The Role Of MSP/RON Signaling Pathway In Lipopolysaccharide-mediated Dendritic Cells Maturation

ObjectiveInvestigate the role of macrophage-stimulating protein/tyrosine kinase receptor RON(MSP/RON)signaling pathway in the maturation of LPS-mediated dendritic cells and its possible molecular mechanism.MethodsBone marrow derived dendritic cells(BMDCs)were cultured in vitro from C57/B6mice.RT-PCR,flow cytometry,and Western blot were used to detect the expression of tyrosine kinase receptor RON in mature BMDCs and immature BMDCs.The RON agonist Macrophage stimulating protein(MSP)or inhibitor BMS777607 was used to change the activity of RON in LPS-stimulated dendritic cells maturation,and the activation of downstream pathways Erk1/2 and Akt was detected.Flow cytometry was used to detect CD86 and MHC II expression after inhibiting or activating RON with MSP or BMS777607 for 6h,12h and 24h in LPS-stimulated BMDCs and RT-PCR was used to detect the m RNA of CD86 and CIITA(MHC class II transactivator).RT-PCR was used to detect the expression of March-I and March-8 gene in each group,and immunoprecipitation was used to detect the ubiquitination level of CD86 and MHC II molecules.Elisa was used to detect the IL-1?and IL-12p70 in the supernatants of each group.According to the mixed lymphocyte reaction protocol,the CD3~+T cells sorted from Balb/c mice spleen by magnetic beads were mixed with the stimulated BMDCs,and the T cell proliferation was detected by flow cytometry.Results1. The tyrosine kinase receptor RON is expressed in BMDCs,and the expression of RON is not changed after stimulation by LPS.2. RON activation on BMDCs can still activate downstream pathways Akt and Erk1/2.3. The inhibition of RON receptors significantly reduced the expression of CD86 and MHC II in LPS-stimulated BMDCs,but the m RNA of CD86 did not change,and the expression of CIITA increased.4. Inhibition of RON resulted in increased expression of E3 ligase March-1 while the expression of E3 ligase March-8 remained unchanged. Immunoprecipitation experiments showed that inhibition of RON led to increased ubiquitination of CD86 and MHC II.5. Inhibition of RON results in a weakened ability of mature BMDCs to release IL-1?and IL-12p70.6. The mixed lymphocyte reaction showed the ability of mature DC stimulated T lymphocyte proliferation decreased after RON inhibition.ConclusionThe tyrosine kinase receptor RON protects LPS-induced dendritic cell maturation through the E3 ligase March-1.