Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20

Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting ‘autophagy receptors’ in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF–TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.     

Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin

The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were blocked by GF-109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization and androgen responses in target cells. Received 31 July 2001; received after revision 18 September 2001; accepted 30 October 2001     

Activation of protease-activated receptor (PAR) 1 by frog trefoil factor (TFF) 2 and PAR4 by human TFF2

Trefoil factors (TFFs) promote epithelial cell migration to reseal superficial wounds after mucosal injury, but their receptors and the molecular mechanisms underlying this process are poorly understood. In this study, we showed that frog TFF2 activates protease-activated receptor (PAR) 1 to induce human platelet aggregation. Based on this result, we further tested the involvement of PARs in human TFF2 (hTFF2)-promoted mucosal healing. hTFF2-stimulated migration of epithelial HT-29 cells was largely inhibited by PAR4 depletion with small interfering RNAs but not by PAR1 or PAR2 depletion. The PAR4-negative epithelial cell lines AGS and LoVo were highly responsive to hTFF2 as assessed by phosphorylation of ERK1/2 and cell migration upon PAR4 expression. Our findings suggest that hTFF2 promotes cell migration via PAR4. These findings will be helpful in further investigations into the functions and molecular mechanisms of TFFs and PARs in physiology and disease.