Study On The Mechanism Of CD44-RhoA-YAP Signaling In The Activation Of Fibroblast And Progression Of Experimental Silicosis

Objective: Silicosis is a type of pneumoconiosis,usually caused by long-term exposure to crystalline silica.Silicosis is characterized by chronic inflammation and irreversible pulmonary fibrosis due to excessive deposition of extracellular matrix by activated fibroblasts.Although significant inflammatory responses are maintained throughout the course of silicosis,once fibrosis is established,anti-inflammatory therapy alone cannot postpone or even worsen the fibrosis process.Recent studies have confirmed that excessive extracellular matrix deposition plays a positive role in promoting the continuous fibrosis mediated by fibroblast proliferation activation,but the specific molecular mechanism remains to be further explored.In this project,starting from the positive feedback regulation between the activation of fibroblasts and the fibrosis of silicosis,we explored the regulatory mechanism of extracellular matrix synthesis and increased matrix stiffness on the progression of silicosis fibrosis,so as to provide new theoretical basis and potential molecular targets for delaying the progression of silicosis fibrosis.Methods: In this study,an experimental silicosis mouse model was established by intratracheal instillation of crystalline silica suspension.Western blot and immunohistochemical analysis were used to detect the changes in the expression and localization of YAP in the lung tissues of mice exposed to silica.The mouse fibroblast line NIH-3T3 was used for in vitro experiments.NIH-3T3 was cultured in soft and stiff gel to mimic the normal and injured fibrotic lung tissue in vivo.Immunofluorescence analysis was used to further verify the effect of matrix stiffness on YAP expression and localization.Through bioinformatics analysis,we predicted proteins related to matrix stiffness regulation of YAP and targeted it to construct the CD44-RhoA-F-actin-YAP signaling pathway.Immunofluorescence and western blot were used to detect the effect of progressively silencing CD44 and RhoA,inhibiting the polymerization of F-actin on the downstream signaling pathway and effector factor YAP;We co-transfected constitutively activated RhoA plasmid and CD44 siRNA in fibroblasts cultured in stiff gel,and treated NIH-3T3 cells transfected with constitutively activated RhoA plasmid with F-actin inhibitor.The expression and localization changes of YAP were detected by immunofluorescence and western blot to further confirm the upstream and downstream relationship of the pathway.The effect of upstream and downstream blocking on YAP localization was detected by immunofluorescence;The influence of upstream and downstream blocking on NIH-3T3 migration capacity was examined by wound-healing and Trans-well experiments;Western blot,CCK8 assay and flow cytometry were used to detect the effects of upstream and downstream blockade on NIH-3T3 proliferation and apoptosis;Luciferase reporter assay was used to detect the changes in transcriptional activity of Smads gene caused by upstream and downstream blockade,further confirming the changes in TGF-Smad signaling pathway caused by blockade processing;Realtime-PCR was used to detect the changes of proinflammatory and profibrotic genes by blocking treatment.Mice of experimental silicosis were given CD44 neutralizing antibody,verteporfin(VP),YAP small molecule inhibitor,and gradient-dose DHI,respectively,to establish the upstream and downstream blocking models of the signaling pathway.Nuclear and cytoplasmic extracts were extracted from whole lung tissues,and the expression of YAP was detected by western blot;Total extracts of mice lung tissues were extracted and the effects of blocking treatment on Smads family related proteins were detected by western blot;By recording the relevant indicators of lung function in mice,the improvement of damaged lung function in silicosis mice by upstream and downstream blocking was observed dynamically;Masson staining and collagen 1 and fibronectin immunohistochemical analysis were used to observe the effects of upstream and downstream blocking on pulmonary fibrosis in silicosis mice;The change of fibrosis level in mice was confirmed by the determination of strong proline;HE staining was used to observe the effect of blocking treatment on lung inflammation in silicosis mice;The changes of the relevant pro-inflammatory factors in bronchoalveolar lavage fluid were detected by ELISA to help confirm the changes of mouse inflammation level.Results: 1.The localization and expression of YAP were enhanced in the lung tissues of experimental mice with silicosis.Western blot and immunohistochemical analysis were used to observe the location and expression of YAP in lung tissue of silica injured mice.The results showed that nuclear expression of YAP increased significantly in the silica injured lung tissues at day 56(P < 0.05).Immunohistochemical localization analysis showed that the nuclear localization of YAP was more pronounced in spindle-shaped cells in silicotic lesions suggesting the high expression of YAP in fibroblasts.2.Extracellular matrix proteins can regulate the activation of RhoA protein family through CD44,thus affecting the localization and expression of YAP.By bioinformatics analysis,we found the key target protein CD44 involved in the regulation of matrix protein and YAP interaction.In vivo and in vitro immunofluorescence results showed that the increase of matrix stiffness enhanced the expression of CD44.Immunofluorescence and western blot were used to detect the changes of the downstream pathway and target protein YAP by progressively silencing CD44 and Rho family proteins(RhoA,Rac1 and Cdc42).The results