Adverse effects of cigarette smoke extract on the human A549 cell line

Smoking is directly related to the pathogenesis of chronic lung diseases. Cigarette with more than 7000 harmful substances in its content can stimulate epithelial-mesenchymal transition (EMT). EMT has a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease and lung cancer. Studies have shown that vitamin D has anti-fibrotic effects in fibrotic tissues and EMT regressive effects in cancer. It is also known that IPF patients have vitamin D deficiency. In the present study, we tested EMT-mediated myofibroblast differentiation whether or not could be prevented by exogenous vitamin D (cholecalciferol) in cigarette extract (CSE) and TGF-β-stimulated A549 cells. Additionally, interactions of EMT-associated transforming growth factor beta (TGF-β), Wnt / β-catenin signaling pathways and various proteins with vitamin D were investigated. One group of human A549 cells was stimulated with 5% CSE and 5% CSE + 5ng / ml TGF-β. Second group was treated with 50 nM vitamin D half an hour before stimulating with 5% CSE and 5% CSE + TGF-β. All cells were collected at 24, 48 and 72 hours following 5% CSE and TGF-β stimulation. The nontoxic dose of vitamin D was determined by MTT analysis. Changes in levels of E-cadherin, α-SMA, p-VDR, p-SMAD2/3, SMAD4, SMAD7, active β-catenin, DKK1, p120ctn, Kaiso, and NEDD9 proteins were determined by Western impregnation method from cell lysates. Cellular localizations of P-VDR/Kaiso and p-VDR/p-SMAD2/3 proteins were determined by immunofluorescence method. The physical interactions between p-VDR and active β-catenin, Kaiso and p120ctn proteins were examined by co-immunoprecipitation in fibrotic (IPF, n = 3) and non-fibrotic (n = 4) human lung samples. In the A549 cells that were stimulated with 5% CSE and 5% CSE + TGF-β, p-VDR and E-cadherin protein levels decreased and α-SMA levels increased; TGF-β signaling pathway elements that p-SMAD2/3 and SMAD4 levels increased while SMAD7 levels decreased when time progresses. It was found that active β-catenin levels increased and DKK1 levels decreased from Wnt signaling pathway elements. In addition, the levels of Kaiso and p120ctn, which are involved in the stabilization of E-cadherin and NEDD9 levels which is responsible for the destruction of E-cadherin were also increased in these cells when time progresses. When the A549 cells were treated with 50 nM vitamin D half an hour before CSE and TGF-β stimulation, induced p-VDR signaling increased the levels of E-cadherin and reduced α-SMA levels, causing EMT regression. In addition, TGF-β signaling pathway activation decreased due to reducedlevels of p-SMAD2/3 and SMAD4 protein with elevatedlevels of SMAD7 protein; Wnt signaling pathway activation was also decreased by reducedlevels of active β-catenin protein and elevated DKK1 levels. In addition, vitamin D applications prevented the destruction of E-cadherin by reduced p120ctn, Kaiso and NEDD9 protein levels in A549 cells that were stimulated with 5% CSE and 5% CSE+TGF-β. Immunofluorescence analysis showed that in CSE and TGF-β treated groups, the p-VDR immunoreactivity (IR) in the cytoplasm and nucleus of the cell is decreased; furthermore, it was seen that p-SMAD2/3-IR was increased in nucleus but Kaiso-IR was increased in cytoplasm, mostly around nucleus periphery. In vitamin D treated groups, while cytoplasmic and nuclear p-VDR-IR was increased, p-SMAD2/3-IR was increased in cytoplasm but Kaiso-IR concentrated in the nucleus and nucleus periphery. Co-immunoprecipitation analysis revealed that in human lungs with IPF, the p-VDR protein could physically interact with Kaiso and active β-catenin and also that p-VDR did not interact with p120ctn. Due to decreased p-VDR signaling in A549 cells, applications of 5% CSE and 5% CSE+TGF-β stimulated the activation of TGF-β and Wnt/β-catenin signaling pathways along with expression of p120ctn and NEDD9 proteins which regulate the E-cadherin stabilization and degradation. This lead the A549 cells to decrease the amount of E-cadherin protein and start to produce α-SMA protein, which then caused EMT-mediated myofibroblast differentiation. These results clearly show that smoking and excessive activation of TGF-β signaling pathway associated with smoking, together withdecreased VDR signaling might be important risk factors for IPF development. In addition, the results of the current study showed that, when treated with 50 nM vitamin D of A549 cells half an hour before CSE and TGF-β administration, the increase in VDR signaling in these cells was mediated by DKK1, active β-catenin and Kaiso proteins, and the increase of vitamin D signalling reduced the activation of TGF-β and Wnt/β-catenin pathways thus protecting the A549 cells against EMT. In addition, VDR signaling regulated the activation of the Wnt/β-catenin signaling pathway in cells by controlling the transcriptional activity of β-catenin and Kaiso. Smoking, overactivation of the TGF-β signaling pathway associated with smoking, and decreased VDR signaling may lead to IPF development, leading to EMT. In addition to the therapeutics used in the treatment of IPF patients with vitamin D deficiency, the use of vitamin D as additional support may be recommended.