Simvastatin decreases IL-6 and IL-8 production in epithelial cells

Abstract

Many cardiovascular studies have suggested that 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) have anti-inflammatory effects independent of cholesterol lowering. As a chronic inflammatory disease, periodontitis shares some mechanisms with atherosclerosis. Since oral epithelial cells participate importantly in periodontal inflammation, we measured simvastatin effects on interleukin-6 and interleukin-8 production by cultured human epithelial cell line (KB cells) in response to interleukin-1alpha. Simvastatin decreased production, an effect reversed by adding mevalonate or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate. Simvastatin was found to reduce NF-kappaB and AP-1 promoter activity in KB cells. Dominant-negative Rac1 severely inhibited interleukin-1alpha-induced NF-kappaB and AP-1 promoter activity. Our results may indicate an anti-inflammatory effect of simvastatin on human oral epithelial cells, apparently involving Rac1 GTPase inhibition.

Figures

Figure 1

Dose-dependent effects of simvastatin on KB cells. (A) Cellular expression of IL-6 and IL-8 is shown in cultures after treatment with the indicated concentrations of simvastatin for 7 hrs, and/or IL-1α (1 ng/mL) for the last 5 of these hrs. Data are expressed as means ± SD (n = 4). *P < 0.01 and **P < 0.001 vs. control (treated with IL-1α). (B) Evaluation of cytotoxicity from simvastatin according to release of LDH into medium was carried out after 24 hrs of culture. LC, low control (medium alone); HC, high control (1% Triton X100).

Figure 2

Reversal of inhibitory effect of simvastatin on KB cells by co-treatment with downstream metabolites of HMG-CoA reductase. (A) Inhibitory effects of simvastatin (10−6 M) on KB cells were reversed by co-treatment with mevalonate (10−4 M) or GGPP (5 × 10−6 M), but not with FPP (5 × 10−6 M). Data are expressed as means ± SD (n = 4). *P < 0.001 vs. control (treated with IL-1α). (B) Schematic representation of the mevalonate pathway. Statins block conversion of HMG-CoA to mevalonate. This leads to reduced synthesis of cholesterol and decreased prenylation of proteins such as small GTPases. Isopentenyl-PP, isopentenyl pyrophosphate; Geranyl-PP, geranyl pyrophosphate.

Figure 3

Suppression of NF-κB and AP-1 activity in IL-1α-stimulated KB cells by simvastatin (10−6 M). KB cells were transiently co-transfected with pNF-κB-luc or pAP-1-luc, together with pCMV-κgal. The cells were analyzed 48 hrs later, with five-hour stimulation with IL-1α (1 ng/mL). All results were normalized for transfection efficiency using expression of β-galactosidase. Data are expressed as means ± SD (n = 4). *P < 0.001 vs. control (treated with IL-1α).

Figure 4

Effects of Rho family GTPases on IL-1α-mediated transactivation of NF-κB and AP-1. (A) IL-1α-induced NF-κB and AP-1 promoter activity of the transient transfectants of N17Rac1 was greatly inhibited, and IL-1α-induced NF-κB and AP-1 promoter activities of transiently transfected cells (N17Cdc42 and N19RhoA) also were inhibited. Mock, transient transfectants of empty vector. Data represent means ± SD (n = 4). *P < 0:01 and **P < 0.001 vs. control (mock with IL-1α). (B) Model depicting contributions of Rho family GTPases to NF-κB and AP-1 activation by IL-1. Rho, Rho family GTPases; GG-Rho, geranylgeranylated Rho family GTPases.