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. 2013 Apr 30;288(23):16282–16294. doi: 10.1074/jbc.M113.464230

FIGURE 6.

FIGURE 6.

Both IL-6 and IL-8 up-regulation occurs via NF-κB, but IL-8 is also negatively regulated by TGFβ and PAR-2. A, ELISA measurements of IL-6 and IL-8 protein levels after incubation of MDA-MB-231 clonal isolates stably transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant (EA) MMP-8 after incubation with 10 μm BAY 11-7082, an NF-κB inhibitor, for 48 h. DMSO, dimethyl sulfoxide. B, ELISA measurements of IL-6 and IL-8 protein levels after incubation of MDA-MB-231 clonal isolates stably transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant MMP-8 after incubation with 10 μm SB431542, a TGFβR1 inhibitor, for 48 h. C, RNA quantification by real-time TaqMan RT-PCR of PAR-2 in MDA-MB-231 clonal isolates stably transfected with pcDNA4 empty vector, wild-type MMP-8, and E198A mutant MMP-8. D, top panel, Western blot analysis and RNA quantification by real-time TaqMan RT-PCR of PAR2 to confirm the knockdown of PAR-2 after siRNA knockdown for 48 h in wild-type MMP-8 overexpressing MDA-MB-231 clonal isolate 1. Center panel, RNA quantification by real-time TaqMan RT-PCR of IL-6 and IL-8 in cells after PAR-2 siRNA knockdown. Bottom panel, ELISA measurement on serum-free conditioned media of IL-6 and IL-8 protein levels after PAR-2 siRNA knockdown. *, p < 0.05; **, p < 0.01, ***, p < 0.001; ns, not significant. E, schematic showing the relationships between MMP-8, IL-6, and IL-8 in a self-reinforcing loop and pathways that may contribute to this system in breast cancer cells. On acute exposure to catalytically active WT MMP-8, breast cancer cells up-regulate the expression of IL-6 and IL-8 (black lines and arrows). IL-6 is also able to induce MMP-8 expression. Red lines and arrows show the pathways that are evident in breast cancer cells that adapt to long-term expression of WT MMP-8. Not shown on the diagram is that IL-6 and IL-8 expression is also dependent on NF-κB signaling, which may be activated by PAR-2 as well as other signaling pathways, including autocrine loops resulting from IL-6 and IL-8 induction.