Inhibition of interleukin 1β–stimulated interleukin‐6 production by cranberry components in human gingival epithelial cells: effects on nuclear factor κB and activator protein 1 activation pathways

Abstract

In periodontitis, gingival epithelial cells can produce interleukin (IL)-6, a regulator of osteoclastic bone resorption, in response to IL-1β. IL-1β regulates cytokine expression via signaling pathways, including nuclear factor (NF)-κB and mitogen activated protein kinase (MAPK)/activator protein (AP)-1. Cranberry proanthocyanidins (PACs) inhibit IL-1β-stimulated IL-6 production, but specific mechanisms are unclear. The objectives of this study were to determine effects of cranberry PACs on NF-κB and MAPK/AP-1 activation of IL-1β-stimulated IL-6 production in gingival epithelial cells. Cranberry high molecular weight non-dialyzable material (NDM), rich in PACs, was derived from cranberry juice. Human gingival epithelial cells [Smulow-Glickman (S-G)] were incubated with IL-1β in the presence or absence of NDM or inhibitors of NF-κB, [nemo-binding domain (NBD) peptide] or AP-1 (SP600125), and IL-6 levels were measured by ELISA. Effects of NDM on IL-1β-activated NF-κB and AP-1 and phosphorylated intermediates in both pathways were measured in cell extracts via binding to specific oligonucleotides and specific sandwich ELISAs, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. IL-1β (≥ 0.1 nm) caused a time- and dose-dependent stimulation of S-G epithelial cell IL-6 production (p < 0.005). This was significantly decreased in a dose-dependent manner by NBD peptide or SP600125 [maximum inhibition ~30-40% (p < 0.02)], and together, the two inhibitors decreased IL-6 by ~80%, similar to the inhibition caused by NDM (p < 0.001). IL-1β stimulated NF-κB and AP-1 activation (p < 0.003), which was inhibited by NDM (p < 0.0001). NDM did not significantly affect IL-1β-stimulated levels of phosphorylated intermediates in the NF-κB pathway (IκBα) or the AP-1 pathway (c-Jun, ERK1/2). In S-G epithelial cells, IL-1β appeared to upregulate IL-6 production via activation of both NF-κB and MAPK/AP-1 signaling pathways because cranberry NDM decreased nuclear levels of IL-1β-activated NF-κB (p65) and AP-1 (phospho-c-Jun) and strongly inhibited IL-6 production. Lack of inhibition of phosphorylation of IκBα, c-Jun or ERK1/2 suggested that NDM might affect both pathways downstream from those points in S-G cells, such as ubiquitination and proteosomal degradation of IκBα, or inhibition of nuclear activity of c-Jun and/or ERK1/2. Defining these points of inhibition precisely may help identify molecular targets of cranberry polyphenols.