Nintedanib Regulates TGF-β₁-induced 16HBE Epithelial-mesenchymal Translation

Background and Objective:Idiopathic pulmonary fibrosis(IPF)is the most common among the idiopathic interstitial pneumonias and has the worst prognosis,with a median survival of 3-5 years.Epithelial to mesenchymal cell transition(EMT),whereby fully differentiated epithelial cells transition to a mesenchymal phenotype,has been implicated in the pathogenesis of idiopathic pulmonary fibrosis(IPF).Nintedanib(BIBF1120)is a multiple tyrosine kinase inhibitor recently approved by the U.S.Food and Drug Administration for the treatment of IPF.However,its role in EMT remains unclear.We sought to investigate whether transforming growth factor-β1(TGF-β1 stimulates human bronchial epithelial cells(16HBE)to undergo EMT,and whether this transition can be abrogated by BIBF1120.Methods:1)Cell viability of human bronchial epithelial(16HBE)was assessed by MTT.2)Observing cellular morphology changes by inverted phase-contrast microscope.3)Cells were stimulated with 5ng/mL TGF-β1 for 48h to induce EMT,with or without BIBF1120 pretreatment,and assayed for epithelial or mesenchymal markers using qRT-PCR and Western blot.The mRNA levels and protein expression of α-SMA and E-cadherin was determined with qRT-PCR and Western blot.4)To investigate the effects of TGF-β1 on Smad3,Erk1/2,P38,AKT,Smad2 and JNKactivation in 16HBE.The protein expression levels of phosphorylated Smad3,Erk1/2,P38,AKT,Smad2 and JNK were detected by Western blot.5)The mechanisms,to investigative the signaling events,we tested P38 inhibitor SB203580,Erk1/2 inhibitor U0126 and Smad3 inhibitor SIS3 in TGF-β1-stimulated 16-HBE cells.Results:1)We found that BIBF1 120 differently induced toxicity in a concentration-dependent manner.2)Treatment of 16HBE cells with TGF-β1 for 48h induced EMT as reflected by conversion to the spindle-like morphology,loss of E-cadherin,and acquisition of α-SMA.3)Induced by TGF--β1,cellular morphology of 16HBE cells turn from pebble to fusiform shape.The connetion between cells becomes loose.BIBF1120 could maintain epithelial morphology of 16HBE cells induced by TGF--β1,inhibiting turning to mesenchymal morphology.4)Pretreatment of 16HBE cells with BIBF1120 preserved the epithelial-like morphology,restored the expression of E-cadherin,and abolished the activation of α-SMA.5)This effect may dependent on Smad3,P38,Erk1/2 signaling pathway.Conclusion:Based on these results,the present study suggested that TGF-β1 induces EMT by the Smad3/P38/Erk1/2-dependent manner.BIBF1120 reduces TGF-β1-stimulate d the bronchial epithelial to mesenchymal transition via the inhibition of Smad3/P38/E-rk1/2-dependent signal pathway.