Gene expression profile of human gingival fibroblasts induced by interleukin-1beta

Objectives: IL-1b is a key cytokine inducing numerous inflammatory mediators in the pathogenesis of periodontitis. We investigated effects of IL-1b on gene-expression in immortalized-human-gingival-fibroblasts (HGFs) using GeneChip-expression-arrays sampling transcript levels for 40,000 genes.

Methods: Gene-expression-analyses were performed using GeneChip®-U133-plus-2-arrays with biological replications of IL-1b- and mock-treated cells. Data were statistically analyzed by Cyber-t program. Confirmation of expression was carried out using quantitative-RT-PCR and results were compared with DDCt-method. Phospho-IkBa-protein was determined by Western analysis.

Results: 382-probe-sets of 270-genes were differentially expressed in IL-1b-induced cells (P< 0.001). 226-genes were up-regulated and 42-genes were down-regulated. AP-1 transcription-factor members (cJun, Junb, Fos and ATF3), NF-kB pathway members, (NF-kB1, NF-kB2, IkBa, IkBe, IkBz, REL, RELB, TA-NFKBH) and other transcription factors including EGR-3, ETS1, STAT5A, IRF1 were up-regulated. Expression of TNF members (TNFa, FAS, TNFIP-1, -2, -3, -6, -8, TRAF1, TRAF4, TIFA), IL-6, IL-8, IRAK2, MMP3, MMP12 and COX-2 were up-regulated. Chemokines (CCL2, CCL20, CCL5, CXCL1, CXCL2, CXCL3, CXCL6, CXCL10), interferon-induced proteins, anti-apoptotic genes (BCL3, BCL2A1, BIRC-2,-3,-5), membrane receptors (BRKRB1, 2, ICAM1, VCAM1, CD44) and colony-stimulating factors were up-regulated after 2hours-IL-1b-induction. Of the 31 genes validated by Q-RT-PCR, 29 showed concordance between two experiments (93.5%). Of the IL1b-induced genes, 27 were down-regulated by pre-treatment with NF-kB inhibitor-BAY 11-7085. Pre-treatment of IL-1b-induced cells with NF-kB-inhibitor decreased the number of viable cells suggesting an anti-apoptotic role for NF-kB in IL-1b-induced-HGFs. IkBa, inhibitor of NF-kB activation, was phosphorylated by IL-1b-induction indicating NF-kB activation by IL-1b.

Conclusion: IL-1b-induced numerous genes of pro-inflammatory pathways which may be relevant to responses in HGFs leading to pathological status of periodontitis. IL-1b may regulate gene expression predominantly by stimulation of NF-kB pathway in HGFs and NFkB-inhibition may be a good strategy for controlling IL-1-mediated inflammation in periodontitis. Gene-expression-arrays applied to HGFs may be a useful tool for investigating new treatments targeting IL-1b-regulated-inflammatory genes.