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• W2897547577 abstract "•NLRP3 inflammasome activation couples SREBP2 maturation•SCAP-SREBP2 translocation and S1P are required for optimal NLRP3 inflammasome activity•SCAP escorts both NLRP3 and SREBP2 by forming a ternary complex•SCAP-SREBP2 inhibition protects mice from systemic inflammation Cholesterol metabolism has been linked to immune functions, but the mechanisms by which cholesterol biosynthetic signaling orchestrates inflammasome activation remain unclear. Here, we have shown that NLRP3 inflammasome activation is integrated with the maturation of cholesterol master transcription factor SREBP2. Importantly, SCAP-SREBP2 complex endoplasmic reticulum-to-Golgi translocation was required for optimal activation of the NLRP3 inflammasome both in vitro and in vivo. Enforced cholesterol biosynthetic signaling by sterol depletion or statins promoted NLPR3 inflammasome activation. However, this regulation did not predominantly depend on changes in cholesterol homeostasis controlled by the transcriptional activity of SREBP2, but relied on the escort activity of SCAP. Mechanistically, NLRP3 associated with SCAP-SREBP2 to form a ternary complex which translocated to the Golgi apparatus adjacent to a mitochondrial cluster for optimal inflammasome assembly. Our study reveals that, in addition to controlling cholesterol biosynthesis, SCAP-SREBP2 also serves as a signaling hub integrating cholesterol metabolism with inflammation in macrophages. Cholesterol metabolism has been linked to immune functions, but the mechanisms by which cholesterol biosynthetic signaling orchestrates inflammasome activation remain unclear. Here, we have shown that NLRP3 inflammasome activation is integrated with the maturation of cholesterol master transcription factor SREBP2. Importantly, SCAP-SREBP2 complex endoplasmic reticulum-to-Golgi translocation was required for optimal activation of the NLRP3 inflammasome both in vitro and in vivo. Enforced cholesterol biosynthetic signaling by sterol depletion or statins promoted NLPR3 inflammasome activation. However, this regulation did not predominantly depend on changes in cholesterol homeostasis controlled by the transcriptional activity of SREBP2, but relied on the escort activity of SCAP. Mechanistically, NLRP3 associated with SCAP-SREBP2 to form a ternary complex which translocated to the Golgi apparatus adjacent to a mitochondrial cluster for optimal inflammasome assembly. Our study reveals that, in addition to controlling cholesterol biosynthesis, SCAP-SREBP2 also serves as a signaling hub integrating cholesterol metabolism with inflammation in macrophages. The immune and metabolic systems are highly integrated with one another, and this is critical for immune cells to fulfill their functions. A hallmark of pro-inflammatory macrophages is the increased anabolic demand in response to pathogen- or damage-associated molecular patterns (PAMPs or DAMPs) (O’Neill et al., 2016O’Neill L.A. Kishton R.J. Rathmell J. A guide to immunometabolism for immunologists.Nat. Rev. Immunol. 2016; 16: 553-565Crossref PubMed Scopus (1465) Google Scholar, Pearce and Pearce, 2013Pearce E.L. Pearce E.J. Metabolic pathways in immune cell activation and quiescence.Immunity. 2013; 38: 633-643Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar). The NLRP3 inflammasome is a molecular platform that modulates innate immune functions via the maturation of caspase-1 and via the cleavage of cytokine substrates such as pro-interleukin (IL)-1β and pro-IL-18 (Rathinam and Fitzgerald, 2016Rathinam V.A. Fitzgerald K.A. Inflammasome complexes: emerging mechanisms and effector functions.Cell. 2016; 165: 792-800Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Recent studies have implicated several cellular metabolites and metabolic enzymes, traditionally linked to metabolism but not immunity, in modulation of NLRP3 inflammasome activation (Próchnicki and Latz, 2017Próchnicki T. Latz E. Inflammasomes on the crossroads of innate immune recognition and metabolic control.Cell Metab. 