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Alternative splicing generates a diacylglycerol kinase ? transcript that acts as a dominant-negative modulator of superoxide production in localized aggressive periodontitis.

ABSTRACT: Diacylglycerol (DAG), levels of which are tightly regulated by diacylglycerol kinases (DGKs), is a lipid mediator linked to key biologic functions. Members of the DGK family undergo alternative splicing, generating the protein diversity necessary to control different intracellular DAG pools. DGK? function is altered in polymorphonuclear neutrophils (PMNs) of patients with localized aggressive periodontitis (LAgP), suggesting a genetic basis. Here, the authors assess DGK? spliced transcripts in human LAgP neutrophils.In an expression library of a patient with LAgP, PMNs were screened for different DGK? transcripts. Real-time polymerase chain reaction and in vitro expression assays were performed to assess the fate of different transcripts on protein translocation and superoxide production in human leukemia cells (HL-60) and COS-7 cells.A DGK? transcript that lacks exon 10 (DGK??10) and generates a premature stop codon and a truncated protein was identified as being upregulated in LAgP neutrophils. In vitro assays revealed that DGK??10 translocation occurred even in the absence of important regulatory motifs. Transfection of HL-60 neutrophil-like cells with the DGK??10 spliced variant induced an increase in the stimulated production of superoxide anion replicating the phenotype of LAgP PMNs.DGK??10 can act as a dominant-negative transcript that can modulate superoxide production and provides an example of genetic regulation of the inflammatory response that may be relevant to human inflammatory diseases such as LAgP.

SUBMITTER: Batista EL

PROVIDER: S-EPMC4527154 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Alternative splicing generates a diacylglycerol kinase α transcript that acts as a dominant-negative modulator of superoxide production in localized aggressive periodontitis.

Batista Eraldo L EL Kantarci Alpdogan I AI Hasturk Hatice H Van Dyke Thomas E TE

Journal of periodontology 20131030 7

<h4>Background</h4>Diacylglycerol (DAG), levels of which are tightly regulated by diacylglycerol kinases (DGKs), is a lipid mediator linked to key biologic functions. Members of the DGK family undergo alternative splicing, generating the protein diversity necessary to control different intracellular DAG pools. DGKα function is altered in polymorphonuclear neutrophils (PMNs) of patients with localized aggressive periodontitis (LAgP), suggesting a genetic basis. Here, the authors assess DGKα splic ...[more]

PMID: 24171497

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Project description:Localized aggressive periodontitis (LAP) patients possess a systemic hyperinflammatory response after lipopolysaccharide stimulation. However, the levels of inflammatory and bone biomarkers in plasma, as well as possible associations between local and plasma biomarkers, are unknown in LAP. This cross-sectional study aimed to characterize gingival crevicular fluid (GCF) and plasma biomarker profiles in LAP patients, their healthy siblings (HS), and healthy unrelated controls (HC). Fifty-eight LAP subjects, 33 HS, and 49 HC (African Americans, aged 5 to 25 y) were included. Following collection of clinical parameters with GCF and plasma samples, levels of 16 inflammatory and bone resorption biomarkers were determined with Milliplex. Univariate and correlation analyses were performed among all clinical and laboratorial parameters. Discriminant analyses were used to investigate groups of biomarkers discriminating LAP from HS and HC in GCF and plasma. GCF levels of multiple cytokines and chemokines and RANKL:OPG ratio (receptor activator of nuclear factor kappa-B ligand:osteoprotegerin) were higher in LAP disease, most of which positively correlated with probing depth and attachment loss of sampled sites. A group of IL-12p40, IL-6, IL-12p70, IL-2, and MIP-1? discriminated LAP diseased sites from twheir healthy sites, as well as from HS and HC healthy sites. In plasma, only RANKL levels were increased in LAP versus controls, which positively correlated with the percentage of affected sites and deep/bleeding sites. A plasma inflammatory profile including MIP-1?, IL-8, IL-10, and INF-? could significantly discriminate LAP patients from HS and HC. No correlations were found between GCF and plasma levels of biomarkers. In conclusion, an inflammatory profile including groups of specific biomarkers in GCF and plasma may significantly discriminate LAP from healthy individuals. The hyperinflammatory response previously found in the peripheral blood of LAP patients is dependent on lipopolysaccharide stimulation, apparently resulting mostly in local tissue destruction and changes in biomarker profile, with a slight influence in the systemic inflammatory profile (ClinicalTrials.gov NCT01330719). Knowledge Transfer Statement: The results of this study can be possibly used by clinicians in the future as diagnostic tools for localized aggressive periodontitis. Thus, in the future, with proper consideration of cost, patient preference, chair-side feasibility and ultimately further studies validating the role of GCF markers for disease progression and response to treatment, this information could lead to more appropriate therapeutic decisions and the development of preventive approaches for susceptible patients.

Project description:AimThe purpose of this study was to test for the role of the P2X7 receptor in localized aggressive periodontitis (LAP).MethodsPeripheral blood was obtained from 95 subjects with LAP and 76 healthy unrelated controls (HUCs). Three P2RX7 single-nucleotide polymorphisms (rs1718119, rs2230911, and rs3751143) were genotyped from these subjects, and their peripheral blood samples were stimulated with lipopolysaccharide (LPS) from Escherichia coli and tested for inflammatory markers. The 3 P2RX7 single-nucleotide polymorphisms were in found to be in perfect linkage disequilibrium, and a total of 4 haplotypes and 9 diplotypes were identified among all subjects. For both subject populations, the 9 diplotypes were grouped into 4 functional groups and tested for association with subject inflammatory response. To specifically study the effects of extrinsic activation of the P2X7 receptor in LAP, peripheral blood samples from were stimulated under 3 treatments: LPS, LPS + ATP, and LPS +ATP+ P2X7 selective inhibitor. The effects of these treatments on P2X7 receptor activity were measured through Luminex protein assay. Last, to test whether receptor stimulation was related to P2RX7 expression, relative mRNA levels of P2RX7 were quantified with real-time quantitative polymerase chain reaction.ResultsSeveral associations between the P2RX7 diplotypes and LPS-stimulated blood chemokine/cytokine levels were found between the LAP and HUC populations (P < 0.05). P2X7 activation resulted in statistically significant differences in IL-1β and IL-12p40 concentrations for both subject populations. The relative P2RX7 mRNA levels increased significantly after addition of its inhibitor for both LAP and HUC populations.ConclusionsThis study detected an association between P2RX7 functional diplotypes and in vitro immune response of whole blood from subjects with LAP. In addition, we found that inhibition of the activated P2X7 receptor leads to increased P2RX7 mRNA levels, suggesting a feedback loop ( ClinicalTrials.gov NCT01330719).Knowledge transfer statementThe results of this study suggest that P2RX7 functional diplotypes are associated with LAP and their in vitro immune response to bacteria. Ongoing studies to uncover the mechanistic link between P2RX7 and LAP phenotypes could lead to the development of preventive approaches for susceptible subjects.

Dataset Information

Alternative splicing generates a diacylglycerol kinase ? transcript that acts as a dominant-negative modulator of superoxide production in localized aggressive periodontitis.

Publications

Alternative splicing generates a diacylglycerol kinase α transcript that acts as a dominant-negative modulator of superoxide production in localized aggressive periodontitis.

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