Extracellular Heat Shock Protein 90α Mediates Cellular Senescence In Pulmonary Fibrosis

BackgroundIdiopathic Pulmonary Fibrosis(IPF)is a chronic,progressive,fibrotic interstitial lung disease with unknown mechanisms.IPF significantly impacts aging population globaly which is characterized by worsening respiratory symptoms and reduced forced vital capacity(FVC).Cellular senescence is a state of irreversible cell-cycle arrest accompanied by an abnormal secretory profile and is thought to play a critical role in age-ralated fibrotic diseases.Accumulated senescent cells was observed in lung tissue from IPF patient.Extracellular Heat Shock Protein 90 Alpha(eHSP90α)has various roles,including promoting inflammation,wound healing,tumour metastasis,and regulating cellular metabolic homeostasis.Previous research has shown that eHSP90αpromotes the progression of pulmonary fibrotic diseases.This project aims to elucidate the role of eHSP90α in IPF,explore its impact on cellular senescence during pulmonary fibrotic diseases,and investigate its molecular mechanisms.Methods1.Determine the expression levels of eHSP90α in the progression of bleomycininduced murine pulmonary fibrosis:Construct a murine model of pulmonary fibrosis induced by bleomycin(BLM).Assess the levels of HSP90α in bronchoalveolar lavage fluid(BALF)of the model mice at postoperative days 7,14,and 21,concurrently measuring the expression of fibrosisrelated proteins and cellular senescence markers.2.Elucidate the regulatory role of eHSP90α in cellular senescence during the pulmonary fibrosis process:(1)Establish an in vitro senescent cell model in human lung fibroblast cell line IMR90 using etoposide.Observe the protein expression levels of HSP90α in the intracellular and extracellular cells.(2)Co-stimulate fibroblasts using F-5(a functional fragment of eHSP90α)and 1G6-D7 antibody(specific antagonist of eHSP90α)and assess changes in senescencerelated markers(β-galactosidase activity,p16,and p21).(3)Apply 1G6-D7 treatment to BLM-injured mice and assess changes in lung function,collagen deposition,fibrosis-related proteins(α-SMA,collagen Ⅰ,fibronectin),and senescence-related markers(p16 and p21).3.Explore the molecular mechanisms of eHSP90α-induced cellular senescence:(1)Co-stimulate with F-5 and 1G6-D7 and examine the activation of TGF-βsignalling pathway.Also,assess the activation of TGF-β signalling pathway in the 1G6-D7-treated lung fibrosis mouse model.(2)Observe the effect of eHSP90α on cellular senescence through the regulation of SMAD complexes:Combine F-5 and RepSox(a TGF-β signalling pathway inhibitor)and test the senescence-related markers.Utilize Chromatin Immunoprecipitation(ChIP)technology to investigate the transcriptional regulation of senescence genes(p16,p21,p53)by SMAD complexes.(3)Assess the mitochondrial function in eHSP90α-induced senescent fibroblasts(4)Investigate the role of mtROS in the eHSP90α-TGF-β pathway:Combine F-5 and Mito-Tempo(a mtROS scavenger)and examine the expression of essential proteins in the TGF-β signalling pathway.4.Validate the role of eHSP90α in age-related pulmonary fibrosis:Induce lung injury in 2-month-old and 18-month-old mice using BLM and administer 1G6-D7 treatment,evaluating changes in collagen deposition and fibrosisrelated proteins(α-SMA,fibronectin).Key Results1.eHSP90α was elevated in the BLM-induced lung fibrosis mouse model and correlated with fibrosis and cellular senescence.The level of HSP90α in the BALF from BLM-injuried mice was increased.Upregulated expression of p16 and p21was observed.Transcript levels of IL6,TGF-β,PAI1 were increased.2.eHSP90α regulated fibroblast senescence and contributed to fibrotic lung disease.(1)eHSP90α expression was increased in etoposide-induced senescent cells,while intracellular HSP90α remaind unchanged.(2)F-5 fragment treatment stimulated protein expression of p16 and p21 and activity of SA-β GAL,and treatment with 1G6-D7 markedly reduced the cellular senescence phenotype.(3)1G6-D7 monoclonal antibody attenuated BLM-induced weight loss,lung function decline,collagen deposition,and reduced α-SMA,Fibronectin and collagen Ⅰexpression.(4)Intervention with 1G6-D7 monoclonal antibody in lung fibrosis model mice resulted in a significant downregulation of senescent proteins p16 and p21.3.eHSP90α,through the classical TGF-β/SMAD signalling,regulates cellular senescence,causes mitochondrial dysfunction,and promotes the progression of lung fibrosis disease.(1)The F-5 fragment stimulated the expression of TGF-β/SMAD signaling pathway proteins(p-SMAD2,p-SMAD3,and SMAD4)in fibroblasts while 1G6-D7 inhibited TGF-β/SMAD signaling activation.In vivo,1G6-D7 monoclonal antibody intervention reduced the expression of p-SMAD2 and p-SMAD3.(2)RepSox,a TGF-β signalling specific inhibitor,reduced the effect of F-5 on senescence-associated β-galactosidase activity and p16 and p21 protein expression.ChIP showed that with the stimulation of eHSP90α,the SMAD complex was mainly enriched in the promoter regions of p21 and p53.(3)eHSP90α induced mitochondrial dysfunction accompanied by changes in mitochondrial morphology and quality and the accumulation of mitochondrial ROS.(4)Mito-Tempo,a mitochondrial ROS scavenger,inhibited eHSP90α-induced TGF-β/SMAD pathway activation.4.Blocking the eHSP90α with 1G6-D7 attenuated age-related lung fibrosis.(1)The levels of HSP90α was increased in 18-month-old mice compared to 2month-old mice.(2)Both 2-month-old and 18-month-old mice received BLM administration.18month-old mice exhibited increased collagen deposition and expression of α-SMA and fibronectin.(3)Intervention with 1G6-D7 reduced collagen deposition and the protein expression of α-SMA and fibronectin in 18-month-old mice.ConclusionExtracellular HSP90α may promote fibroblast senescence through the TGF-βsignaling pathway in pulmonary fibrosis.Specific targeting eHSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF.