The Effect Of TGF-β1 And Smad7 Gene Transfer On The Phenotypic Changes Of Rat Type Π Alveolar Epithelial Cells

IntroductionIn recent years, with the use of bleomycin (BLM), an anticancer drug, and the prolongation of life expectancy, the incidence of pulmonary fibrosis has been increasingly elevated. Although there has been a large body of research related to pulmonary fibrosis at present, the exact pathogenesis of pulmonary fibrosis has not been elucidated without any effective measures of both prevention and treatment clinically.In the past, studies of scholars both at home and abroad including our study group had found that at pulmonary fibrosis, the hyperplastic mesenchymal cells had marked heterogeneity in immune label, light microscope, electronic microscope and morphology. Due to limited experiment condition and theory, the proliferative mesenchymal cells at pulmonary fibrosis were thought to be primarily Myofibroblasts (MFb) which came from the inherent mesenchymal cells of the lung. After the year 2000, some scholars had proposed the theory of multiple origin of MFb in organic fibrotic foci. As for pulmonary fibrosis, some reports claimed that except the inherent mesenchymal cells of the lung, MFb might also come from peripheral blood (bone marrow-derived). Recently, there have been reports that typeⅡalveolar epithelial cells within the pulmonary fibrotic foci may be one of the sources of MFb through epithelial-mesenchymal transition (EMT) . Transforming growth factor TGF-β1 plays a very important role in EMT. Recent researches have also found that Smad (Sma/Mothers Against Decapentaplegic) protein is a extremely important inducing molecule in the process of signal transduction of TGF-β1 within the cells. The combination of TGF-β1 with its receptors on cell membrane surface may activate both receptors of Smad2 and Smad3 which form a trimer with the common pathway of Smad4 and get into the nucleus, thus involving in the upregulation of the expression of some EMT-relevant genes together with some relevant factors within the nucleus. During this process, some researches demonstrated that the epidermal growth factor (EGF) can cooperate with TGF-β1 to induce the hologenesis of the EMT of nephric tubule through the action of promoting growth and inhibiting apoptosis. In the first place, this study is to observe the effect of TGF-β1 on the phenotype change of RLE-6TN of typeⅡalveolar epithelial cells and its relevant mechanism. Then, the stable transgenic technology using vitro cells was employed to observe the effect of TGF-β1 on the phenotype of RLE-6TN which had been transfected by Smad7 and the effect and the mechanism of the combination of TGF-β1 and EGF on the phenotype and function changes of RLE-6TN, so as to investigate the possible epithelial source of myofibroblast within the lung when pulmonary fibrosis occurred and to explore the role of the signal pathway of TGF-β1/Smad in the pulmonary fibrosis. Consequently, the pathogenesis of pulmonary fibrosis is to be further elucidated for laying a solid foundation for the prevention and treatment of pulmonary fibrosis clinically.PartⅠTGF-β1 Induced Epithelial-Mesenchymal Transition of TypeⅡAlveolar Epithelial CellsPurpose To investigate whether or not transforming growth factor-β3 (TGF-β1) could induce epithelial-mesenchymal transition (EMT) of typeⅡalveolar epithelial cells (RLE-6TN) in vitro and its possible mechanism.Methods After the in vitro cultured RLE-6TN were treated with TGF-β1 (3ng/mL) at different time points, expression of the markers of epithelial cell and mesenchymal cell was assayed using Western blot and Real-time PCR analysis. The expression ofα-SMA protein was examined by fluorescene microscope. Western blot was also used to check the effect of TGF-β1 on the p-SAPK/JNK, p-p38MAPK, p-ERK, p-Akt and poSmad2 expression of RLE-6TN. Morphological alterations were examined by phase-contrast