G?i Proteins Mediate TNF-?(Tumor Necrosis Factor ?)-Induced Signaling And Pro-inflammatory Response

Objective:Human immune system is an extreme complex network,which not only protects our bodies from external attacks by microbes,but also protects internal damaged or dysfunctional tissues.In the network,the inflammatory response is one of the first protective barriers.Inflammation is the primary mechanism of innate immunity and is modulated in many processes,such as cytokine networks.Importantly,tumor necrosis factor ?(TNF-?)plays a key role in the cytokine networks.TNF-? can regulate the progression of various diseases such as cancer and autoimmune diseases,as well as the growth,differentiation and death of cells.G protein is a kind of signal transduction protein that has the activity of GTP hydrolase and can bind with GDP,which plays a vital role in the intracellular signal transduction pathway.Recently,it has been found that Gai proteins are required for mediating the initiation and progression of various inflammation by activating related signaling pathways,suggesting that G?i proteins may be a potential target for inflammation modulation.In this study,we explored the relevant molecular mechanisms of G?i proteins involved in mediating TNF-? signaling in MEFs cells,and further clarify the role of G?il/3 in inflammation through the model of THP-1 and BMDM cells,in order to find new targets for the treatment of inflammation in some diseases.Methods:We cultured WT,Gail/3 DKO,G?il KO,G?i3 KO,Gai2 KO MEFs and Gab1 KO MEFs.The shRNA strategy was used to knockdown Gail and/or G?i3 in WT MEFs and stable cell lines are established.CRISPR/Cas9 strategy was used to knockout Gail and Gai3 in WT MEFs.Gail/3 adenovirus constructs were used to re-express the Gail and/or Gai3 in DKO MEFs and increase the expression of G?il/3 in WT MEFs.Western blotting analysis detected the expression of major proteins(Akt-mTORC1?MAPK and NF-?B pathways)and Gab1 protein in MEFs.The shRNA strategy was used to knockdown Gail and G?i3 in THP-1 and BMDM cells,in order to establish the stable G?il/3-shRNA THP-1 and G?il/3-shRNA BMDM cells.We detected the expression of inflammatory factors in G?il/3-shRNA THP-1 and G?il/3-shRNA BMDM cells.The activation levels of Akt-mTORC1 and MAPK pathways were detected in G?i1/3-shRNA BMDM cells.Results:Western Blot analysis showed that in MEFs,G?il/3 double knock(DKO)blocked TNF-?-induced activation of AKT-mTOR and MAPK signaling pathways.In addition,the activation levels of AKT-mTOR and MAPK signaling pathways were largely decreased in G?il/3-shRNA?G?il-KO?G?i3-KO MEFs,but not significantly in G?i2-KO MEFs.However,re-expression of Gail or Gai3 partially rescued TNF-?-induced Akt-mTORC1 and MAPK activation in DKO MEFs;and overexpression of Gail/3 significantly increased the activation levels of AKT-mTOR and MAPK pathways.The activation levels of TNF-?-induced AKT-mTOR and MAPK signaling pathways were also significantly decreased in Gab1-KO MEFs.Further studies showed that knockdown of G?il/3 significantly suppressed the expression of inflammatory factors in THP-1 and BMDM cells.The activation levels of AKT-mTOR and MAPK pathways were largely decreased in Gail/3-shRNA BMDM cells.Conclusion:Gail/3 is critical for the activation of AKT-mTORC1 and MAPK pathways induced by TNF-?.Meanwhile,Gail/3 is required for the regulation of TNF-?-induced macrophage inflammation.This study enriches the understanding of the mechanism of TNF-?-mediated inflammatory response.In view of the important role of TNF-? in inflammation,Gail/3 may become a new target for the treatment of inflammatory diseases.