NR4a1 promotes the activation of cAMP-PKA signaling through down-regulating PED4B expression.
Our previous results implied a key role of PED4B in NR4a1-regulated inflammatory macrophage. First, we established the NR4a1 silenced or over-expressed RAW264.7 macrophage by transfecting pcDNA3.1-NR4a1 or siNR4a1 (Fig S1 ). The expression of PDE4B was appraised by western blot and RT-PCR. As shown in Fig3 (A,B), It can be seen that LPS prominently elevated the protein and mRNA level of PED4B. Compared to the LPS group, pcDNA3.1-NR4a1 group exhibited the decreased PED4B expression and the NR4a1 silenced group showed the increased. We further evaluated the components of cAMP signaling pathway by western blot(Fig2 C,D ). LPS stimulation could significantly decrease the cAMP, p-PKA protein level. pcDNA3.1-NR4a1group exhibited higher cAMP, p-PKA protein level and the siNR4a1 group showed lower. However, cotransfection of pcDNA3.1-PDE4B and pcDNA3.1-NR4a1 group exhibited the decreased cAMP, p-PKA protein level compared to pcDNA3.1- NR4a1 group(Fig2 E,F ) .
NR4a1 supresses the LPS induced activation of NFκB signaling pathway and the IL-6 and IL-1β mRNA level by down-regulating PED4B expression
Our previous discussion has been illustrated that the activation of NFκB signaling pathway regulated by NR4a1 and cAMP activated PKA. NF-κB, acting as a transcription factor, could manipulate a number of proinflammatory cytokines expression such as IL-6 IL1β [31,32]. We further detected the effects of NR4a1 and PDE4B on LPS induced activation of NFκB and the expression of IL-6 and IL1β. As depicted inFig4(A,B,E), pcDNA3.1NR4a1 group displayed the decreased protein level of p-p65 and p-IκBα and mRNA level of IL-6 and IL-1β and the siNR4a1 group showed higher compared to LPS treated group. However, the protein level of p-p65 and p-IκBα and mRNA level of IL-6 and IL-1β of co-transfected group were higher than the pcDNA3.1-NR4a1 group(Fig4 C,D,F ).