TNF induced inhibition of Cirbp expression depends on RelB NF-κB signalling pathway

Abstract

The circadian clock is required for the rhythmic expression of a plethora of genes that orchestrate metabolism, sleep-wake behaviour and the immune response to pathogens. The cold-inducible RNA binding protein (CIRBP) is required for high amplitude expression of clock genes. Moreover, CIRBP protects the expression of clock genes from the inhibitory effects of tumour necrosis factor (TNF). However, since TNF represses Cirbp expression, the protective effect of CIRBP is lost. Here, we show that the TNF effect on Cirbp requires the non-canonical NF-κB signalling pathway. While a knock down of RelA does not alter the effects of TNF on Cirbp, a knock down of RelB represses this effect. In addition, the data indicate that p50 and p52 are required in the TNF induced inhibition of Cirbp. These results show that Cirbp expression in TNF treated cells is regulated via the non-canonical NF-κB pathway.

Keywords: Circadian rhythm; Cytokine; Gene transcription; Inflammation; Signalling pathway.

Fig. 1

Inhibition of IKKα and IKKβ prevents TNF mediated inhibition of Cirbp expression. (A) NIH3T3 cells were treated with the IKKα and β inhibitors IKK III and IKK VII in the presence or absence of TNF (10 ng/ml). Data show the expression of Cirbp mRNA (A) and protein (B). As a control for the effectiveness of the NF-κB blockers the expression of IκB was used, Data of RT-qPCR assays show the mean +/− SEM (error bars) of biological triplicates from three independent experiments. Significance of grouped results were calculated with one way-ANOVA and Bonferroni post-hoc test; ^⁎⁎⁎p<0.001. Western blots were quantitated by densitometric analysis; the respective data are given above the western blots.

Fig. 2

Knockdown of RelB, but not of RelA prevents from the inhibitory effect of TNF on Cirbp expression. Cells transfected with siRNA against RelA (A and C) or siRNA against RelB (B and D) were left untreated or treated with TNF (10 ng/ml) for 4 h. Data show the expression of Cirbp (A and B) and the effectiveness of siRNA treatment against RelA (C) and RelB (D). Knockdown reduces RelA expression by 77% and RelB by 70%. Data of RT-qPCR assays show the mean +/−SEM (error bars) of biological triplicates from three independent experiments. Significance of grouped results were calculated with one way-ANOVA and Bonferroni post-hoc test; significance for siRNA effectivity was calculated with a two-tailed Student's t-test; ^⁎p<0.01 ^⁎⁎⁎p<0.001.

Fig. 3

Knockdown of p50 and p52 reduces the TNF induced inhibition of Cirbp expression. NIH3T3 cells were transfected with siRNA against p50, p52 or both. Thereafter, cells were treated with TNF (10 ng/ml). Expression of mRNA (Fig. 3A) and protein (Fig. 3B) was assessed after 4 h and 6 h of TNF treatment, respectively. Compared to nontransfected control cells knockdown of p52 reduces Cirbp mRNA expression by 42%. Densitometric analysis shows data for CIRBP and p50. Since the antibodies to p52 proved not to work reliably, the respective data are not included. Data of RT-qPCR assays show the mean +/− SEM (error bars) of biological triplicates from four independent experiments. Significance of grouped results were calculated with one way-ANOVA and Bonferroni post-hoc test; ^⁎⁎p<0.01, ^⁎⁎⁎p<0.001; ^⁎ without line significance compared to untreated NIH3T3; # gives significance compared to NIH3T3+TNF.