Studies On The Role Of NF-?B Pathway In Lung Cancer In Gprc5a- Knockout Mice And The Effect Of Mesenchymal Stem Cells (MSC) On IPF-like Lung Injury In Mice

GPRC5A is a member of GPCR,a target gene of retinoic acid,and preferentially expressed in lung tissue.Gprc5 a gene deletion or repression leads to spontaneous lung cancer development,suggesting Gprc5 a as a lung tumor suppressor gene.However,the mechanisms underlying Gprc5a-deficiency-induced lung cancer remain elusive.Early studies showed that lung tumor development in Gprc5a-ko mice is strongly associated with pulmonary inflammation,suggesting that lung tumor development is associated with inflammation.Thus,inflammation is implicated to promote lung tumorigenesis,which resembles the pathological features in human lung cancer.However,the causal relationship between two are not clear.Preliminary study showed that,GPRC5 A repression is associated with inflammation or NF-kB activation.The results of lung tumor development suggest that NF-kB is essential for lung tumorigenesis.The role of inflammation on GPRC5 A expression remains unclear.Lung injury,pulmonary fibrosis or COPD are severe lung diseases that seriously threaten human health.Idiopathic Pulmonary Fibrosis(IPF)is the most serious one among lung disease,progressive,exhaustive and deadly.There is still no effective way to treatment these disease in clinic.Mesenchymal stem cells(MSC)are of multipotency,capable to divide into multiple types of cells.MSC based cell therapy provides a promising approach for IPF treatment in clinic.However,there are many aspects to be improved.To determine if the inflammatory pathway or NF-kB pathway is essential for lung tumorigenesis.The regulatory role of NF-kB on GPRC5 A expression.Based on Bleomycin-induced lung injury mouse model,we intend to improve and modify the method of MSC therapy approach.By cross-breeding Gprc5a-ko mice with SPC-dn-IkBa mice,we developed Gprc5a-ko/SPC-dn-IkBa mice,or NF-kB pathway is specifically suppressed in the lung tissue of Gprc5a-ko mice.Lung tumorigenesis and inflammation were examined.In vivo,LPS injection is performed for induction of lung inflammation and GPRC5 A expression was analyzed by Westernblot analysis.In vitro analysis,TNFa was used to induce NF-kB activation in cell culture,Westernblot and RT-PCR analysis were used for determination of GPRC5 A levels at protein and mRNA levels.GPRC5 A promoter-driven luciferase(pGL3-5A-luc.)and NF-kB-driven luciferase(kB-luc.)were used for transfection of cells,luciferase activity were measured as the transcription activity.GFP-p65 and GPRC5A-myc were used for transfection and immunefluorescent staining analysis.We develop a lung injury mouse model via bleomycin.Then we treated those mice with MSCs via different routes,intratracheal inoculation or intravenous injection.NF-kB signaling is greatly suppressed in lung tissues of Gprc5a-ko/SPC-dnIkBa mice compared to Gprc5a-ko mice.Lung tumor development is in Gprc5a-ko mice at age of 12 months,whereas lung tumor development in Gprc5a-ko/SPC-dnIkBa mice at age of 18 months,6 month delay in lung tumor development.In vivo,inflammatory signal or LPS induces Gprc5 a suppression;whereas in vitro,TNFa treatment,or NF-kB activation,significantly suppress GPRC5 A.NF-kB-mediated suppression occurred at transcriptional level in the promoter of GPRC5 A.Intratracheal inoculation of MSCs resulted in better local therapeutic effect;whereas intravenous injection of MSCs resulted in better overall therapeutic effect.Since MSC from wild-type(wt)mice were used for treatment of lung injury in Gprc5a-ko mice,the feature of Gprc5 a expression in wt-MSC can be used to trace MSC in injured lungs.Indeed,wt-MSC was found in the injury area in lung tissues of Gprc5a-ko mice via IHC staining Gprc5 a,in which they are differentiated into lung epithelial cells.Importantly,the injury area is greatly repaired following MSC treatment.This suggests that inflammation or NF-kB activation is essential for lung tumor development,or targeting NF-kB is a promising strategy for lung cancer prevention and therapy.Inflammation or NF-kB activation suppresses Gprc5 a expression.Thus,inflammation inhibition can improve the preventive or therapeutic effect by restoration of GPRC5 A expression.MSC therapy can significantly repair lung injury in Bleomycin-induced lung injury mouse model,which provides the basis for further improvement of lung injury therapy.MSC therapy can significantly repair lung injury in Bleomycin-induced lung injury mouse model,which provides the basis for further improvement of lung injury therapy.