Role and mechanism of CD36/FAK/mTORC1 pathway in LPS-induced inflammatory cytokine expression in human mammary epithelial cells

Abstract

OBJECTIVE: To investigate the role and mechanism of the CD36/FAK/mTORC1 pathway in expressing inflammatory cytokines induced by lipopolysaccharide (LPS) in human mammary epithelial cells (MCF-10A).METHODS: Cells were infected with Escherichia coli using LPS, and the expression of CD36, activation of the FAK/mTORC1 pathway and secretion of inflammatory cytokines were measured. The cell membrane receptors CD36 and TLR4 were subsequently blocked with anti-CD36 and anti-TLR4, and the activation of the CD36/mTORC1 pathway was assessed. The expression of inflammatory cytokines and the activation of the FAK/mTORC1 pathway in LPS-treated cells were evaluated after FAK signal inhibition using TAE226, a specific chemical inhibitor of FAK. The expression of inflammatory cytokines and the activation of the mTORC1 pathway in cells treated with or without LPS were also evaluated after mTORC1 signal inhibition using RNAi.RESULTS: Western blot revealed that CD36 expression was significantly elevated at the mRNA and protein levels after treatment with LPS (p<0.05), and the activation of the FAK/mTORC1 pathway and related transcription factors was notably increased (p<0.05). ELISA showed an increase in the number of inflammatory cytokines TNF-α and IL-6 (p<0.05). The levels of phosphorylated FAK, S6 and 4EBP1 in LPS-stimulated cells were significantly attenuated (p<0.05) when CD36 was blocked compared to the levels in the control group. Further, the levels of phosphorylated FAK, S6, 4EBP1 and STAT1 decreased significantly when CD36 and TLR4 were blocked compared to the levels in the control group. TAE226 remarkably inhibited the activation of the FAK/mTORC1 pathway and the expression of inflammatory cytokines induced by LPS stimulation (p<0.05). The RNAi method was used to alter the expression of the key component of mTORC1, Raptor. The results were consistent with previous findings in Rapamycin-treated cells, in which the mTORC1 pathway was weakened, and the levels of TNF-α and IL-6 decreased.CONCLUSION: CD36 is a co-receptor for LPS and binds to mTORC1 and FAK to form the CD36/FAK/mTORC1 pathway.