Systems biology unravels novel biomarkers in heterogeneous PgLPS-HGF interaction

Abstract

OBJECTIVES: Porphyromonas gingivalis lipopolysaccharide (PgLPS) is a crucial virulence factor strongly associated with chronic periodontitis. It displays significant amount of lipid A structural heterogeneity, containing both tetra- (LPS1435/1449) and penta-acylated (LPS1690) isoforms. We recently showed these isoforms evoked an opposing immuno-inflammatory response in human gingival fibroblasts (HGFs) (IADR/Barcelona, 2010). In the present study we have for the first time employed a systems biology approach using advanced tools in transcriptomics, proteomics, metabolomics and bioinformatics to determine novel molecules induced in the PgLPS-HGF Interaction. METHODS: Primary HGFs were stimulated with 1 µg/ml PgLPS isoforms for 24h using E. coli LPS as the reference. Cellular proteins were separated by 2D-PAGE coupled with quantitative fluorescent dyes. Differentially expressed proteins ≥ 2 folds (P < 0.05) were identified by tandem mass spectrometry (MS/MS). Tryptic digested secretory proteins were identified by affinity chromatography combined Nano-LC-MALDI TOF/TOF MS/MS. Generic and specific biomarkers were cataloged using bioinformatics pathways. HGF RNA was subjected to transcriptomic analysis using toll-like receptor pathway gene-arrays. RESULTS: Overall, 32 differentially expressed cell-bound proteins and 176 secretory proteins (metabolome) were identified in HGF by using LPS1435/1449 and LPS1690. Both induced the expression of cathepsins, osteonectin, ADAM-28 and TGF-β binding protein. Conversely, PgLPS1690 induced the expression of IL-6, IL-8, GM-CSF, GCSF and hitherto underscribed small molecular weight proteins. In contrast, PgLPS1435/1449 induced the expression of anti-inflammatory molecules such as SOCS box proteins, IL-20, TNF-α inhibitor and down-regulated the expression of lipid peroxidation pathway molecules perilipin, superoxide dismutase, peroxiredoxin-6 and thioredoxin. PgLPS1690 activated the apoptosis-related pathway (Ras-Bax), whereas Pg LPS1435/1449 inhibited it. CONCLUSION: The present study unraveled novel biomarkers with immuno-inflammatory agonist-antagonist activities released by HGFs in their interaction with the heterogeneous PgLPS isoforms, which may lay the foundation for the development of novel diagnostic and therapeutic approaches to control periodontal diseases (GRF HKU766909M to LJ JIN).