SHP-1 Regulates Pulmonary Fibrosis Through Inhibition Of β-catenin Activation In Lung Epithelial Cells

Rationale:Accumulating evidence suggests that the incidence and mortality from idiopathic pulmonary fibrosis(IPF)are increasing worldwide,whereas the mechanisms in IPF are still not completely clear.The activation of Wnt/β-catenin signaling pathway has been implicated in IPF.Tyrosine phosphorylation of β-catenin is one potential mechanism for its activation.However,the regulatory mechanisms that regulateβ-catenin activation are not defined.Src homology region 2 domain-containing phosphatase-1(SHP-1)regulates cytokine and growth factor signaling and plays a role in allergic inflammation in the lung.SHP-1 deficient mice develop severe lung inflammation and fibrosis.We investigated whether SHP-1 expression is altered in pulmonary fibrosis and sought to define the role of SHP-1 in regulating β-catenin in fibrogenesis.Methods:Microarray data from control subjects(CTRL)and IPF patients were analyzed for SHP-1 mRNA expression.Immunoblot and immunostain were performed to determine SHP-1 protein expression and distribution in lung tissues.In vivo,SHP-1 deficient mev/mev mice were compared with wild type(WT)mice;Sftpc-CreERT2(tamoxifen inducible Cre)and SHP-1flox/flox mice were cross-bred to generate SPC-SHP-1 mice with type Ⅱ alveolar epithelial cell-specific deletion of SHP-1;WT,heterozygous mev/+,and SPC-SHP-1 mice were challenged with Bleomycin(BLM,1.5mg/kg,i.t.)for 7-28 days,and then lung samples were analyzed for inflammation and fibrosis.In vitro the effects of SHP-1 inhibition on epithelial cells response to transforming growth factor-β1(TGF-β1)were analyzed in A549 cells.Co-immunoprecipitation was used to evaluate the interaction between β-catenin and SHP-1 in A549 cells.Immunostain,immunoblot and quantitative real time polymerase chain reaction(qRT-PCR)were used to investigate the expression and activation of fibrogenic factors and signaling molecules.Results:(1)Microarray analysis showed that SHP-1 mRNA expression was significantly decreased in IPF patients(n=255)compared to CTRL(n=137).By immunoblot and immunostain,SHP-1 protein was significantly decreased in IPF patients compared to CTRL,SHP-1 was readily seen in lung epithelial cells of CTRL albeittoo low to be detected in IPF samples.Decreased E-cadherin and increased α-smooth muscle actin(α-SMA)and p-Tyrosine 654(Y654)-β-catenin were seen in the IPF lung by immunostain and immunoblot.(2)Histology showed that mev/mev mice developed severe lung fibrosis,while no abnormalities were observed in WT mice.Isolated lung fibroblasts from mev/mev mice showed remarkably accelerated proliferation and migration.Immunostain and immunoblot of lung tissues and proteins showed that p-STAT3,p-Akt,α-SMA,Collagen1A1,fibrinogen,and vimentin were significantly upregulated,but E-cadherin was downregulated.Further analysis showed increased p-Y654-β-catenin.qPCR analysis showed differential mRNA expression of E-cadherin,Collagen1A1,Twist and Snail in the mev/mev lung.BLM-challenged mev/mev mice showed enhanced inflammation and fibrosis compared to WT mice.After tamoxifen induction,SHP-1 expression in the lung epithelial cells was undetectable in SPC-SHP-1 mice and BLM challenge resulted in significantly exaggerated inflammation and fibrosis with increased p-Y654-β-catenin and α-SMA together with decreased E-cadherin in the lung.(3)Loss of SHP-1 expression or activity was effective to amplified TGF-β1-induced epithelial-mesenchymal transition(EMT)in A549 cells,which might be associated to enhanced p-Y654-β-catenin.Crosslinking of β-catenin with the SHP-1 was natural happening in A549 cells that could be increased under pervanadate stimulation.Conclusions:These findings demonstrate that SHP-1 is down-regulated in human IPF;SHP-1 plays a critical role in lung epithelial cells;and SHP-1 inhibits the phosphorylation of β-catenin tyrosine in type Ⅱ AEC,which may be an important mechanism for the protection of lung epithelial injury and fibrosis.