2017; 26: 71-93Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Cholesterol is an essential lipid involved in diverse biological processes (Tall and Yvan-Charvet, 2015Tall A.R. Yvan-Charvet L. Cholesterol, inflammation and innate immunity.Nat. Rev. Immunol. 2015; 15: 104-116Crossref PubMed Scopus (794) Google Scholar). Excess cholesterol can form cholesterol crystals, which directly activate the NLRP3 inflammasome and underlie the pathogenesis of atherosclerosis (Duewell et al., 2010Duewell P. Kono H. Rayner K.J. Sirois C.M. Vladimer G. Bauernfeind F.G. Abela G.S. Franchi L. Nuñez G. Schnurr M. et al.NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.Nature. 2010; 464: 1357-1361Crossref PubMed Scopus (2570) Google Scholar). Cholesterol accumulation in dendritic cells accelerates the development of autoimmunity via the NLRP3 inflammasome, possibly at the transcriptional level (Ito et al., 2016Ito A. Hong C. Oka K. Salazar J.V. Diehl C. Witztum J.L. Diaz M. Castrillo A. Bensinger S.J. Chan L. Tontonoz P. Cholesterol accumulation in CD11c+ immune cells is a causal and targetable factor in autoimmune disease.Immunity. 2016; 45: 1311-1326Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, Westerterp et al., 2017Westerterp M. Gautier E.L. Ganda A. Molusky M.M. Wang W. Fotakis P. Wang N. Randolph G.J. D’Agati V.D. Yvan-Charvet L. et al.Cholesterol accumulation in dendritic cells links the inflammasome to acquired immunity.Cell Met. 2017; 25: 1294-1304Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, the molecular mechanisms that directly link cholesterol metabolic signaling and NLRP3 inflammasome activation remain poorly defined. The sterol regulatory element-binding proteins (SREBPs) are master regulators of lipid homeostasis (Goldstein et al., 2006Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar). Mammals have two SREBP-encoding genes that express three SREBPs: SREBP1a, SREBP1c, and SREBP2. SREBP2 principally regulates genes involved in cholesterol metabolism, whereas genes involved in fatty-acid metabolism are preferentially regulated by SREBP1. They are synthesized as inactive precursors localized to the endoplasmic reticulum (ER) bound to SREBP cleavage-activating protein (SCAP). SCAP escorts SREBPs via coat protein complex II (COPII) vesicles to the Golgi apparatus where two steps of proteolytic cleavage mediated by Site-1 protease (S1P) and Site-2 protease (S2P) releases the N terminus of SREBP protein and allows its entry into the nucleus (Goldstein et al., 2006Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar). In the current study, we found that SCAP-SREBP2 promoted NLRP3 inflammasome activation, which was mainly dependent on its ER-to-Golgi translocation, rather than by affecting cholesterol homeostasis. By inhibiting this translocation, synthetic SCAP-SREBP2 inhibitors and endogenous sterols including cholesterol and 25-HC suppressed NLRP3 inflammasome activation both in vitro and in vivo. In contrast, cholesterol depletion or statins promoted NLRP3 inflammasome activation by enforcing this process. Collectively, we unveil an important role of SCAP-SREBP2 that is directly used by NLRP3 for inflammasome activation, thus suggesting an intimate metabolic-inflammatory crosstalk in pro-inflammatory macrophages. Upon nigericin stimulation in lipopolysaccharide (LPS)-primed THP1 macrophages, we found that the SREBP2 inactive precursor (pre-SREBP2) was clearly proteolytically processed to the N-terminal mature form (n-SREBP2) (Figure 1A). To determine whether this nigericin-induced SREBP2 processing occurred via the well-established SCAP-, S1P-, and S2P-dependent pathway, we used several synthetic inhibitors and endogenous sterols targeting this pathway (Figure 1B). Betulin, a small molecule that specifically inhibits SCAP-SREBP2 activation by interacting with SCAP (Tang et al., 2011Tang J.J. Li J.G. Qi W. Qiu W.W. Li P.S. Li B.L. Song B.L. Inhibition of SREBP by a small molecule, betulin, improves hyperlipidemia and insulin resistance and reduces atherosclerotic plaques.Cell Metab. 