Abstract
Protein SUMOylation has been reported to play a role in innate immune response, but the enzymes, substrates, and consequences of the specific inflammatory signaling events are largely unknown. Reactive oxygen species (ROS) are abundantly produced during macrophage activation and required for Toll-like receptor 4 (TLR4)–mediated inflammatory signaling. Previously, we demonstrated that SENP3 is a redox-sensitive SUMO2/3 protease. To explore any links between reversible SUMOylation and ROS-related inflammatory signaling in macrophage activation, we generated mice with Senp3 conditional knock-out in myeloid cells. In bacterial lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models, we found that SENP3 deficiency markedly compromises the activation of TLR4 inflammatory signaling and the production of proinflammatory cytokines in macrophages exposed to LPS. Moreover, Senp3 conditional knock-out mice were significantly less susceptible to septic shock. Of note, SENP3 deficiency was associated with impairment in JNK phosphorylation. We found that MKK7, which selectively phosphorylates JNK, is a SENP3 substrate and that SENP3-mediated deSUMOylation of MKK7 may favor its binding to JNK. Importantly, ROS-dependent SENP3 accumulation and MKK7 deSUMOylation rapidly occurred after LPS stimulation. In conclusion, our findings indicate that SENP3 potentiates LPS-induced TLR4 signaling via deSUMOylation of MKK7 leading to enhancement in JNK phosphorylation and the downstream events. Therefore this work provides novel mechanistic insights into redox regulation of innate immune responses.
Highlights
Protein SUMOylation has been reported to play a role in innate immune response, but the enzymes, substrates, and consequences of the specific inflammatory signaling events are largely unknown
The results of quantitative reverse transcription PCR showed that the mRNA transcriptional levels of IL-6, TNF␣, and IL-1 were significantly lower in SENP3 knock-down cells compared with nonspecific small interfering RNA (siRNA) control cells (Fig. 1A)
Correlations between SUMOylation and inflammatory signaling have been widely proposed in various types of cells (18 –20, 23–25, 28, 29, 31, 48 – 64), but studies conducted in real bone marrow-derived macrophages (BMDM) or RAW macrophages in innate immune contexts are scarce [18, 20, 23, 24, 28, 29]
Summary
We examined the expression of the major inflammatory cytokines in macrophages exposed to 100 ng/ml LPS for 6 h. When we overexpressed SUMO2/3 in these SENP3 knockdown cells, a more remarkable increase of JNK phosphorylation could be observed in K18R mutant-expressing cells, compared with that in WT-expressing cells (Fig. 4C) This might be attributed to that more SUMO2/3 conjugation to the WT sustained a difference in effects of a Lys-18 –SUMO-less MKK7 over a SUMOylated one. Given that a Lys-18 –SUMO-less MKK7 mutant increased JNK phosphorylation, we speculated that SUMO conjugation at this region might hinder the JNK binding moiety of MKK7 To test this hypothesis, an MKK7–SUMO3 fusion protein with a FLAG tag was overexpressed in TLR4 293 cells. The abundance of basal and LPS-triggered endogenous SUMO-conjugation of MKK7 appeared robustly in SENP3 knock-out BMDM, indicating a remarkable deSUMOylation otherwise occurred during LPS exposure (Fig. 5F) These results suggested that SENP3 potently deconjugates SUMO from MKK7 under LPS-induced inflammation scenarios. These data demonstrated that the cKO mice had generally less severe systemic inflammation
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