Lipopolysaccharide induces overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells: possible key phenomenon of hepatolithiasis

Abstract

Bacterial infection, bile stasis, mucin hypersecretion, and an alteration of the mucin profile such as an aberrant expression of gel-forming apomucin (MUC2 and MUC5AC) in the intrahepatic biliary tree are thought to be important in the lithogenesis of hepatolithiasis. So far, there have been no detailed studies linking bacterial infection to altered mucus secretion of biliary epithelium. In this study, the influence of lipopolysaccharide (LPS), a bacterial component, on apomucin expression in cultured murine biliary epithelial cells was examined with emphasis on the participation of tumor necrosis factor (TNF)-alpha. It was found that LPS up-regulated the expression of MUC2 and MUC5AC in cultured murine biliary epithelial cells. LPS also induced the expression of TNF-alpha in biliary epithelial cells and its secretion into the culture medium. The up-regulation of these apomucins was inhibited by pretreatment with TNF-alpha antibody. TNF-alpha alone also induced the overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells. This overexpression was inhibited by pretreatment with calphostin C, an inhibitor of protein kinase C. These findings suggest that LPS can induce overexpression of MUC2 and MUC5AC in biliary epithelial cells via synthesis of TNF-alpha and activation of protein kinase C. This mechanism might be involved in the lithogenesis of hepatolithiasis.

Figure 1.

Expression of CD14, CD120a, and CD120b in cultured murine BECs. A: RT-PCR revealed mRNA expression of CD14 (453 bp), CD120a (200 bp), and CD120b (380 bp). B: Western blot analysis revealed protein expression of CD14 (53 kd), CD120a (55 kd), and CD120b (75 kd).

Figure 2.

A: RT-PCR of MUC1, MUC2, MUC3, and MUC5AC mRNAs in the cultured BECs. B: Expression levels of these apomucin mRNAs in BECs with or without LPS and TNF-α antibody were compared. No significant changes were observed among the three groups in MUC1. LPS significantly enhanced the expression of MUC2, MUC3, and MUC5AC. In BECs treated with LPS and TNF-α antibody, the LPS-induced expression of MUC2 and MUC5AC was partially inhibited, but MUC3 expression was not. The ratio of the band density of apomucin mRNA to β-actin mRNA was taken as the expression level. Results are expressed as a ratio of each expression level to the expression level of control BECs, and the data represent the mean ± SEM. β-actin was used as an internal marker. Control, BECs incubated with basic medium; LPS, BECs incubated with LPS alone; LPS + TNF-α, BECs incubated with LPS and TNF-α antibody; RELC, ratio to the expression level of control. *, P < 0.05.

Figure 3.

Immunoblot analysis of MUC1, MUC3, and MUC5AC. Signals were detected in the high molecular weight region greater than 200 kd that consisted of mucin glycoproteins. Signals for MUC1 were almost of the same intensity in the three groups. MUC3 and MUC5AC were overproduced in the LPS-treated group. In BECs incubated with LPS and TNF-α antibody (TNF-α Ab), the signal for MUC3 was comparable to that of the LPS-treated group, and the signal for MUC5AC was weaker than that of the LPS-treated group. Control, BECs incubated with basic medium; LPS, BECs incubated with LPS alone; LPS + TNF-α Ab, BECs incubated with LPS and TNF-α antibody.

Figure 4.

A: RT-PCR of TNF-α, IL-1α, and IL-1β mRNAs in BECs with or without LPS treatment. BECs expressed these three inflammatory cytokines but the expression of IL-1α and IL-1β was weak compared to that of TNF-α. B: Time-course of TNF-α mRNA expression in BECs cultured with or without LPS. TNF-α expression was significantly increased by addition of LPS in the early phase. The ratio of the band density of cytokine mRNA to β-actin mRNA was taken as the expression level, and the expression level at time 0 (just before the addition of LPS) was regarded as the expression level of control. Results are expressed as a ratio of each expression level to the expression level of the control and the data represent the mean ± SEM. β-actin was used as an internal marker. RELC, ratio to the expression level of control. Control, BECs incubated with basic medium; LPS, BECs incubated with LPS. *, P < 0.05 versus controls.

Figure 5.

ELISA of TNF-α in the culture medium. TNF-α in the medium of BECs treated with LPS was significantly increased at 6 to 48 hours after addition of LPS compared to nontreated BECs. TNF-α was quantified using a mouse TNF-α antigen standard curve. Results represent the mean ± SEM. Control, the culture medium of nontreated BECs; LPS, the culture medium of LPS-treated BECs. *, P < 0.05 versus controls.

Figure 6.

Northern blot analysis of MUC2 (A) and MUC5AC (B) mRNA expression in BECs cultured with LPS, TNF-α, TNF-α antibody, and calphostin C. A and B: In BECs cultured with TNF-α (1.0 ng/ml and 10.0 ng/ml), signals for MUC2 and MUC5AC mRNA were detected, and their levels showed a dose-dependent manner. In BECs cultured with TNF-α and calphostin C, MUC2 showed no signal and MUC5AC a very weak signal. In BECs cultured with LPS, signals for MUC2 and MUC5AC were detected. In BECs cultured with LPS and TNF-α antibody or calphostin C, no band of MUC2 and MUC5AC was observed. Signals for MUC2 or MUC5AC mRNA were confirmed using RNA extracted from murine colon or stomach. Lane 1, control; lane 2, TNF-α (0.1 ng/ml); lane 3, TNF-α (1.0 ng/ml); lane 4, TNF-α (10.0 ng/ml); lane 5, TNF-α (10.0 ng/ml) and calphostin C; lane 6, LPS (100 μg/ml); lane 7, LPS (100 μg/ml) and TNF-α antibody; lane 8, LPS (100 μg/ml) and calphostin C; lane 9, stomach; lane 10, colon. C: In ethidium bromide-stained gel, RNA was applied in each lane is approximately equal amounts.

Figure 7.

PKC assay of cultured BECs. In BECs incubated with TNF-α, PKC was significantly activated compared to control BECs, and in a dose-dependent manner. In BECs cultured with TNF-α and calphostin C, TNF-α-induced activation of PKC was inhibited. In BECs cultured with LPS, PKC was significantly activated, which was inhibited in BECs treated with LPS and TNF-α antibody or LPS and calphostin C. Control, BECs incubated with basic medium; TNF, BECs incubated with TNF-α; TNF + CP; BECs incubated with TNF-α and calphostin C; LPS, BECs incubated with LPS; LPS + TNF-Ab, BECs incubated with LPS and TNF-α antibody; LPS + CP, BECs incubated with LPS and calphostin C; *, P < 0.05 versus control; **, P < 0.05 versus TNF (10.0 ng/ml); ***, P < 0.05 versus LPS.

Figure 8.

Mechanism of MUC2 and MUC5AC overproduction and secretion in BECs induced by LPS. LPS binds to CD14 expressed on BECs, which is followed by TNF-α synthesis and secretion. Secreted TNF-α reacts with TNF receptors (CD120a and CD120b) in an autocrine or paracrine manner. TNF-α activates PKC, which mediates the up-regulation of MUC2 and MUC5AC transcription and the secretion.