2011; 13: 44-56Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), suppressed this nigericin-induced cleavage of SREBP2 (Figure 1C). Desmosterol binds to SCAP and prevents it from interacting with COPII proteins, which results in the retention of SCAP-SREBP2 in the ER. In contrast, the oxysterol 25-HC binds to the ER-retention proteins Insig1 and/or Insig2, thereby causing SCAP-SREBP2 to also be retained in the ER (Spann and Glass, 2013Spann N.J. Glass C.K. Sterols and oxysterols in immune cell function.Nat. Immunol. 2013; 14: 893-900Crossref PubMed Scopus (204) Google Scholar). We found that either desmosterol or 25-HC suppressed this nigericin-induced SREBP2 maturation (Figure 1D). Furthermore, S1P inhibition by PF-429242 or AEBSF and S2P inhibition by 1,10-phenanthroline (Feng et al., 2007Feng L. Yan H. Wu Z. Yan N. Wang Z. Jeffrey P.D. Shi Y. Structure of a site-2 protease family intramembrane metalloprotease.Science. 2007; 318: 1608-1612Crossref PubMed Scopus (188) Google Scholar, Okada et al., 2003Okada T. Haze K. Nadanaka S. Yoshida H. Seidah N.G. Hirano Y. Sato R. Negishi M. Mori K. A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6.J. Biol. Chem. 2003; 278: 31024-31032Crossref PubMed Scopus (172) Google Scholar, Shao and Espenshade, 2014Shao W. Espenshade P.J. Sterol regulatory element-binding protein (SREBP) cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-activating protein (SCAP).J. Biol. Chem. 2014; 289: 7547-7557Crossref PubMed Scopus (52) Google Scholar) were capable of suppressing this nigericin-induced SREBP2 maturation (Figure 1E). Altogether, these findings suggested that this SREBP2 maturation occurring during NLRP3 inflammasome activation also relied on SCAP and the sequential processing by S1P and S2P. In contrast, treatment with the caspase-1 inhibitor z-YVAD-fmk or the pan-caspase inhibitor z-VAD-fmk had limited effects on this SREBP2 maturation (Figure 1F). To further strengthen these results in primary macrophages, we measured the mRNA expression of SREBP2 target genes as indicative of SREBP2 maturation, including Srebf2 which is a direct target of itself (Sato et al., 1996Sato R. Inoue J. Kawabe Y. Kodama T. Takano T. Maeda M. Sterol-dependent transcriptional regulation of sterol regulatory element-binding protein-2.J. Biol. Chem. 1996; 271: 26461-26464Crossref PubMed Scopus (226) Google Scholar). Our data showed that the five measured genes involved in cholesterol metabolism, Srebf2, Hmgcr, Ldlr, Hmgcs1, and Insig1, were modestly increased in LPS-primed bone marrow-derived macrophages (BMDMs) and then further enhanced upon ATP or nigericin stimulation (Figures 1G and S1A). Similar results were obtained in LPS-primed mouse peritoneal macrophages upon ATP stimulation (Figure 1H). In contrast, the caspase-1 inhibitor VX765 had no effect on the upregulation of these SREBP2 target genes (Figure S1A). Consistently, SCAP or S1P deficiency in BMDMs blocked this upregulation of SREBP2 target genes, whereas NLRP3, ASC, or Caspase-1 deficiency showed less effect on that, further suggesting that SREBP2 maturation may not depend on NLRP3 inflammasome activation in macrophages (Figure 1I). In contrast, the six measured genes involved in fatty-acid metabolism, Srebf1, Acly, Acaca, Fasn, Acss2, and Gpam, were less affected during NLRP3 inflammasome activation in BMDMs (Figure S1B). In addition, Scap and cholesterol efflux genes including Abca1 and Abcg5 were largely unchanged (Figure S1C). Taken together, our data demonstrate that activation of the NLRP3 inflammasome is coupled with SCAP ER-to-Golgi translocation and SREBP2 maturation. We next examined the role of SCAP-SREBP2 in NLRP3 inflammasome activation by acute inhibition of its ER-to-Golgi translocation (Figure 2A). Betulin, which inhibits SCAP-SREBP2 ER-to-Golgi translocation by interacting with SCAP, markedly suppressed ATP-induced caspase-1 maturation and IL-1β or IL-18 secretion in a dose-dependent manner (Figures 2B and 2C). Apart from ATP, betulin also inhibited the caspase-1 maturation and IL-1β secretion triggered by the NLRP3 agonists nigericin, monosodium urate crystals (MSU), and alum (Figures 2D and 2E). In addition, ASC (apoptosis-associated speck-like protein containing a CARD) nucleation-induced oligomerization and ASC-speck formation were also inhibited by betulin (Figures 2F and 2G). In contrast, betulin had no effect on activation of the AIM2 and NLRC4 inflammasomes, which are triggered by poly(dA:dT) transfection and Salmonella typhimurium infection, respectively (Figures 2H and 2I). Another selective synthetic inhibitor of SCAP-SREBP2 ER-to-Golgi translocation via binding to SCAP, fatostatin (Kamisuki et al., 2009Kamisuki S. Mao Q. Abu-Elheiga L. Gu Z. Kugimiya A. Kwon Y. Shinohara T. Kawazoe Y. Sato S. Asakura K. et al.A small molecule that blocks fat synthesis by inhibiting the activation of SREBP.Chem. Biol. 2009; 16: 882-892Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), similarly inhibited activation of the NLRP3 inflammasome (Figures 2J and 2K). We reasoned that the suppressed NLRP3 inflammasome activity might be explained by a perturbation in cellular cholesterol homeostasis. However, the cellular cholesterol amounts and viability remained unchanged after treatment of these inhibitors (Figures S2A and S2B). We also examined these inhibitory effects of betulin and fatostatin on NLRP3 inflammasome activation in human monocyte-derived macrophages and found similar results (Figures S2C and S2D). We next investigated this regulation of the NLRP3 inflammasome using two endogenous sterols that directly inhibit SCAP-SREBP2 ER-to-Golgi translocation: 25-HC and cholesterol itself. We found that 25-HC dose dependently repressed ATP- or nigericin-induced caspase-1 maturation, IL-1β production, ASC oligomerization, and ASC-speck formation (Figures 2L, 2M, S2E, and S2F). Although cholesterol crystal has been shown to promote inflammasome activation and atherogenesis (Duewell et al., 2010Duewell P. Kono H. Rayner K.J. Sirois C.M. Vladimer G. Bauernfeind F.G. Abela G.S. Franchi L. Nuñez G. Schnurr M. et al.NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.Nature. 2010; 464: 1357-1361Crossref PubMed Scopus (2570) Google Scholar, Sheedy et al., 2013Sheedy F.J. Grebe A. Rayner K.J. Kalantari P. Ramkhelawon B. Carpenter S.B. Becker C.E. Ediriweera H.N. Mullick A.E. Golenbock D.T. et al.CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation.Nat. Immunol. 2013; 14: 812-820Crossref PubMed Scopus (614) Google Scholar), under physiological conditions, the ER contains a delicate feedback system that senses the excess cholesterol to inactivate SCAP-SREBP2 translocation and thus maintains the intracellular cholesterol within narrow limits (Goldstein et al., 2006Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar, Radhakrishnan et al., 2008Radhakrishnan A. Goldstein J.L. McDonald J.G. Brown M.S. Switch-like control of SREBP-2 transport triggered by small changes in ER cholesterol: a delicate balance.Cell Metab. 2008; 8: 512-521Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, Spann and Glass, 2013Spann N.J. Glass C.K. Sterols and oxysterols in immune cell function.Nat. Immunol. 2013; 14: 893-900Crossref PubMed Scopus (204) Google Scholar). As predicted, acute treatment with 20–60 μg/mL methyl-β-cyclodextrin (MCD)-cholesterol during activation stage of the NLRP3 inflammasome, concentrations typically used to inhibit SCAP-SREBP2 ER-to-Golgi translocation, clearly suppressed, rather than promoted, NLRP3 inflammasome activation in a dose-dependent manner (Figures 2N and 2O). Moreover, 60 μg/mL cholesterol did not directly activate the NLRP3 inflammasome in LPS-primed BMDMs as a danger signal like crystalline cholesterol (Figures 2N and 2O). Importantly, this inhibitory effect of cholesterol on NLRP3 inflammasome activation was prompt (Figure S2G), which is in line with the report that MCD-cholesterol is transferred rapidly from the plasma membrane to the ER by vesicular or non-vesicular carriers (Prinz, 2007Prinz W.A. Non-vesicular sterol transport in cells.Prog. Lipid Res. 2007; 46: 297-314Crossref PubMed Scopus (75) Google Scholar). Furthermore, we also examined this regulatory effect of cholesterol on NLRP3 inflammasome with liposomal-cholesterol and obtained similar results (Figure S2H). SCAP-SREBP2 plays a well-defined role in the transcriptional regulation of enzymes involved in the cholesterol-biosynthetic mevalonate pathway. However, consistent with the previous studies showing that blockade of the mevalonate pathway by statins enhances NLRP3 inflammasome activation (Próchnicki and Latz, 2017Próchnicki T. Latz E. Inflammasomes on the crossroads of innate immune recognition and metabolic control.Cell Metab. 