Roles Of MAPK Signaling Pathway And Effects Of Thalidomide In The Transforming Growth Factor-β1-induced Alveolar Epithelial-mesenchymal Transition

Objective:To investigate the roles of p38mitogen-activated protein kinases (p38MAPK) and c-Jun N-terminal kinases (JNK) in the transforming growth factor-β1(TGF-β1)-induced epithelial to mesenchymal transition (EMT) of type II alveolar epithelial cells, and the effects of thalidomide on the EMT which induced by TGF-(31. This study was also designed to explore the effects of thalidomide on the p38MAPK and JNK expression during EMT and the possible mechanism of idiopathic pulmonary fibrosis (IPF), in order to find a new treatment target of IPF.Method:Part One A549cells were incubated with TGF-β1at a final concentration of3ng/ml for48h. In experiments using inhibitors, the cells were pre-incubated for1h with a JNK inhibitor SP-600125(Sigma, St. Louis, MO, USA) at5μM or a p38MAPK inhibitor SB-203580(Sigma, St. Louis, MO, USA) at5μM before treatment with exogenous TGF-β1. In gene silencing experiments, after shRNAs transfection the cells were stimulated with TGF-β1at a final concentration of3ng/ml in serum free0.1%BSA/DMEM for48h. AECs were transfected with100nM of p38MAPK shRNA and JNK shRNA or control shRNA using the LipofectamineTM2000according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Following incubation, transfection efficiency was evaluated by examining fluorescence intensity under a fluorescence microscopy. Cells were cultured in growth medium for48h and the knockdown efficiencies of the target genes were confirmed by determining decreases in the total expression levels of p38MAPK or JNK. Cells and media were collected for phospho-JNK and phospho-p38MAPK in30min after TGF-pM incubation. The morphological evaluation and cell markers detection were carried out in48hours after TGF-β1incubation. Protein expression of p38MAPK, JNK, desmin, vimentin, a-SMA, E-cadherin, zonula occludens-1(ZO-1) and aquaporin-5(AQP-5) were detected by Western-blot. Positive band was analyzed by gel photodensitometry analysis software Gel pro4.5(Media Cybertic). p38MAPK and JNK expression levels for the genes of interest were analyzed via quantitative real-time RT-PCR using a Bio-Rad iCycler system (Bio-Rad, Hercules, CA, USA).Total RNA was reverse-transcribed into cDNA using an Omniscript RT kit. GAPDH primer was included in every plate as an internal loading control. The mRNA level of each sample for each gene was normalized against that of GAPDH mRNA. The relative mRNA level was determined as2[(Ct/GAPDH-Ct/gene of interest)]. All data are presented as the mean±S. E. M of three separate experiments.Part Two Thalidomide was diluted by dimethyl sulfoxide (DMSO). A549cells were cultured with TGF-β1(3ng/ml). Cells were randomly divided into three groups:A Control group; B TGF-β1+DMSO group;C TGF-β1+thalidomide (10μg/ml). Then cells were cultured for another48hours. Cells and media were collected for phospho-JNK and phospho-p38MAPK in30min after TGF-(31incubation. The morphological evaluation and cell markers detection were carried out in48hours after TGF-β1incubation. Protein expression of p38MAPK, JNK, desmin, vimentin, a-SMA, E-cadherin, zonula occludens-1(ZO-1) and aquaporin-5(AQP-5) were detected by Western-blot, In Western-blot, GAPDH was used as a control for equal protein loading. Positive band was analyzed by gel photodensitometry analysis software Gel pro4.5(Media Cybertic).Results:Part oneTGF-β1significantly (P<0.05) decreased the expression of AQP5, ZO-1and E-cadherin, In parallel with the marked decrease in the AQP5, ZO-1and E-cadherin, TGF-β1significantly (P<0.05) induced the expression of desmin, vimentin and a-SMA. The treatment with TGF-β1also resulted into a significant (P<0.05) increased expression of phospho-p38MAPK and phospho-JNK at30min after TGF-β1incubation. In addition to the changes in the phenotypic markers expressed in A549cells after TGF-β1stimulation, the cells also underwent morphological changes. A549cells cultured in the absence of TGF-β1maintained a cobblestone epithelial morphology, but after stimulation with TGF-β1for48h, the cells adopted a more fibroblast-like morphology and reduced their cell-cell contact.In our study, JNK inhibitor SP-600125and p38MAPK inhibitor SB-203580, significantly (P<0.05) attenuated TGF-β1-induced desmin, vimentin and a-SMA expression when added to the cultures1h prior to TGF-β1. These two inhibitors also prevented the decrease in AQP5, ZO-1and E-cadherin production induced by TGF-β1. The activated forms of MAPK (phospho-JNK and phospho-p38MAPK) were significantly decreased at30min after TGF-β1incubation. JNK inhibitor SP-600125and p38MAPK inhibitor SB-203580separately inhibited the morphological transformation from epithelial phenotype to fibroblast-like cell morphology in TGF-β1-treated A549cells.The most effective shRNAs p38MAPK-H-999and JNK-H-888were separately selected to silence p38MAPK and JNK gene. p38MAPK and JNK gene silencing reduced the decreased expression of AQP5, ZO-1and E-cadherin induced by TGF-β1treatment. Correspondingly, the expression of desmin, vimentin and a-SMA was inhibited by p38MAPK and JNK gene silencing. Quantitative real-time PCR and western-blot analysis respectively revealed that total p38MAPK and JNK mRNA and protein expressions were significantly (P<0.01) inhibited by gene silencing in TGF-β1-treated cells, while negative control shRNAs had no detectable effects on protein expression. Interestingly, shRNA-mediated MAPK gene silencing presented a possible "cross-talk" phenomenon. Each specific shRNA could simultaneously inhibit both the expression of p38MAPK and JNK with a different extent in suppression. In addition, shRNA-mediated gene silencing inhibited the morphological transformation from epithelial phenotype to fibroblast-like cell morphology in TGF-P1-treated A549.Part Two Thalidomide could inhibit the EMT of A549cells. This study showed that Thalidomide could significantly mitigate the inhibition of the expression of AEC markers. The expression of desmin, vimentin and a-SMA was significantly lower in the B(TGF-β1+DMSO)group than that in C(TGF-β1+thalidomide) group (p<0.05). In addition, thalidomide also induced the expression of mesenchymal markers:E-cadherin, zonula occludens-1(ZO-1)and aquaporin-5(AQP-5) level in C(TGF-β1+thalidomide) group was significantly lower than that in B(TGF-β1+DMSO)group (p<0.05). Furthermore, the phosphorylation of p38MAPK and JNK during TGF-β1-induced EMT was significantly inhibited by thalidomide, the phosphorylation level of p38MAPK and JNK was significantly lower in C(TGF-β1+thalidomide) group than that in B(TGF-β1+DMSO)group (p<0.05).Conclusion:1.TGF-β1could induces A549alveolar epithelial cells (AECs) to undergo epithelial to mesenchymal transition (EMT).2. TGF-β1could induces A549alveolar epithelial cells (AECs) to undergo epithelial to mesenchymal transition (EMT) partially via p38MAPK and JNK activation.3. Thalidomide could significantly mitigate the inhibition of the expression of AEC markers and significantly inhibit the expression of mesenchymal markers during EMT, via inhibiting the phosphorylation of p38MAPK and JNK, which means that thalidomide could inhibit the EMT of A549cells via interfering the activation of MAPKs.