Experimental Study On Effects Of 17?-estradiol On MTOR Signaling Pathways In Human Osteoarthritis Chondrocytes

Objective:To confirm whether 17?-estradiol(E2) activates mammalian target of rapamycin(mTOR) signaling path-way in huaman chondrocytes and how activate or in what way activates mTOR.This new molecular mechanism might reveal the physiological effects of estrogen on the chondrocytes. Our study might provide insight into mTOR as a potential target for OA therapy and provide references for the research of useful inhibitors of mTOR.Methods:Human immortalized chondrocytes cell lines TC28a2 and C28/I2E2 were subjected to incubate with or without E2, LY294002(the inhibitor of PI3K), rapamycin(the inhibitor of mTOR), or E2 in combination with LY294002 or rapamycin. Thereafter, protein levels of S6K1, p-S6K1, protein kinase B(AKT), and p-AKT were determined by Western blot analysis. Matrix metallopeptidase(MMP) 3 or MMP13 mRNA levels were evaluated by quantitative real-time PCR(qRT-PCR),Co-immunoprecipitation and Western blot analysis were performed to verify the interaction between ER? and mTOR.Results:Both p-S6K1 and p- AKT protein levels in TC28a2 and C28/I2E2 cells were significantly increased by incubation with E2(0.5 h and 1 h)(P < 0.05). Ra-pamycin did not affect the levels of p- AKT, but were significantly reduced by LY294002 or E2 in combination with LY294002. The levels of p-S6K1 were significantly decreased byincubation with LY294002, but the effect could be reversed by E2 in combination with LY294002. Rabbit anti-mTOR antibody was able to immunoprecipitate ER ? after incubation with E2. Moreover, E2 inhibited the mRNA levels of MMP3 and MMP13 by mTOR pathway.Conclusion:E2 actives mTOR in human chondrocytes through AKT-dependent and independent ways.