2017; 26: 71-93Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, Spann and Glass, 2013Spann N.J. Glass C.K. Sterols and oxysterols in immune cell function.Nat. Immunol. 2013; 14: 893-900Crossref PubMed Scopus (204) Google Scholar), NB-598 targeting downstream squalene epoxidase also slightly increased IL-1β production (Figures S2I and S2J). Taken together, our results suggest a role of SCAP-SREBP2 ER-to-Golgi translocation in activation of the NLRP3 inflammasome. We next adopted multiple genetic strategies to investigate the role of SCAP-SREBP2 in NLRP3 inflammasome activation. First, silencing of Scap (encoding SCAP) or Srebf2 (encoding SREBP2) by small-interfering RNAs (siRNAs) blocked optimal NLRP3 inflammasome activation without affecting TNF-α production in peritoneal macrophages (Figures 3A and 3B ). Second, we generated J774.1 and THP1 macrophages stably expressing shRNAs targeting Scap and SREBF2, respectively. Similarly, J774.1-SCAP-shRNA and THP1-SREBP2-shRNA macrophages showed greatly impaired NLRP3 inflammasome activity (Figures S3A–S3E). In contrast, TNF-α production and NLRC4 inflammasome activation remained unaffected in these cells (Figures S3F–S3H). In line with a previous study showing that SCAP deficiency in BMDMs had limited effect on total amounts of lipids including cholesterol (York et al., 2015York A.G. Williams K.J. Argus J.P. Zhou Q.D. Brar G. Vergnes L. Gray E.E. Zhen A. Wu N.C. Yamada D.H. et al.Limiting cholesterol biosynthetic flux spontaneously engages type I IFN signaling.Cell. 2015; 163: 1716-1729Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), these stable Scap-silenced macrophages showed minimally affected cellular cholesterol amounts (Figure S3I), suggesting that SCAP may control NLRP3 inflammasome activation without affecting cellular cholesterol amounts. We also confirmed these observations with macrophage-specific deletion of SCAP using the Lyz2-cre model (Lyz2-cre-Scapf/f). As expected, BMDMs from Lyz2-cre-Scapf/f mice had impaired NLRP3 inflammasome activation triggered by different NLRP3 agonists compared with control mice (Figures 3C–3E). Moreover, compared with wild-type mice, the remaining NLRP3 inflammasome activity in BMDMs from Lyz2-cre-Scapf/f mice caused by the residual SCAP expression showed a similar kinetic response upon ATP stimulation (Figure 3D), suggesting that SCAP deficiency may not affect response sensitivity of the NLRP3 inflammasome. In contrast, TNF-α production was unaffected in the absence of SCAP (Figure 3E). To further examine whether any potential changes in cholesterol homeostasis contribute to this suppressed NLRP3 activity caused by SCAP deficiency, we supplemented different concentrations of MCD-cholesterol in these macrophages. In line with the results that cholesterol suppressed NLRP3 inflammasome activation (Figures 2O and S2F), supplementation of MCD-cholesterol suppressed, rather than restored, IL-1β production with a similar kinetic pattern in both wild-type and SCAP-deficient BMDMs (Figure 3F). These results together suggest that SCAP may play a direct role in NLRP3 inflammasome activation rather than change cholesterol homeostasis. Previous studies have demonstrated that overexpression of SCAP or SREBP2 leads to spontaneous SCAP-SREBP2 ER-to-Golgi translocation (Sakai et al., 1998Sakai J. Nohturfft A. Goldstein J.L. Brown M.S. Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence from in vivo competition studies.J. Biol. Chem. 1998; 273: 5785-5793Crossref PubMed Scopus (191) Google Scholar). Consistently, stably transfected THP1 macrophages overexpressing SCAP or SREBP2 showed increased NLRP3 inflammasome activity (Figures 3G–3J). A similar result was also obtained in immortalized BMDMs stably overexpressing SCAP (Figure S3J). To further confirm that this promoting effect of SCAP on the NLRP3 inflammasome occurs at the activation stage, we reconstituted NLRP3 inflammasomes in HEK293T cells without expression of pro-IL-1β. Notably, overexpression of SCAP promoted nigericin-induced caspase-1 maturation (Figure 3K). Besides overexpressing their components, we further assessed the effect of SCAP-SREBP2 on the NLRP3 inflammasome by using a “SCAP-SREBP2 activating medium” which constitutively transports SCAP-SREBP2 from the ER to the Golgi apparatus caused by the sterol depletion (Goldstein et al., 2006Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar). We found that this medium enhanced nigericin-induced caspase-1 maturation as well as IL-1β and IL-18 secretion compared with the control medium (Figures 3L and 3M), while the production of TNF-α was less affected (Figure 3M). More importantly, without an NLRP3 agonist, SCAP-SREBP2 activating medium alone promoted the maturation of IL-1β (Figures 3M and S3K). In addition, we induced acute SCAP-SREBP2 ER-to-Golgi translocation by sterol extraction with methyl-β-cyclodextrin (Goldstein et al., 2006Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar, Gurcel et al., 2006Gurcel L. Abrami L. Girardin S. Tschopp J. van der Goot F.G. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival.Cell. 2006; 126: 1135-1145Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). As expected, ATP-induced IL-1β production was augmented (Figure S3L). Similarly, MCD alone spontaneously triggered the maturation of IL-1β in LPS-primed macrophages, and this effect was impeded by inhibiting SCAP-SREBP2 translocation with 25-HC (Figure S3L). These results together suggest that enforced cholesterol biosynthetic signaling in macrophages promotes activation of the NLRP3 inflammasome. To address the role of S1P- and S2P-mediated two-step cleavage in NLRP3 inflammasome activation, we first silenced the expression of S1P and S2P with siRNAs targeting Mbtps1 (encoding S1P) and Mbtps2 (encoding S2P) in peritoneal macrophages (Figures S4A–S4C). Our results revealed that specific siRNA-mediated reduction of S1P but not S2P resulted in marked suppression of NLRP3 inflammasome activation (Figures 4A–4D, S4D, and S4E). These results are in line with the report that the release of SCAP from the Golgi depends on the cleavage of SREBP protein by S1P but not S2P (Shao and Espenshade, 2014Shao W. Espenshade P.J. Sterol regulatory element-binding protein (SREBP) cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-activating protein (SCAP).J. Biol. Chem. 2014; 289: 7547-7557Crossref PubMed Scopus (52) Google Scholar), suggesting a requirement of SCAP release from the Golgi for optimal NLRP3 inflammasome activation. We next generated mice deficient in S1P in macrophages (Lyz2-cre-Mbtps1f/f) and found that the S1P-deficient BMDMs had greatly impaired NLRP3 inflammasome activity triggered by different NLRP3 agonists (Figures 4E, 4F, and S4F). Consistently, supplementation of different concentrations of cholesterol did not restore the decreased NLRP3 inflammasome activity in S1P-deficient BMDMs, but rather suppressed its residual NLRP3 inflammasome activity in a similar kinetic pattern to wild-type BMDMs (Figure 4G). In addition, we investigated the involvement of S1P in NLRP3 inflammasome activation using PF-429242 and AEBSF, two S1P inhibitors that do not affect S2P activity (Okada et al., 2003Okada T. Haze K. Nadanaka S. Yoshida H. Seidah N.G. Hirano Y. Sato R. Negishi M. Mori K. A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6.J. Biol. Chem. 2003; 278: 31024-31032Crossref PubMed Scopus (172) Google Scholar, Shao and Espenshade, 2014Shao W. Espenshade P.J. Sterol regulatory element-binding protein (SREBP) cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-activating protein (SCAP).J. Biol. Chem. 2014; 289: 7547-7557Crossref PubMed Scopus (52) Google Scholar). Our results revealed that acute treatment of either AEBSF or PF-429242 greatly suppressed the NLRP3 inflammasome activity (Figures 4H–4J), without affecting cellular cholesterol amounts and viability (Figures S2A and S2B). Brefeldin A (BFA), which causes the Golgi to collapse into the ER and results in constitutive processing of SREBP2 in the ER by relocating S1P to the ER (DeBose-Boyd et al., 1999DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi.Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), spontaneously induced IL-1β secretion without an NLRP3 agonist (Figures 4K and 4L). Importantly, this secretion was suppressed by S1P inhibition with AEBSF (Figures 4K and 4L), suggesting that enforced S1P-mediated cleavage of SREBP2 promoted NLRP3 inflammasome activation. In contrast, the production of TNF-α was not affected in these experiments (Figures 4K and 4L). In addition to BFA, we retrovirally transduced Lyz2-cre-Scapf/f BMDMs with a mutant form of S1P that contains an ER retention and retrieval signal, Lys-Asp-Glu-Leu (KDEL). This also causes relocation of S1P from the Golgi to the ER and bypasses the SCAP requirement for SREBP2 cleavage (DeBose-Boyd et al., 1999DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi.Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Compared with the empty vector, S1P-KDEL enhanced NLRP3 inflammasome activation but not TNF-α production in Lyz2-cre-Scapf/f BMDMs, while the inactive S1P-KDEL-S414A mutant had no effect (Figures 4M and S4G). Together, these results suggest that S1P-mediated SREBP2 cleavage promotes NLRP3 inflammasome activation. The requirement of SCAP-SREBP2 for optimal NLRP3 inflammasome activation prompted us to investigate the possibility that NLRP3 associates with this complex. Co-immunoprecipitation analysis in LPS-primed BMDMs and THP1 macrophages stably expressing V5-SCAP revealed the association of NLRP3 with SCAP upon ATP and nigericin stimulation (Figures 5A and 5B ). Similarly, NLRP3 also associated with SREBP2 in LPS-primed BMDMs and THP1 macrophages stably expressing Flag-SREBP2, and this was further enhanced by stimulation with an NLRP3 agonist (Figures 5C and 5D). To further confirm these interactions, we performed GST pull-down assay and found the direct interaction of NLRP3 with either SCAP or SREBP2 (Figures 5E and 5F). To further delineate the association of NLRP3 with the SCAP-SREBP2 complex, we introduced various truncated mutants of these proteins according to their domain structures into HEK293T cells (Figure 5G). As predicted, NLRP3 associated with the SCAP WD40-repeat domain but not the N-terminal transmembrane domain, mainly through its NACHT domain (Figures 5H, 5I, S5A, and S5B). In addition, the NLRP3 NACHT domain and the SREBP2 C-terminal regulatory domain were mainly required for the association between NLRP3 and SREBP2 (Figures 5J, 5K, S5C, and S5D). These results presented thus far support a scenario in which NLRP3 associates with both SCAP and SREBP2, so we hypothesized that a ternary complex might be formed with these three proteins. To test this hypothesis, we performed co-immunoprecipitation experiments in HEK293T cells transfected with V5-SCAP, HA-NLRP3, and Flag-SREBP2. Immunoprecipitation of SREBP2 pulled down both SCAP and NLRP3, while immunoprecipitation of SCAP pulled down both NLRP3 and SREBP2 (Figure 5L). Furthermore, we performed a two-step co-immunoprecipitation assay in these cells. As expected, NLRP3 was present in the final immunoprecipitate but not in the control samples (Figure 5M). In contrast, SCAP and SREBP2 did not associate with other essential components of the NLRP3 inflamma" @default.
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• W2897547577 date "2018-11-01" @default.
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• W2897547577 title "Cholesterol Homeostatic Regulator SCAP-SREBP2 Integrates NLRP3 Inflammasome Activation and Cholesterol Biosynthetic Signaling in Macrophages" @default.
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• W2897547577 doi "https://doi.org/10.1016/j.immuni.2018.08.021" @default.
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