confirmed that CD44 regulated the RhoA subfamily and affected the location and expression of YAP,while the Rac1 and Cdc42 subfamily had no significant effect in this process.3.RhoA directly regulates YAP activity through influencing F-actin cytoskeleton polymerization/depolymerization.Silencing of Rho family proteins(RhoA,Rac1 and Cdc42)respectively,immunofluorescence and western blot results showed that only silencing of RhoA could affect the expression of downstream F-actin and effector YAP.After treatment with Factin inhibitor Lat A,immunofluorescence and western blot were used to detect the changes of Hippo pathway kinases Mst 1,Lats 1 and effector protein YAP.The results showed that F-actin directly regulates the location and expression of YAP independent of Hippo pathway kinases.4.Upstream and downstream reverse experiments confirm that matrix stiffness regulates the location and expression of YAP through CD44-RhoA-F-actin signaling pathway.Constitutively activated RhoA plasmid and CD44 siRNA were co-transfected into NIH-3T3 cells;Continuously activate Rho A NIH-3T3 cells were treated with Lat A.Through immunofluorescence and western blot,the upstream and downstream relationship of CD44-RhoA-F-actin signaling pathway was further verified.CD44 regulates the activation of RhoA,thereby affecting the polymerization and depolymerization of F-actin,which is involved in the positive feedback regulation of matrix stiffness increase and YAP activation.5.Upstream and downstream blocking of CD44-RhoA-YAP signaling pathway can inhibit the function activation of fibroblast induced by matrix stiffness.CD44 neutralizing antibody was used to block the upstream pathway;Verteporfin,YAP small molecule inhibitor,and gradient-dose DHI,were used to block the downstream pathway.YAP siRNA was used as a positive control.The results of woundhealing and Trans-well experiments showed that upstream and downstream blocking could inhibit the migration of fibroblasts induced by matrix stiffness;Western blot analysis of Collegen1,MMP2 and its inhibitor TIMP2 showed that blocking treatment reduced the expression of Collegen 1 and MMP2 and increased the expression of TIMP2,which was statistically significant(P < 0.05);CCK8 assay and flow cytometry results showed that the down-regulation of fibroblast proliferation level was mainly mediated by the blocking of the downstream pathway,while the up-regulation of apoptosis level was mainly mediated by the blocking of the upstream pathway(P < 0.05);The results of luciferase reporter assay showed that upstream and downstream blocking could inhibit the transcriptional activity of Smads in fibroblasts(P < 0.05).6.Upstream and downstream blocking of CD44-RhoA-YAP signaling pathway can inhibit the expression of pro-inflammatory and pro-fibrotic cytokines.CD44 neutralizing antibody,verteporfin and gradient-dose DHI were used to block the signaling pathway from upstream to downstream.Realtime-PCR analysis showed that blocking treatment could down-regulate the expression of pro-inflammatory and pro-fibrosis factors Tgf-β1,Pai,Il-6,Il-8,Fibronectin and Collagen-1a1(P < 0.05).7.Upstream and downstream blocking of CD44-RhoA-YAP signaling pathway can regulate the nuclear/cytoplasmic expression level of YAP.Silica injured mice were treated with CD44 neutralizing antibody,verteporfin and gradient-dose DHI,and the expression level of YAP in the nuclear and cytoplasmic extracts in lung tissue was performed by western blot.The results showed that upstream and downstream blocking could reduce the expression of YAP in nuclear protein and correspondingly increase the expression of YAP in cytoplasmic protein(P < 0.05).8.Upstream and downstream blocking of CD44-RhoA-YAP signaling pathway can down-regulate the expression level of Smads protein in lung tissues of experimental silicosis model mice.Silica injured mice were treated with CD44 neutralizing antibody,verteporfin and gradient-dose DHI,and the expression level of Smads family protein in the total extracts in lung tissue was performed by western blot.The results showed that upstream and downstream blocking inhibited the phosphorylation of Samd2 and increased the expression of inhibitory Smad7 protein(P < 0.05).9.Upstream and downstream blocking of CD44-RhoA-YAP signaling pathway can improve lung function and inflammatory/fibrosis level in experimental silicosis model mice.The results showed that blocking treatment could improve the lung function level of silicosis mice by recording the lung function indicators of mice in real time,including minute volume(MV),tidal volume(TV)and breathing frequency(f).HE and Masson staining detected inflammatory infiltration and collagen deposition in lung tissue.The results showed that,compared to crystalline silica group,blocking treatment could reduce inflammatory and fibrosis symptoms.ELISA showed that blocking treatment reduced the expression of pro-inflammation cytokines TNF-α,IL-6,and IL-1β expression in BALF(P < 0.05),supporting the HE staining results.Immunohistochemical test results showed that blocking treatment could reduce the expression of Fibronectin and Collagen-1 in lung tissue,and HYP showed that blocking treatment could reduce the content of hydroxyproline in lung tissue,supporting Masson staining results.Conclusion: 1.CD44-RhoA-YAP signaling pathway is involved in the regulation of matrix hardness mediated fibroblast activation.2.Blocking this signaling pathway upstream and downstream can affect the activation and function of fibroblasts.3.Blocking this signaling pathway upstream and downstream can improve lung function damage,pulmonary inflammation and fibrosis symptoms caused by crystalline silica in mice,and provide a potential therapeutic strategy for the treatment of silicosis.