TLR Regulation of SPSB1 Controls Inducible Nitric Oxide Synthase Induction

Abstract

The mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens via the TLRs. TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing IFN-β (TRIF), which results in the transcription of a small array of genes, including IFN-β. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli, including cytokines and microbes. NO is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. We have recently identified iNOS as a target of the mammalian SPSB2 protein. The SOCS box is a peptide motif, which, in conjunction with elongins B and C, recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin ligase complex. In this study, we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterize the interaction between SPSB1 and iNOS. Through the use of SPSB1 transgenic mouse macrophages and short hairpin RNA knockdown of SPSB1, we show that SPSB1 controls both the induction of iNOS and the subsequent production of NO downstream of TLR3 and TLR4. Further, we demonstrate that regulation of iNOS by SPSB1 is dependent on the proteasome. These results suggest that SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production.

Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity

“…These observations suggest that arctigenin could manifest its effect on iNOS activation through multiple mechanisms. However, recent studies have shown that CHIP is not the only E3 ubiquitin ligase capable of mediating downregulation of iNOS [31,32]. Thus, involvement of other E3 ligases in arctigenin-induced degradation of iNOS needs to be further studied.…”

The role of oxidative stress during inflammatory processes

“…Increasing evidence is indeed accumulating showing that the formation of ROS represents an essential pathway of TLR-dependent signaling in cells from immune and non-immune origin, which occurs mainly through the activation of various NOX isoforms (Tsung et al, 2007;Ogier-Denis et al, 2008;Gill et al, 2010). In addition, the pro-inflammatory signaling cascades triggered by TLR engagement enhance the expression of iNOS, and thus promotes the generation of NO (Lewis et al, 2011;Shweash et al, 2011). In turn, the concomitant generation of O 2 .-(by NOXs) and NO .…”

The SOCS box—Adapting proteins for ubiquitination and proteasomal degradation

“…SPSB1, 2 and 4 are similar both in amino acid sequence and in function. We, and others, have recently shown that inducible nitric oxide synthase (iNOS/NOS2) is a physiological target of the mammalian SPSB1 and SPSB2 proteins, suggesting a role for these proteins in regulating the innate immune response (70)(71)(72). SPSB1, 2 and 4 interact via their SPRY domain with a DINNN motif in the N-terminal region of iNOS and through the SOCS box-associated CRL ubiquitinate iNOS causing it to be degraded by the proteasome.…”