Cylindromatosis (CYLD), a Deubiquitinase, Attenuates Inflammatory Signaling Pathways by Activating Toll-Like Receptor 3 in Human Mesangial Cells

Abstract

Background/Aims: Cylindromatosis (CYLD), a deubiquitinase, negatively regulates nuclear factor-κB in various cells. However, its potential roles in glomerular inflammation remain unclear. Because the activation of the Toll-like receptor 3 (TLR3)/type I interferon (IFN) pathways plays a pivotal role in chronic kidney diseases (CKD), we examined the role of CYLD in the TLR3 signaling in cultured human mesangial cells (MCs). Methods: We stimulated CYLD-silenced MCs with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of dsRNA, and studied representative TLR3/IFN-β pathways (i.e., TLR3/IFN-β/retinoic acid-inducible gene-I (RIG-I)/CCL5, and TLR3/IFN-β/melanoma differentiation associated gene 5 (MDA5)/CXCL10 axes) using RT-PCR, western blotting, and ELISA. We also used immunofluorescence staining and microscopy to examine mesangial CYLD expression in biopsied specimens from patients with CKD. Results: CYLD silencing resulted in an increase of poly IC-induced RIG-I and MDA5 protein levels and increased CCL5 and CXCL10 mRNA and protein expression, but unexpectedly decreased mRNA expressions of RIG-I and MDA5. Interestingly, CYLD silencing did not affect IFN-β or the phosphorylated STAT1 (signal transducers and activator of transcription protein 1). CYLD was highly expressed in biopsied specimens from patients with proliferative lupus nephritis (LN). Conclusion: CYLD inhibits post-transcriptional regulation of RIG-I and MDA5 expression following TLR3 activation in MCs. CYLD may be involved in the pathogenesis of CKD, especially pathogenesis of LN.

Cytosolic Sensors of Viral RNA Are Involved in the Production of Interleukin-6 via Toll-Like Receptor 3 Signaling in Human Glomerular Endothelial Cells

“…Experiments using siRNA confirmed that this response was mediated by signaling via TLR3. Concerning type I IFNs, we have shown in prior works using cultured human GECs and mesangial cells that IFN-β synthesized de novo following TLR3 activation is a key in regional inflammatory cascades [8, 13-15, 20]. Indeed, induction of RIG-I, MDA5, and IL-6 expression was lagged behind the increase in IFN-β levels observed here, and knockdown of IFN-β resulted in decreased expression of these molecules.…”

“…Furthermore, we previously noted intense glomerular expressions of RIG-I and MDA5, cytosolic sensors of viral RNA, in biopsy specimens from patients with proliferative LN, but only MDA5 and not RIG-I expression in specimens from patients with IgA nephropathy [19, 20]. Thus, activation of innate immunity, including the TLR3/RIG-I/MDA5 signaling axis, in the inflammatory process in residual glomerular cells is likely to be involved in the pathogenesis of some forms of GN, especially LN [5-8, 15]. With respect to the recruitment of circulating neutrophils by GECs, we previously observed that activation of TLR3/IFN-β signaling induces endothelial expression of CXCL1 and E-selectin [8], although this finding remains preliminary.…”

“…Small-interfering RNAs (siRNAs) against TLR3 (SI02655156), MDA5 (SI03649037), and RIG-I (SI03019646) and a non-silencing negative-control siRNA (1027281) were obtained from Qiagen (Hilden, Germany). The IFN-β-targeting siRNA used has been described previously [8, 13-15]. An illustra RNA spin kit was supplied by GE Healthcare (Little Chalfont, UK).…”

Regulatory interplay between deubiquitinating enzymes and cytokines

“…CYLD is another DUB that removes K63-Ub chain from RIG-I to decrease the IFN production [26,27], but TBK1 and IKKε were also identified as the target of the deubiquitination of CYLD in 293 EBNA cells [27], resulting in the same effect. CYLD also interacted with IPS-1 to negatively regulate it, but did not deubiquitinate it [27].…”

“…Schmid et al found that in brain and peripheral blood of C57BL/6, the mRNA level of IFN-γ gene decreased with the knockdown of CYLD, while the serum concentration of IFN-γ increased [28]. A study conducted using human kidney mesangial cells (MC) showed slightly different results: silencing CYLD in MC cells and stimulating them with poly IC increased the toll-like receptor 3 (TLR3)-induced activation of RIG-I and MDA5 [26]; however, the level of mRNA of RIG-I and MDA5 actually decreased [26]. The authors speculated this difference to be caused by the change in cell line used [26], but further study is necessary to determine the cause.…”