Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Abstract

Toll-like receptors (TLR) are sentinel receptors capable of recognizing pathogen-associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG-containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram-positive and Gram-negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up-regulation of co-stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up-regulated following LPS stimulation. The up-regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor-alpha induced by LPS plus CpG ODN. While inhibition of extracellular signal-related kinase and NF-kappaB activation suppressed the up-regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up-regulation of TLR2 and TLR4 mRNA expression but enhanced the up-regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up-regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.

Figure 1

Up-regulation of TLR2, TLR4 and TLR9 mRNA expression by LPS in mouse immature DC. Mouse immature DC were stimulated with 10 ng/ml LPS for 1, 2, 3, 4 and 5 hr. After LPS stimulation, total RNA was prepared and TLR gene expression was examined by semiquantitative RT-PCR. β-actin expression was used as control. The signal for TLR gene expression was integrated on a Gel Doc 1000 Mini-Transilluminator (Bio-Rad). Relative gene expression of TLR over that of β-actin was plotted again the time (in hours) the cells were treated with constitutive TLR expression expressed as 100%. Similar results were observed in three independent experiments.

Figure 2

Induction of TNF-α in mouse DC by CpG ODN and LPS. Mouse immature DC were stimulated with 3·0 µg/ml CpG ODN, 10 ng/ml LPS, or 3·0 µg/ml CpG ODN plus 10 ng/ml LPS for 18 hr. Supernatants were measured using an ELISA kit for TNF-α. Data are shown as an average from duplicate wells. Similar results were obtained in three independent experiments.

Figure 3

Involvement of ERK, p38 kinase and NF-κB pathways in the regulation of TLR2, TLR4 and TLR9 mRNA expression by LPS in mouse immature DC. (a) Mouse immature DC were pretreated with 30 µm PD98059, 30 µm SB203580, or 15 µm PDTC for 30 min and followed by stimulation with LPS for 1 hr. Total RNA was prepared and expression of TLR was analysed by semiquantitative RT-PCR. β-actin mRNA expression was used as control. Relative TLR expression was shown with constitutive TLR expression expressed as 100%. Similar results were obtained in three independent experiments. (b) DC were treated as described in Fig. 3(a). Thirty micrograms of total RNA isolated from the treated cells was loaded per lane to be fractioned. The levels of TLR2, TLR4 and TLR9 were determined by Northern blot. One representative ethidium bromide (EtBr)-stained gel (bottom) is shown to indicate relative amounts of RNA loaded per lane. (c) LPS-induced activation of ERK and p38 kinase in DC was inhibited by specific inhibitors. Upper panel: mouse immature DC were pretreated with PD98059 followed by stimulation with LPS for the indicated times. LPS-mediated ERK phosphorylation was detected by Western blot using anti-phospho-ERK mAb. Lower panel: mouse immature DC were pretreated with SB203580 followed by stimulation with LPS for the indicated time. LPS-mediated p38 kinase phosphorylation was detected using anti-phospho-p38 mAb. The results of a representative experiment of three are presented. (d) PDTC inhibited LPS-induced NF-κB nuclear translocation. Mouse immature DC were pretreated with 15 µm PDTC for 30 min followed by stimulation with LPS for the indicated time. Nuclear extracts were prepared and nuclear translocated NF-κB p65 subunit was measured by Western blot using an anti-p65 antibody.