TXNIP interacts with various proteins including NLRP Yoshiha

TXNIP interacts with various proteins, including NLRP3 (Yoshihara et al., 2014, Zhou et al., 2010). Emerging evidence suggests that TXNIP plays a role in ER stress-mediated cell death and in the NLRP3 inflammasome activation step (Abderrazak et al., 2015, Lerner et al., 2012, Oslowski et al., 2012, Zhou et al., 2010). Our results demonstrate that Fxr KO mice displayed robust changes in TXNIP levels and severe liver injury in response to ER stress. Consistently, FXR activation attenuated the ER stress-mediated induction of TXNIP. Thus, the ability of FXR to inhibit TXNIP may contribute to the inhibition of the NLRP3 inflammasome. We also showed that ER stress induced TXNIP through the PERK-CHOP pathway in hepatocytes. However, TXNIP seemed to not be directly regulated by CHOP, as suggested by the lack of CHOP binding sites within the −5-kb human TXNIP promoter region. TXNIP belongs to the ATF5-specific genes (Teske et al., 2013). So, CHOP may indirectly control TXNIP through ATF5 (Teske et al., 2013). In contrast to NLRP3, TXNIP was also affected by IRE1 pathway in hepatocytes, which is consistent with the finding that IRE1 destabilized miR-17 for TXNIP in pancreatic beta cells (Lerner et al., 2012). These findings indicate that NLRP3 and TXNIP expression might be differentially controlled by UPR signaling. TXNIP serves as an inhibitory partner of thioredoxin, regulating redox homeostasis in cells (Lu and Holmgren, 2014, Yoshihara et al., 2014). Thus, the overexpression of NLRP3 and its binding partner TXNIP might contribute to ER stress-induced hepatocyte injury and be augmented by FXR deficiency. NCK1 is the SRC homology-domain-containing adaptor protein, functionally linking cell surface receptors with the tcs products cytoskeleton (Li et al., 2001). NCK1 interacts with PERK through protein-protein binding, inhibiting the activity of PERK (Yamani et al., 2014, Yamani et al., 2015). In this study, we proposed that NCK1 has a functional role in the FXR regulation of the UPR pathway in hepatocytes (i.e., that FXR inhibits PERK through NCK1 expression). Our data show that ER stress inhibited NCK1 and that this effect was overcome by FXR activation. Moreover, our results show that the siRNA-mediated knockdown of Nck1 attenuated the inhibitory effects of FXR on NLRP3, TXNIP, and CHOP expression under ER stress condition. Consistently, the rescuing effect of FXR agonist on cell viability was diminished by Nck1 silencing. Thus, the inhibition of the NLRP3 inflammasome by FXR, and the consequent attenuation of hepatocyte injury in response to ER stress, may depend on NCK1, indicating that a rheostatic balance might exist between NCK1 and PERK. miR-186 has been studied in the field of cancer biology (Cai et al., 2013, Ruan et al., 2016, Zhu et al., 2016). Here, we report a role for miR-186 in the regulation of ER stress-induced NLRP3 inflammasome activation. Of the putative microRNAs targeting NCK1, FXR specifically regulated miR-186 in our study. Consistently, the ligand-mediated activation of FXR prevented ER stress from causing an increase in miR-186 levels. Our results indicate that miR-186 might inhibit NCK1, as supported by the experiments where we used a miR-186 mimic or inhibitor (ASO). In these experiments, transfecting hepatocyte cell models with a miR-186 mimic prevented NLRP3 inflammasome downregulation by FXR under ER stress condition. By the same token, the knocking down of Nck1 reversed the inhibitory effect of miR-186 ASO on the NLRP3 inflammasome, indicating that a functional association might exist between these molecules and NLRP3 inflammasome activation in hepatocytes. Our time course study also verified that a temporal relationship exists between the expression of miR-186, CHOP, and NLRP3. In our supplementary analysis, a p53 binding site was predicted in the promoter region of miR-186. Hence, we do not exclude the possibility that small heterodimer partner-mediated p53 inhibition (Lee et al., 2010) contributes to the regulation of miR-186.