JAK1/STAT3 Interacts With Canonical TGFβ Pathway To Modulate Myofibroblast Transdifferentiation And Fibrosis

Objective:Idiopathic pulmonary fibrosis(IPF)is a progressive,irreversible condition with no unifying mechanism and a median survival of 2-3 years from the time of diagnosis.As to current treatment options,modestly improve lung function and reduce exacerbation frequency but have limited or no impact on survival.Therefore,in order to develope novel target treatments,it is extremely vital to investigate the pathogenesis of IPF.The histopathological of IPF is usual interstitial pneumonia,which is characterized by collagenized fibrosis,foci of smooth muscle expressing myofibroblast proliferation alternating with active fibroblast foci.Current thinking supports the concept that myofibroblasts are the primary cell source of excessive secretion and deposition of collagen and other extracellular matrix that lead to lung scarring and fibrosis.Alpha smooth muscle actin(SMA)is a widely used myofibroblast marker.One of the most well-known pathways is the transforming growth factor beta(TGF-β)and SMAD-dependent signaling pathway,which plays a key role in IPF pathogenesis.Recently,molecules belonging to the Janus kinase(JAK)and Signal Transducers and Activators of Transcription(STAT)pathway have been identified in IPF lung tissues and postulated to have a direct role in the pathogenesis of pulmonary fibrosis.In addition,cross-communication between TGF-β/SMAD and JAK/STAT pathways has also been reported,but the interaction between these two pathways and their roles in fibrosis are still unknown.The purpose of this study was to specifically explore the role of the JAK1/STAT3 axis in TGF-β induced fibroblast trans-differentiation and pulmonary fibrosis.Materials and Methods:The interaction between JAK1 and TGF-β canonical pathway:(1)transcriptional silencing of JAK1 on NHLF to detect it’s effect on TGF-β-induced fibroblast transdifferentiation,cell proliferation and extracellular matrix expression;(2)inhibiting JAK1 with JAK1 selective inhibitor-Upadacitinib on NHLF to detect it’s effect on TGF-β-induced fibroblast transdifferentiation and cell proliferation;(3)NHLF with siRNA-mediated stable knockdown of JAK1(siJAK1)and non-targeting(si Con)pre-treated with TβRI inhibitor SB431542 to detect it’s effect on TGF-β-induced fibroblast transdifferentiation;(4)Western blot of SMA and RT-PCR of TGF-β in NHLF growing on 0.2Kpa gel with siRNA-mediated stable knockdown of JAK1(siJAK1)and non-targeting(si Con)stimulated with TGF-β(2.5ng/ml);(5)WB and immunofluorescence were used to detect the expression of p-TβRI on NHLF with siRNA-mediated stable knockdown of JAK1;(6)the interaction between JAK1 and TβRI was detected by immunoprecipitation and proximity ligation assay;(7)bleomycin-induced fibrosis was generated on C57BL/6 wild mice,and the mice were treated by a selective JAK1 inhibitor(Upadacitinib).Body weight,lung function,survival,pulmonary fibrosis and lung inflammation were evaluated.The interaction between STAT3 and TGF-β canonical pathway:(1)transcriptional silencing of JAK1 on NHLF to detect it’s effect on TGF-β-induced fibroblast transdifferentiation,cell proliferation and downstream p-SMAD3;(2)the expression of p-STAT3 and SMA was detected by immunofluorescence staining on lung tissues of IPF patients;(3)Overexpress of p-STAT3 by Oncostatin M(OSM),and detect the fibroblasts transdifferentiation induced by TGF-β;(4)NHLF were transfected by STAT3 wild type lentivirus to overexpress p-STAT3,explore the fibroblasts transdifferentiation induced by TGF-β;(5)NHLF were treated with PSTAT3 inhibitor C188-9,fibroblasts transdifferentiation induced by TGF-β was evaluated;(6)NHLF with siRNA-mediated stable knockdown of JAK1(siJAK1)and non-targeting(si Con)pre-treated with unphosphorylated STAT3 inhibitor STA-21(2.5μM),following TGF-β stimulation,the fibroblasts transdifferentiation was explored;(7)RNA sequence and Ingenuity Pathway Analysis result of differentially affected Pathways of lung tissues from IPF patients and healthy controls was performed to comfirm the role of JAK1/STAT3 pathway in IPF.Results:1.Transcriptional silencing of JAK1 significantly increased TGF-β induced expression of SMA on NHLF cells without significantly affecting cell proliferation or extracellular matrix protein generation.2.Upadacitinib had no effect on TGF-β-induced SMA expression and TGF-β-induced cell proliferation.3.The observed increase in SMA expression that occurs following transcriptional silencing of JAK1 is reversed by TβRI inhibitor SB431542.4.The increase of SMA that results from transcriptional JAK1 silencing is related to the rigid culture environment not the autocrine of TGF-β.5.The increase of SMA induced by transcriptional silencing of JAK1 is associated with an increase of p-TβRI.6.JAK1 and TβRI can combine to form a complex.7.Upadacitinib treatment on bleomycin-induced pulmonary fibrosis model did not improve lung function,survival and lung pathological injury.8.JAK1 silencing significantly inhibited the expression of p-STAT3,and the expression of p-STAT3 gradually decreased with the prolonged TGF-β stimulation time.9.High expression of p-STAT3 leads to low expression of SMA and versus vice.10.U-STAT3 can significantly reduce the high expression of SMA induced by JAK1 through reducing p-SMAD3.11.Gene sequence analysis showed that JAK/STAT pathway and STAT3 pathway were inhibited in IPF patients.Conclusion:JAK1 can inhibit the fibroblasts transdifferentiation by binding to TβRI to form a complex and inhibit the phosphorylation of TβRI,resulting in the weak signal of TGF-β/SMAD3,and the regulation of JAK1 on TGF-β/SMAD signal is related to stiffness.However,Upadacitinib,a selective JAK1 inhibitor,did not significantly attenuate lung fibrosis.Two forms of STAT3(phosphorylated p-STAT3 and unphosphorylated U-STAT3)play different roles in TGF-β signal.Our results suggest that p-STAT3 is JAK1 dependent,and p-STAT3 can negatively regulate myofibroblast transdifferentiation by inhibiting the activation of SMAD3.Meanwhile,U-STAT3 is an important signal transducer in TGF-β/SMAD pathway and can partially promote the activation of SMAD3.Pathway analysis showed that JAK/STAT3 pathway and STAT3 pathway was inhibited in IPF patients compared to control group.