microscope, and ultrastructure changes by electron microscope. The migration of cells was assayed using transwell system.Results Incubation of RLE-6TN with TGF-β1 resulted in the up-regulation of the expression of the total mesenchymocyte markers, of which Vimentin's expression upregulated at 1, 3 and 24h and was 4.03 times (P＜0.01), 3.37 times (P＜0.05) and 2.43 times that of the control group, respectively. The expression ofα-SMA mRNA upregulated at all the time points following the treatment of TGF-β1, especially at 1 and 24 h, and was 7.39 times (P＜0.01) and 8.37 times (P＜0.01) of the control group at 1 and 24 h respectively. The expression of FN mRNA upregulated at 1, 3 and 6h, and the highest expression at 3h was 2.43 times (P＜0.01) that of the control group. Under the treatment of TGF-β1, the expression of the total epithelial markers was down-regulated, of which the expression of E-cad mRNA down-regulated from 3 h, especially at 6 and 12 h, and was both 66% (P＜0.05) of that of the control group at these hours. The expression of CK19 mRNA down-regulated from 1h, especially at 3 and 24 h, and was 46% (P＜0.01) and 12% (P＜0.01) of the control group at these hours respectively. In addition, under the treatment of TGF-β1, the expression of autocrine TGF-β1 mRNA of RLE-6TN upregulated, especially at 1, 3 and 24 h, and was 4.38 times (P＜0.01), 4.34 times (P＜0.01) and 3.23 times that of the control group at these hours respectively. The expression of EGFR mRNA of RLE-6TN upregulated at 1, 3, 6 and 24 h, especially at 1h, and was 2.92 times (P＜0.01) that of the control group at 1 h. Incubation of RLE-6TN with TGF-β1 could also lead to the apparent up-regulation of the expression of p-p38MAPK, p-ERK, p-Akt and p-Smad2 and the transition of RLE-6TN to myofibroblast-like cells morphologically. In ultrastructure, TGF-β1 exposure resulted in the disappearance of the osmiophilic multilamellar bodies specific to RLE-6TN. In addition, TGF-β1 treatment could lead to the expression ofα-SMA by some RLE-6TN cells and the number of cells migrating to the microporous filtration membrane were greatly increased in vitro (P＜0.01).Conclusions TGF-β1 can induce EMT of RLE-6TN in vitro, and this transition is the common results of the initiated Smad signals pathway and downstream protein of EGFR signals pathway.PartⅡThe Effect of Smad7 Overexpression on TGF-β1-induced Phenotypic Changes of RLE-6TN CellsPurpose To investigate the effect of Smad7 overexpression on the TGF-β1-induced phenotypic changes of RLE-6TN cells and the possible signaling mechanism.Methods Cultured rat' RLE-6TN were transfected stably with recombinant plasmids of pcDNA3.0-mSmad7 by Lipofectamine^TM 2000. After the plasmid was purified and identified, the positive clones were determined by semiquantitative RT-PCR, Western blot analysis and immunoflurescence respectively. Prior to and following the transfection, the markers of epithelial and mesenchyumal cells were assayed using Western Blot. Following the TGF-β1 treatment of the positive cloned cells cultured in vitro, the cells were gathered at different time points, and the expression of markers of the epithelial cell (E-cad and CK19) and mesenchymal cell (FN,α-SMA and Vimentin) at the level of both mRNA and protein was assayed using Real-time PCR analysis and Western Blot, respectively. Indirect immune fluorescence method was used to examine theα-SMA. Morphological alterations were examined by phase-contrast microscope while the in vitro migration of cells was by transwell system. Western Blot was also used to check the effect of TGF-β1 on the p-Smad2/3 expression of RLE-6TN with or without Smad7 gene transfer.Results Smad7 was successfully transfected to RLE-6TN (two positive clones were named ST1 and ST6 respectively). After transfection, the expression of Smad7 mRNA and protein of ST1 cells were greatly increased. Compared with RLE-6TN cells and mock, the expression of E-cad