Abstract
The NLRP3 inflammasome is a central regulator of inflammation in many common diseases, including atherosclerosis and type 2 diabetes, driving the production of pro-inflammatory mediators such as IL-1β and IL-18. Due to its function as an inflammatory gatekeeper, expression and activation of NLRP3 need to be tightly regulated. In this study, we highlight novel post-transcriptional mechanisms that can modulate NLRP3 expression. We have identified the RNA-binding protein Tristetraprolin (TTP) as a negative regulator of NLRP3 in human macrophages. TTP targets AU-rich elements in the NLRP3 3'-untranslated region (UTR) and represses NLRP3 expression. Knocking down TTP in primary macrophages leads to an increased induction of NLRP3 by LPS, which is also accompanied by increased Caspase-1 and IL-1β cleavage upon NLRP3, but not AIM2 or NLRC4 inflammasome activation. Furthermore, we found that human NLRP3 can be alternatively polyadenylated, producing a short 3'-UTR isoform that excludes regulatory elements, including the TTP- and miRNA-223-binding sites. Because TTP also represses IL-1β expression, it is a dual inhibitor of the IL-1β system, regulating expression of the cytokine and the upstream controller NLRP3.
Highlights
The NLRP3 inflammasome is a central regulator of inflammation in many common diseases, including atherosclerosis and type 2 diabetes, driving the production of pro-inflammatory mediators such as IL-1 and IL-18
During detailed analysis of the human NLRP3 sequence, we noticed that expressed sequence tags (ESTs) indicated alternative 3Ј-ends of transcripts, even though only one 3Ј-untranslated region (UTR) is annotated in RefSeq (Fig. 1a)
Polyadenylation sites are located at the 3Ј-end of both isoforms, suggesting that they could arise from alternative polyadenylation
Summary
The NLRP3 3-UTR is alternatively polyadenylated and contains a repressive AU-rich element. To verify expression of the isoforms experimentally, we analyzed expression of NLRP3 in monocytes and macrophages by 3Ј-RACE This successfully amplified two isoforms of the expected lengths, ϳ600 bp for the long and 300 bp for the short isoform (Fig. 1b). A main ARE consisting of three overlapping AUUUA pentamers is present in this region, in close proximity to the target site for miR-223 [15] (supplemental Fig. S2d) These features are only partially conserved in the mouse gene. In agreement with a weak proximal polyadenylation site, we could only detect one isoform of the murine Nlrp3 3Ј-UTR by 3Ј-RACE (supplemental Fig. S2c). Relative NLRP3 mRNA Normalized luciferase expression (relative to empty vector) Relative expression a long: full length short: proximal half end: distal half
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