Arl8a与树突状细胞TLR4两条下游途径的相关性

Crosstalk between Arl8a and TLR4 Signaling in Dendritic Cells

为探讨Arl8a(ADP-ribosylation factor-like 8A)与树突状细胞(dendritic cells,DCs)TLR4两条下游信号途径的关系,用Arl8a和GEFH1(guanine nucleotide-exchange factors H1)的siRNA转染来自野生型小鼠的DC,进行LPS刺激或未刺激处理后,检测TLR4-TRIF途径中RhoB靶蛋白MYH9的mRNA表达。然后从野生型和IFNα/β受体基因敲除小鼠中分离和培养DC,LPS刺激后收集细胞扩增总cDNA,通过实时定量PCR检测Arl8a的mRNA表达。再用Arl8a的siRNA转染DC,LPS刺激后检测IL-6和IL-12a的mRNA表达。结果表明,Arl8a和GEFH1的siRNA均能显著抑制LPS介导的MYH9的mRNA表达(P<0.01),而且在LPS刺激后,Arl8a的mRNA表达在野生型小鼠的DC中增加,在IFNα/β受体基因敲除小鼠的DC中则未被上调。此外,Arl8a的siRNA对IL-6和IL-12a的mRNA表达没有显著效应。以上结果提示,在转录水平,Arl8a和GEFH1均对MYH9的表达有影响,并且Arl8a基因的表达与TRIF-IFNβ途径有关,Arl8a可能与MyD88途径中细胞因子IL-6和IL-12a的表达无关。

To elucidate the crosstalk between Arl8a and two downstream pathways of TLR4 signaling in dendritic cells （DCs）, we silenced guanine nucleotide-exchange factors H1 （GEFHI） and Arl8a in DCs from wild- type （WT） mice with small interference RNAs （siRNA）, and examined the mRNA levels of MYH9 which was targeted by RhoB in TLR4-TRIF pathway, with or without LPS stimulation. Then, we used Real-time PCR to detect Arl8a mRNA level in LPS stimulated DCs isolated from wild-type and IFNα/β receptor knockout mice （IFNα/β RKO）. We also analyzed IL-6 and IL-12a mRNA expression in DCs after Arl8a silenced by siRNA. The results showed that Arl8a and GEFH 1 siRNA significantly suppressed the LPS-mediated up-regulation of MYH9 mRNA （P〈0.01）. In addition, the up-regulation ofArl8a mRNA was observed in DC from WT mice but not in DC from IFNα/β RKO after LPS incubation, indicating that LPS-induced up-regulation of Arl8a was attenuated by knockout of IFNα/β receptor. However, Arl8a siRNA failed to alter IL-6 and IL-12a mRNA level in DC after LPS stimulation. In conclusion, GEFH1 and RhoB were proved to be able to regulate MYH9 expression, while the Arl8a level could be modified by TRIF-IFNβ pathway in DC. There was no evidence that Arl8a is involved in MyD88-dependent signaling induced cytokines IL-6 and IL-12a expression.