarian follicles and early corpus luteum. Previous studies demonstrated that MMP1-mediated degradation of type I collagen plays a functional role in regulating corpus luteum formation, and transforming growth factor β1 (TGF-β1) inhibits luteinization and progesterone production in granulosa cells (GCs). Whether TGF-β1 regulates the expression of MMP1, COL1A1, or the deposition of type I collagen during corpus luteum formation remains to be elucidated. This study aimed to investigate the molecular mechanisms through which TGF-β1 regulates MMP1 expression and type I collagen deposition in GCs. Our results show that TGF-β1 upregulates COL1A1 expressions and downregulates MMP1 expression. Inhibition approaches, including pharmacological inhibitors such as p38 inhibitor (SB203580), ERK1/2 inhibitor (U0126), AKT inhibitor (LY294002), and GSK-3β inhibitor (LiCl), as well as knockdown using siRNA specific to these genes, were used. Our results suggest that TGF-β1 decreases MMP1 production via an ALK5-mediated AKT/GSK-3β-dependent signaling pathway, and a decrease in MMP1 levels and an increase in COL1A1 levels synergistically promote type I collagen deposition in GCs. Collectively, these findings provide novel insights into the underlying molecular mechanisms by which TGF-β1 upregulates type I collagen deposition in GCs.     

TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.     

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence,apoptosis and attenuated TGF-β signaling

Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. Conclusion: In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.     

Cdc42-interacting protein-4 promotes TGF-Β1-induced epithelial-mesenchymal transition and extracellular matrix deposition in renal proximal tubular epithelial cells

Cdc42-interacting protein-4 (CIP4) is an F-BAR (Fer/CIP4 and Bin, amphiphysin, Rvs) family member that regulates membrane deformation and endocytosis, playing a key role in extracellular matrix (ECM) deposition and invasion of cancer cells. These processes are analogous to those observed during the initial epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. The role of CIP4 in renal tubular EMT and renal tubulointerstitial fibrosis was investigated over the course of the current study, demonstrating that the expression of CIP4 increased in the tubular epithelia of 5/6-nephrectomized rats and TGF-β1 treated HK-2 cells. Endogenous CIP4 evidenced punctate localization throughout the cytosol, with elevated levels observed in the perinuclear region of HK-2 cells. Subsequent to TGF-β1 treatment, CIP4 expression increased, forming clusters at the cell periphery that gradually redistributed into the cytoplasm. Simultaneously, EMT induction in cells was confirmed by the prevalence of morphological changes, loss of E-cadherin, increase in α-SMA expression, and secretion of fibronectin. Overexpression of CIP4 promoted characteristics similar to those commonly observed in EMT, and small interfering RNA (siRNA) molecules capable of CIP4 knockdown were used to demonstrate reversed EMT. Cumulatively, results of the current study suggest that CIP4 promotes TGF-β1-induced EMT in tubular epithelial cells. Through this mechanism, CIP4 is capable of inducing ECM deposition and exacerbating progressive fibrosis in chronic renal failure.     

Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-β1

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-β1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-β1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-β1. Increased expression of TGF-β1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-β1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-β1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-β1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.     

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.     

Role of Smad2/3 and p38 MAP kinase in TGF-β1-induced epithelial-mesenchymal transition of pulmonary epithelial cells

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-β1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-β1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-β1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-β1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-β1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-β1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.     

