| Identifying key genes that affect early lung development is of great significance for understanding lung homeostasis and the development of regeneration medicine.The ADAMTS?A disintegrin and metalloproteinase with thrombospondin motifs?protein superfamily includes 19 secreted metalloproteinases.They have diverse roles in patho-physiological process of embryonic development,tissue morphorgenesis and cancers by the cleavage or modification of extracellular matrix?ECM?.ADAMTS18 is an“orphan ADAMTS”whose function or substrate remains unknown.Our team has long been committed to the study of the biological function of ADAMTS18 and has successfully constructed an Adamts18 knockout(Adamts18-/-)mouse model.In the process of mouse phenotypic analysis,we found that the most prominent changes in Adamts18-/-mice were abnormal lung morphology.Adamts18-/-lungs have sharper edges compared to the round blunt appearance of Adamts18+/+lungs.In pathological analysis,most Adamts18-/-mice showed airspace enlargement after birth.Through in situ hybridization?ISH?,Adamts18 mRNA was found mainly expressed in mouse embryonic lung tissues.The only literature report also shows that ADAMTS18 is highly expressed in human fetal lungs.These findings suggest a potential role of ADAMTS18in early lung development.Using previously constructed Adamts18 knockout mice,we studied the effect of ADAMTS18 on lung physiopathology.The research methods are as followed:1)Determine Adamts18 mRNA spatio-temporal expression in lung tissues by quantative real-time polymerase chain reaction?qRT-PCR?and ISH.2)Determine airway morphology by in vitro lung explant culture and airway cast.3)Compare the histological features and protein distribution patterns of lung sections by hematoxylin and eosin?HE?staining,Hart's staining,sirus red staining,masson staining and immunohistochemistry?IHC?.4)Check the time of inspiration/expiration?Ti/Te?,frequency per minute?f/min?,airway pressure high/mean?Phigh/Pmean?,minute volume?MVb?and peak inspiratory/expiratory flow?PIF/PEF?changes in Adamts18-/-mice through the animal lung fuction detaction system.5)Intraperitoneal lipopolysaccharides?LPS?treatment was used to establish acute lung injury model.Intratracheal bleomycin instillation was used to establish lung fiborosis model.Evaluate the lesions and repairment of the lung sections by histological scores.6)Compare the differences in protein expression between Adamts18+/+and Adamts18-/-lungs by label-free mass spectrometry?MS?.Gene ontology?GO?term and pathway enrichment of significantly changed proteins were analysed by Metascape.7)Diffreientially expressed proteins were verified by Western blot?WB?,transmission electron microscope?TEM?,and qRT-PCR.8)Confirm the interaction between proteins by in vitro expression of ADAMTS18 and target proteins,immunocytochemistry?ICC?,co-immunoprecipitation?Co-IP?and enzyme-linked immunosorbent assay?Elisa?.The results showed:1)Adamts18 mRNAs were detected in the epithelium of distal airways and the mesenchymal cells of lung apexes at embryonic?E9.5-E12.5?and pseudoglandular?E12.5-E16.5?stages.At saccular stage?E17.5-P4?,Adamts18mRNAs decreased in abundance.At alveolarization stage?Postnatal,P5-P21?and in adult,Adamts18 mRNAs were barely detectable in both airway and alveolar cells.2)After anatomical morphological comparison,Adamts18-/-mice showed bilateral tipped lung apexes at E14.5 and 12 weeks.,which were apparently different from the round-edged appearance of Adamts18+/+lungs.3)After the in vitro embryonic lung explant culture,Adamts18 deficiency led to reduced number(Adamts18+/+vs.Adamts18-/-:48h,35.50±0.50 vs.25.00±2.08,P<0.05*;72h,56.50±2.50 vs.41.00±1.16,P<0.05*)and length of bronchi.In adult lung cast,Adamts18-/-mice also had fewer bronchial numbers than normal mice.4)After the alveolization stage,Adamts18-/-mice showed dilated alveoli with decreased number of radical alveolar counts?RACs?and elastin deposition on the alveolar walls.However,they had the normal lung function including pressure,volume and flow rate.5)After LPS treatment,the lungs of Adamts18-/-mice showed more severe pathological injury?e.g.inflammation and bleeding?and higher pathological score than those of Adamts18+/+littermates.Similarly,Adamts18-/-mice exhibited increased mortality in response to bleomycin compared to Adamts18+/+mice.HE and Masson's staining showed that Adamts18-/-lungs had more severe inflammatory reaction and fibrosis after bleomycin challenge.Adamts18 mRNAs were not re-expreesed in the injuried lung tissues.6)Major molecules associated with airway branching,including Fgf10,Wnt,Bmp4,Hhip,Ptch1and Ext1 did not change significantly in Adamts18-/-fetal lung tissues,while the transctiption levels of Fgfr2 and Shh increased in Adamts18-/-mice.7)Embryonic lung proteomes showed expression levels of two major constitutes of microfibrils,fibrillin1and fibrillin2,were upregulated in Adamts18-/-lungs.Several proteins associated with cytoskeleton assembly also had changed significantly.TEM images showed that Adamts18-/-lungs had a thicker layer of microfibrils in the basement membrane surrounding epithelial tubes.8)ADAMTS18 co-localized with exogenous fibrillin1protein in vitro.Co-IP results showed that fibrillin1 was pulled down by ADAMTS18.Recombinant ADAMTS18-C901-1187 fragment showed a high binding to fibrillin-N.9)Blocking excess fibrillin1 restored lung morphogenesis of Adamts18 mutants by adding400 ng/ml anti-fibrillin1 antibody to the culture system in vitro.10)The level of Tyr297-phosphorylated focal adhesion kinase?FAK?was lower in mutant epithelium.Phalloidin staining showed uneven filamentous actin?F-actin?distribution in the epithelial cells in Admats18-/-lungs.In transwell assays,Adamts18-/-mouse embryonic fibroblasts?MEFs?showed a significantly decreased migration ability compared to Adamts18+/+MEFs.Together,ADAMTS18 is secreted by bronchial epithelial cells.Secreted ADAMTS18 binds to fibrillin1 and affects its abundance.ADAMTS18 deficiency causes increased levels of fibrillin1 and fibrillin2,and microfibril accumulation,which weakens FAK signaling,and disrupts normal F-actin organization,resulting in reduced migration of epithelial cells and branching defects.This study confirms for the first time that ADAMTS18 can regulate early lung morphogenesis by affecting fibrillin metabolism,which not only helps to expand the new mechanicm of lung development,but also may provide a new perspective and molecular target for the clinical diagnosis and treatment of deseases related to pulmonary dysplasia. |
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