protein which is the epithelial marker of ST1 upregulated greatly and was 4 times (P＜0.01) that of the control group, whereas the expression of CK19 upregulated obviously and was 4.8 times (P＜0.01) that of the control group. The expression of FN which is the meshenchymal marker was downregulated to be 57.9% (P＜0.05) of the control group, while the expression of Vimentin downregulted to be 37.5% (P＜0.01) of the control group. Following the TGF-β1 treatment, the expression of FN mRNA which is the meshenchymal marker of ST1 cells upregulted to be 1.26 times that of the control group at 1h with the upregulating extent less than that of the RLE-6TN ( which was 1.45 times that of the control group), and downregulated at 3, 6, 12 and 24 h to be 69% (P＜0.05), 27% (P＜0.01), 56% (P＜0.01) and 45% (P＜0.01). The expression of Vimentin mRNA upregulated at 1h to be 1.32 times that of the control group with the upregulating extent less than that of RLE-6TN cells (which was 4.03 times that of the control group), and downregulated at 3, 6, 12 and 24 h to be 97%, 51% (P＜0.01), 76% and 46% (P＜0.01).α-SMA mRNA expression had no obvious change except upregulation at 12h. The expression of the epithelial markers of E-cad and CK19 mRNA upregulated; E-cad mRNA expression was 10.8 (P＜0.01), 1.74, 3.58 and 12.64 (P＜0.01) times that of the control group at 1, 3, 6 and 12h, whereas the expression of CK 19 mRNA was continuosly upregulated (P＜0.01). At protein level, compared with pre-tranfection, the expression of the meshenchymal markers of FN was down-regulated at all the treatment time points,α-SMA mRNA expression upregulated at 24, 48 and 72h to be 2.4 (P＜0.05), 2.1 and 2.1 times that of the control group, with the upregulating extent less than RLE-6TN cells ( to be 13.8, 13.8 and 13 times that of the control group). Vimentin expression upregulated at 24, 48 and 72h with the upregulating extent similar to that of the RLE-6TN cells. The expression of the epithelial markers of E-cad and CK19 proteins had no marked change following the treatment of TGF-β1. Compared with pre-transfection, ST1 cells, under the effect of TGF-β1 treatment, maintained a confluent monolayer of cobblestone specific to the epithelial cells in morphology, but a few cells expressed a little ofα-SMA, with the number of ST1 cells migrating to the the microporous filtration membrane greatly increased. Following the treatment of TGF-β1, the p-Smad2/3 expression in the pre-transfection RLE-6TN cells upregulated and the expression had no change following the transfection.Conclusions Through Lipofectome methods, Smad7 gene has been successfully transfected to rat RLE-6TN cells and two positive clones (ST1, ST6) are gained. Smad7 mRNA and protein expression are greatly enhanced. Transfection of Smad7 gene in vitro can partly stop TGF-β1-induced EMT of RLE-6TN.PartⅢThe Effect of TGF-β1 and EGF on the Phenotype Changes and Function of RLE-6TN CellsPurpose To investigate the effect of TGF-β1 and EGF on the phenotypic changes and function of RLE-6TN cells and its relative mechanism.Methods After the in vitro cultured RLE-6TN grew to confluent state, 0.5% DMEM/F12 cultivated fluid was added for synchronization for 24 h. Then, the cells were divided into four groups: normal RLE-6TN, TGF-β1, EGF and TGF-β1＋EGF. Immune fluorescence method was used to examine the expression ofα-SMA. Expression of markers of the epithelial cell and mesenchymal cell was assayed using Western Blot. Flow cytometer was adopted to estimate the apoptosis of cell and Western blot was also used to examine the expression of CD147, MMP2 and MT1-MMP. The cells synchronized for 24 h were divided into three groups: normal RLE-6TN, TGF-β1 and EGF. Western Blot was used to test the expression of MAPKS (including ERK1/2, SAPK/JNK and p38), Akt and p-Smad2.Results Compared with the control group and under phase contrast microscope, the addition of TGF-β1 enlarged the cellular