KLF2 attenuates bleomycin-induced pulmonary fibrosis and inflammation with regulation of AP-1

Pulmonary fibrosis (PF) is a chronic, fibrosing interstitial pneumonia and devastating disease. Here we investigated the potential roles of Kruppel-like factor 2 (KLF2) on pulmonary fibrosis and inflammation response. A mouse model of pulmonary fibrosis was established by intratracheal injection of bleomycin (BLM). The mRNA and protein levels of KLF2 were assayed by RT-PCR and Western blotting respectively. The extent of lung fibrosis was determined using hematoxylin and eosin (HE) staining and Masson's trichrome staining, and the hydroxyproline content was quantified. RT-PCR was used to evaluate the mRNA expression of collagen type 1a1 (col1a1), col3a1, α-SMA, TNF-α, IL-1β and IL-6. The concentrations of TNF-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) and lung tissue were examined by ELISA. Also, the effects of KLF2 on activator protein-1 (AP-1) were evaluated by measuring the c-Jun and c-Fos protein levels. We found that KLF2 was remarkably downregulated in BLM-treated rats, both in mRNA and protein levels. Additionally, overexpression of KLF2 attenuated the destruction of the alveolar space and pulmonary interstitial collagen hyperplasia, and deposition reduced the expression of col1a1, col3a1, and α-SMA, and blocked the production of TNF-α, IL-1β, and IL-6 in BALF and lung tissue in vivo. Moreover, adenoviral transduction of KLF2 inhibited TGF-β1-induced expression of col1a1, col3a1, and α-SMA in vitro. Mechanically, BLM up-regulated c-Jun and c-Fos expression, which was impeded by KLF2 overexpression. Taken together, our data indicate that KLF2 attenuates pulmonary fibrosis and inflammation, possibly through the regulation of AP-1.     

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy

Transforming growth factor-β (TGF-β) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-β₁ signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-β₁ in cultured mesangial cells. In addition, the expression of TGF-β type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-β₁. An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.     

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G₂/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G₂/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G₂/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G₁/S phase, and increased COL1 expression in the G₂/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G₂/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G₂/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G₂/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G₂/M arrest.     

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis

Background and aims

Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis.

Methods

HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo.

Results

Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice.

Conclusions

Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.     

MiR-448-5p inhibits TGF-β1-induced epithelial-mesenchymal transition and pulmonary fibrosis by targeting Six1 in asthma

MicroRNAs (miRNAs) are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. The aim of this study was to explore how miR-448-5p affects airway remodeling and transforming growth factor-β1 (TGF-β1)-stimulated epithelial-mesenchymal transition (EMT) by targeting Sine oculis homeobox homolog 1 (Six1) in asthma. Asthmatic mice models with airway remodeling were induced with ovalbumin solution. MiRNA expression was evaluated using quantitative real-time polymerase chain reaction. Transfection studies of bronchial epithelial cells were performed to determine the target genes. A luciferase reporter assay system was applied to identify whether Six1 is a target gene of miR-448-5p. In the current study, we found that miR-448-5p was dramatically decreased in lung tissues of asthmatic mice and TGF-β1-stimulated bronchial epithelial cells. In addition, the decreased level of miR-448-5p was closely associated with the increased expression of Six1. Overexpression of miR-448-5p decreased Six1 expression and, in turn, suppressed TGF-β1-mediated EMT and fibrosis. Next, we predicted that Six1 was a potential target gene of miR-448-5p and demonstrated that miR-448-5p could directly target Six1. An SiRNA targeting Six1 was sufficient to suppress TGF-β1-induced EMT and fibrosis in 16HBE cells. Furthermore, the overexpression of Six1 partially reversed the protective effect of miR-448-5p on TGF-β1-mediated EMT and fibrosis in bronchial epithelial cells. Taken together, the miR-448-5p/TGF-β1/Six1 link may play roles in the progression of EMT and pulmonary fibrosis in asthma.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

Combined treatment with zingerone and its novel derivative synergistically inhibits TGF-β1 induced epithelial-mesenchymal transition,migration and invasion of human hepatocellular carcinoma cells

