Dataset Information

Oxysterols Modify NLRP2 in Epithelial Cells, Identifying a Mediator of Ozone-induced Inflammation.

ABSTRACT: Ozone (O3) is a prevalent air pollutant that causes lung inflammation following exposure. Previous studies demonstrate that O3 oxidizes lipids, such as cholesterol, in the airway to produce oxidized cholesterol products (oxysterols), such as secosterol-A (secoA), which form covalent linkages preferentially with lysine residues and consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. Using an alkynyl-tagged form of secosterol-A and shotgun proteomics, we identified NLR Family Pyrin Domain Containing 2 (NLRP2) to be adducted at lysine (K1019) in the terminal leucine-rich-repeat, a known regulatory region for NLR proteins. We characterized NLRP2 expression in airway epithelial cells and used CRISPR-Cas9 knockout and shRNA knockdown of NLRP2 for analysis of inflammasome complex activity and pro-inflammatory mediator production following O3 exposure. We observed an increase in caspase-1 activity in response to O3, which was not altered by knocked down NLRP2 expression. O3-induced pro-inflammatory gene expression for CXCL1, CXCL2, and IL8 was further enhanced in NLRP2 knockout cells. Together, our findings uncover NLRP2 as a highly abundant, key component of pro-inflammatory signaling pathways in airway epithelial cells and formation of oxysterol-NLRP2 adduct as a novel mediator of O3-induced inflammation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Hye-Young Kim

LAB HEAD: Ned A. Porer, Hye-Young H. Kim

PROVIDER: PXD019824 | Pride | 2021-06-21

REPOSITORIES: Pride

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Publications

Oxysterols Modify NLRP2 in Epithelial Cells, Identifying a Mediator of Ozone-induced Inflammation.

Perryman Alexia A Speen Adam M AM Kim Hye-Young H HH Hoffman Jessica R JR Clapp Phillip W PW Rivera Martin William W Snouwaert John N JN Koller Beverly H BH Porter Ned A NA Jaspers Ilona I

American journal of respiratory cell and molecular biology 20211101 5

Ozone (O<sub>3</sub>) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O<sub>3</sub> oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol A (SecoA), which are electrophiles that are capable of forming covalent linkages preferentially with lysine residues and that consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. B ...[more]

PMID: 34126877

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Project description:RELEVANCE: Smith-Lemli-Opitz syndrome (SLOS) is a human disease caused by mutations in the gene coding for the enzyme DHCR7 (7-dehydrocholesterol (7DHC) reductase), which catalyzes the final step of cholesterol biosynthesis. Accumulated 7DHC in tissues and body fluids of SLOS patients gives rise to numerous oxidation products (oxysterols) in situ, some of which are cytotoxic, and which may contribute to the pathophysiology of SLOS. The SLOS phenotype is broad, ranging from death in utero to viable individuals with malformations and malfunctions in numerous organ systems and tissues, including neurological and cognitive defects. The latter presentations suggest that impaired cholesterol synthesis, in particular the generation of toxic oxysterols, has a deleterious impact on the morphogenesis and viability of neurons in the CNS. In a rat model of SLOS (using a small molecule inhibitor of DHCR7), the loss of photoreceptors was also documented, distinct from the continuous viability of other retinal neurons and supporting cells, and this selective cell death was recapitulated in vitro using 661W cells (an immortalized line derived from mouse cones) incubated with purified 7DHC-derived, SLOS-associated oxysterols. Upon exposure to these compounds, cell viability assay results for 661W showed one to two orders of magnitude higher sensitivity with respect to efficacy and potency, and also an accelerated time frame for cell death, compared to retinal Mueller glia and retinal pigment epithelial cells. These findings inspired questions as to the molecular mechanisms underlying oxysterol-induced neuronal cell death. Therefore, we characterized differential gene expression associated with processes and pathways induced by oxysterol treatments, first, to provide insights regarding cell death and dysfunction, not only in SLOS, but also extended to other neurodegenerative diseases (including those affecting the retina), and second, to identify cellular protective responses, with the expectation that these findings would suggest possibilities for future prevention and treatment of neurological disease and damage. INTENT: Generate gene expression array profiles of 661W cells following exposure to either of two oxysterols. The oxysterols are employed at doses already determined to exert full cytotoxicity by 24 hours incubation time; however, for the purposes of the array, stop incubations for each oxysterol at time points preceding global cell death, to harvest still-intact RNA from a majority of cells that have maintained membrane integrity, and are also displaying microscopically observable indications of morphological response to the oxysterol treatments already known to precede the demise of the cells. Therefore, the samples are expected to manifest transcriptomes emblematic of gene expression changes in response to the oxysterols. These changes should fit patterns that correlate with pathways and processes associated with cellular damage and regulated cell death, or with cell survival/protective and repair mechanisms (with expected overlap of the two opposing scenarios). As a negative control, a separate set of replicates are incubated with cholesterol under conditions having no impact on cell viability (23 hours, at a (non-physiological) concentration intermediate between that of the two oxysterols). To identify differentially expressed genes, individual array probe set data for either oxysterol and for cholesterol are matched with those from cells incubated with a vehicle control (for 24 hours), for computing “-fold change” in expression and statistical significance of expression differences. EXPERIMENTAL WORKFLOW: 1) 661W cells were seeded in 100-mm cell culture-treated dishes at a density permitting proliferation to subconfluence, allowing cells to retain neurite-like extensions; includes overnight adaptation to a simplified incubation medium devoid of most growth factors and reagents supporting antioxidant activity. 2) Cells were exposed for a predetermined time period to a single concentration of: i) either of two different oxysterols (EPCD, an endoperoxide specific to SLOS, or 7-ketocholesterol); ii) cholesterol; or iii) vehicle control. 3) Total RNA was harvested, from each triplicate sample representing the above treatments, according to the RNeasy Plus minikit protocol (Qiagen). 4) Final sample preparation (amplification, labeling, fragmentation of cRNA) was carried out a core facility following Affymetrix specifications and protocols. 5) Hybridized chips were scanned to generate raw intensity data for further analysis.

Dataset Information

Oxysterols Modify NLRP2 in Epithelial Cells, Identifying a Mediator of Ozone-induced Inflammation.

Publications

Oxysterols Modify NLRP2 in Epithelial Cells, Identifying a Mediator of Ozone-induced Inflammation.

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