Identification of mRNAs for the various diacylglycerol kinase isoforms in neutrophils from patients with localized aggressive periodontitis

Background: Diacylglycerol kinase (DGK) metabolizes diacylglycerol (DAG), an endogenous activator of protein kinase C, to phosphatidic acid. We have previously reported increased DAG in neutrophils from patients with localized aggressive periodontitis (LAP) associated with reduced DGK activity. This reduction could be related to a mutation, post-translational modification, differential expression, or lack of expression of a particular isoform(s). Objective: The aim of this study was to identify the mRNAs for DGK isoforms in normal and LAP neutrophils. Methods: The α-, γ-, and δ-isoforms of DGK were identified by polymerase chain reaction (PCR) using specific oligonucleotide primers for each isoform. The PCR products were isolated and sequenced for comparison to published sequences to confirm the validity of the PCR reaction. Total RNA was isolated from LAP and normal neutrophils, and northern blotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to examine the level of mRNA for each isoform. Results: No major differences were found in the isoform pattern between resting normal and LAP neutrophils. However, the levels of mRNA for the α- and γ-isoforms of DGK were increased in normal neutrophils while slightly decreased in LAP cells upon stimulation with N-formyl-methionyl-leucyl- phenylalanine (fMLP). Conclusion: These data suggest that alterations in the mRNAs for the various isoforms of DGK during cell stimulation and the involvement of DGK that is expressed in multiple forms are subject to a variety of regulatory/control mechanisms and these mechanisms may explain the role of the 'primed' neutrophil phenotype associated with LAP.