The epithelial–mesenchymal transition (EMT) is an important cellular process during which polarized epithelial cells become motile mesenchymal cells, which promote cancer metastasis. Ginger, the rhizome of Zingiber officinale, is extensively used in cooking worldwide and also as a traditional medicinal herb with antioxidant, anti-inflammatory and anticancer properties. Several pungent compounds have been identified in ginger, including zingerone, which has anticancer potential. However, the role of zingerone in EMT is unclear. We investigated the synergistic effect of zingerone and its derivative on EMT. Transforming growth factor-beta 1 (TGF-β1) induces the EMT to promote hepatocellular carcinoma metastasis, including migration and invasion. To understand the repressive role of the combination of zingerone and its derivative (ZD 2) in hepatocellular carcinoma metastasis, we investigated the potential use of each compound of ginger, such as zingerone, ZD 2 and 6-shogaol, or the mixture of zingerone and ZD 2 (ZD 2-1) as inhibitors of TGF-β1 induced EMT development in SNU182 hepatocellular carcinoma cells in vitro. We show that ZD 2-1, but not zingerone, ZD 2 and 6-shogaol significantly increased expression of the epithelial marker E-cadherin and repressed Snail upregulation and expression of the mesenchymal marker N-cadherin during initiation of the TGF-β1 induced EMT. In addition, ZD 2-1 inhibited the TGF-β1 induced increase in cell migration and invasion of SNU182 hepatocellular carcinoma cells. Furthermore, ZD 2-1 significantly inhibited TGF-β1 regulated matrix metalloproteinase-2/9 and activation of Smad2/3. We also found that ZD 2-1 inhibited nuclear translocation of NF-κB, activation of p42/44 MAPK/AP1 signaling pathway in the TGF-β1 induced EMT. Our findings provide new evidence that combined treatment with ZD 2, novel zingerone derivative, and zingerone synergistically suppresses hepatocellular carcinoma metastasis in vitro by inhibiting the TGF-β1 induced EMT.     

Dioscin suppresses TGF-β1-induced epithelial-mesenchymal transition and suppresses A549 lung cancer migration and invasion

Epithelial-to-mesenchymal transition (EMT), an important cellular process, occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Dioscin is a polyphenolic component isolated from Phyllanthus amarus, which exhibits a wide range of pharmacological and physiological activities, such as anti-tumor, anti-inflammatory, anti-obesity, anti-fungal, and anti-viral activities. However, the possible role of dioscin in the EMT is unclear. We investigated the suppressive effect of dioscin on the EMT. Transforming growth factor-beta 1 (TGF-β1) is known to induce EMT in a number of cancer cell types and promote lung adenocarcinoma migration and invasion. To verify the inhibitory role of dioscin in lung cancer migration and invasion, we investigated the use of dioscin as inhibitors of TGF-β1-induced EMT in A549 lung cancer cells in vitro. Here, we found that dioscin prominently increased expression of the epithelial marker E-cadherin and expression of the mesenchymal marker N-cadherin and Snail during the TGF-β1-induced EMT. In addition, dioscin inhibited the TGF-β1-induced increase in cell migration and invasion of A549 lung cancer cells. Also, dioscin remarkably inhibited TGF-β1-regulated activation of MMP-2/9, Smad2, and p38. Taken together, our findings provide new evidence that dioscin suppresses lung cancer migration, and invasion in vitro by inhibiting the TGF-β1-induced EMT.     

Identification of Epithelial to Mesenchymal Transition as a Novel Source of Fibroblasts in Intestinal Fibrosis

Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin⁺ intestinal epithelial cells activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells that express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, the appearance of LacZ⁺ cells that also express the fibroblast marker FSP1 unequivocally demonstrates EMT. Transforming growth factor (TGF)-β1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells in vitro, and bone morphogenic protein-7, an antagonist of TGF-β1, inhibits EMT and fibrosis both in vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn colitis and that inhibition of TGF-β1 with recombinant human bone morphogenic protein-7 prevents this process and prevents fibrosis.     

The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats

Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-β1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-β1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague–Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-β1 siRNA 0.125 mg/kg treatment group, TGF-β1 siRNA 0.25 mg/kg treatment group and TGF-β1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin–Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-β1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-β1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-β1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-β1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-β1 siRNA negative control group and the TGF-β1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-β1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-β1 can inhibit the expression of TGF-β1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-β1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.