gap and scattered the cells to be singular, with the cell shapes to be fusiform or spidle. Following the treatment of EGF, the cells remained the pepple stone shape with different cells connected closely with one another. Following the treatment of the combination of TGF-β1 and EGF, the cells were scattered to be singular, with the shapes to be long fusiform. Under fluorescence microscope and compared with the control group, the cells had marked expression ofα-SMA in the group treated with TGF-β1, no expression ofα-SMA was seen in the group treated with EGF, and in the group treated with the combination of TGF-β1 and EGF, the cells appeared long fusiform with the expression ofα-SMA. At the protein level, TGF-β1 could induce the expression ofα-SMA (P＜0.01), EGF could not, whereas the treatment of the combination of TGF-β1 and EGF could result in the upregulation ofα-SMA expression (P＜0.01) with the upregulating extent less than that treated with TGF-β1 alone. TGF-β1 could upregulate FN expression (P＜0.01), EGF had no upregulation effect, and the treatment of the combination of TGF-β1 and EGF could result in the upregulation of FN expression (P＜0.01) with the upregulation extent less than that treated with TGF-β1 alone. Both TGF-β1 and EGF could slightly upregulate the expression of Vimentin with no statistical significance, the treatment of the combination of TGF-β1 and EGF could result in the upregulation of Vimentin expression (P＜0.01). TGF-β1 could downregulate E-cad expression (P＜0.05), EGF alone or the combination of TGF-β1 and EGF could slightly downregulate the expression of E-cad with no statistical significance. TGF-β1 and EGF could both slightly downregulte CK19 expression, and the treatment of the combination of the two could lead to the same effect as the treatment of TGF-β1 alone. TGF-β1 could slightly upregulate the expression EGFR, EGF could downregulte its expression (P＜0.05) and the combination of TGF-β1 and EGF could lead to the same effect as the treatment of TGF-β1 alone. The single use of TGF-β1 or EGF could induce the apoptosis of RLE-6TN, and the combination of the two could greatly induce the apoptosis (P＜0.01). The single use of TGF-β1 or EGF could upregulate the expression of MT1-MMP and MMP2, and the combination of the two could reach the same effect as the single use of TGF-β1. TGF-β1 alone or the combination of TGF-β1 and EGF could slightly downregulated the expression of CD147, and the treatment of EGF alone could upregulate its expression with no statistical significance. TGF-β1 and EGF could both upregulate the expression of the EGFR downstream MAPK signaling pathway molecules of p-ERK1/2 and p-p38 and the PI3 kinase pathway signaling molecule p-Akt. TGF-β1 could upregulated p-Smad2 expression.Conclusions EGF alone can not induce EMT of RLE-6TN cells in vitro. The combination of TGF-β1 and EGF has no apparent effect on the induction of EMT but has coordinating role in inducing the cell apoptosis. TGF-β1 induces the EMT of RLE-6TN primarily through EGFR and its downstream signaling molecules to involve in the induction of the MMPs expression.Conclusions1. TGF-β1 can induce EMT of typeⅡalveolar epithelial cells (RLE-6TN) in vitro.2. TGF-β1-induced EMT of RLE-6TN is the common results of the initiated Smad signal pathways and downstream protein of EGFR signal pathways.3. Through Lipofectin methods, Smad7 gene has been successfully transfected to rat RLE-6TN cells and two positive clones (ST1, ST6) are established.4. Transfection of Smad7 gene can partly prevent TGF-β1-induced EMT of RLE-6TN in vitro.5. EGF alone can not induce EMT of RLE-6TN cells in vitro. The combination of TGF-β1 and EGF has no apparent effect on the induction of EMT but has coordinating role in inducing the cell apoptosis.6. TGF-β1 induces the EMT of RLE-6TN primarily through EGFR and its downstream signaling molecules to involve in the induction of the MMPs expression.