Cholesterol Crystals Activate the NLRP3 Inflammasome in Human Macrophages: A Novel Link between Cholesterol Metabolism and Inflammation

Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1beta and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1beta has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway.Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1beta from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1beta was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1beta secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1beta secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization.The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions

Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure

RHO methylation matters: A role for isoprenylcysteine carboxylmethyltransferase in cell migration and adhesion

Numerous proteins involved in diverse aspects of cell biology undergo a process of post-translational modification termed prenylation. The prenylation pathway consists of three enzymatic steps, the final of which is methylation of the carboxyl-terminal prenylcysteine formed in the first two steps by the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt). Due to the prevalence of prenylated proteins in cancer biology and the findings that several of the proteins are involved in processes controlling cell migration and adhesion, we sought to examine the role of Icmt-mediated methylation on cell behavior associated with metastasis. We found that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading were also impaired by Icmt inhibition. Further investigation revealed that inhibition of Icmt significantly decreased the activation of both RhoA and Rac1, which are both prenylated proteins. The data obtained were consistent with the decreased activation being due to an increase in Rho GDP-dissociation inhibitor (GDI) binding by both proteins in the absence of their methylation. Importantly, the addition of exogenous RhoA or Rac1 to cells in which Icmt was inhibited was able to partially, but selectively, rescue directed and random migration, respectively. These results establish a role for Icmt-mediated methylation in cell migration, and point to specific prenylated proteins involved in this biology. The prenylation pathway has been targeted for oncogenic therapies, but the role of methylation in cell motility had been largely unexplored until now. The finding that methylation of Rho family members impacts on a specific component of their function provides an additional avenue through which to interrogate the biology of this important class of regulatory proteins