DUSP-1 Induced by PGE2 and PGE1 Attenuates IL-1β-Activated MAPK Signaling, Leading to Suppression of NGF Expression in Human Intervertebral Disc Cells

Abstract

The molecular mechanism of discogenic low back pain (LBP) involves nonphysiological nerve invasion into a degenerated intervertebral disc (IVD), induced by nerve growth factor (NGF). Selective cyclooxygenase (COX)-2 inhibitors are mainly used in the treatment of LBP, and act by suppressing the inflammatory mediator prostaglandin E₂ (PGE₂), which is induced by inflammatory stimuli, such as interleukin-1β (IL-1β). However, in our previous in vitro study using cultured human IVD cells, we demonstrated that the induction of NGF by IL-1β is augmented by a selective COX-2 inhibitor, and that PGE₂ and PGE₁ suppress NGF expression. Therefore, in this study, to elucidate the mechanism of NGF suppression by PGE₂ and PGE₁, we focused on mitogen-activated protein kinases (MAPKs) and its phosphatase, dual-specificity phosphatase (DUSP)-1. IL-1β-induced NGF expression was altered in human IVD cells by MAPK pathway inhibitors. PGE₂ and PGE₁ enhanced IL-1β-induced DUSP-1 expression, and suppressed the phosphorylation of MAPKs in human IVD cells. In DUSP-1 knockdown cells established using small interfering RNA, IL-1β-induced phosphorylation of MAPKs was enhanced and prolonged, and NGF expression was significantly enhanced. These results suggest that PGE₂ and PGE₁ suppress IL-1β-induced NGF expression by suppression of the MAPK signaling pathway, accompanied by increased DUSP-1 expression.

Keywords: dual-specificity phosphatase (DUSP)-1; interleukin-1β (IL-1β); intervertebral disc (IVD); mitogen-activated protein kinase (MAPK); nerve growth factor (NGF); prostaglandin E1 (PGE1); prostaglandin E2 (PGE2).

Figure 1

Involvement of various mitogen-activated protein kinase (MAPK) pathways in interleukin-1β (IL-1β)-induced nerve growth factor (NGF) expression in human intervertebral disc (IVD) cells. Confluent human IVD cells were serum starved, preincubated with the indicated concentrations of a p38 inhibitor (SB203580) (A), a MEK1/2 (upstream of extracellular signal-regulated kinase [ERK]) inhibitor (U0126) (B), or a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) (C) for 30 min, and then stimulated with IL-1β (10 ng/mL) for 24 h. The relative expression levels of NGF were quantified by real-time PCR. Results are expressed as the mean ± SD (n = 4 individuals) after normalization to GAPDH, and expressed as a relative value to that of IL-1β alone. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. IL-1β alone.

Figure 2

Effects of prostaglandin E₂ (PGE₂) and PGE₁ on IL-1β-induced phosphorylation of MAPKs in human IVD cells. Confluent human IVD cells were serum starved, preincubated with PGE₂ (1 μM) or PGE₁ (1 μM) for 3 h, and then stimulated with IL-1β (10 ng/mL) for 15, 30, and 45 min. The phosphorylation of MAPKs was assessed by Western blotting. Results were reproducible among four individuals, and representative blots are shown.

Figure 3

Effects of PGE₂ and PGE₁ on DUSP-1 expression in human IVD cells. (A) Confluent human IVD cells were serum starved, and then left untreated (circles), treated with PGE₂ (1 μM) (squares), or treated with PGE₁ (triangles) for 0.5, 1, and 3 h. Relative amounts of DUSP-1 were quantified by real-time PCR. Results are shown as the mean ± SD (n = 4 individuals) of DUSP-1 after normalization to GAPDH, and expressed as a relative value to that of untreated cells at each time point. * p < 0.05, ** p < 0.01, and *** p < 0.001 between untreated and PGE₂- or PGE₁-treated cells at each time point. (B) Confluent human IVD cells were serum starved, left untreated (black column), preincubated with PGE₂ (light gray column), or preincubated with PGE₁ (dark gray column) for 3 h (1, 10, or 100 µM), and then stimulated with IL-1β (10 ng/mL) for a further 6 h. Relative amounts of DUSP-1 were quantified by real-time PCR. Results are expressed as the mean ± SD (n = 4 individuals) of DUSP-1 after normalization to GAPDH, and expressed as a relative value to that of untreated cells at each time point. ** p < 0.01 and *** p < 0.001 between untreated and PGE₂ or PGE₁ treated cells at each time point.

Figure 4

DUSP-1 knockdown by the transfection of small interfering RNA (siRNA) into human IVD cells. (A) Transfection efficiency of siRNA in human IVD cells. Semiconfluent human IVD cells were transfected with FITC-labeled siRNA oligonucleotides and cultured for 24 h. Cell images were captured by a digital fluorescence microscope. Transfection efficiency was more than 80% in three independent experiments (n = 3 individuals). Scale bar: 100 μm (B) DUSP-1 siRNA was transfected into semiconfluent human IVD cells to attenuate DUSP-1 expression. The relative amount of DUSP-1 expression was quantified by real-time PCR. Results are expressed as the mean ± SD (n = 3 individuals) of DUSP-1 after normalization to that of GAPDH, and expressed as a relative value to that of untransfected cells. DUSP-1 expression was significantly suppressed by more than 70%. *** p < 0.001 vs. untransfected cells.

Figure 5

Effects of DUSP-1 knockdown on IL-1β-induced MAPK phosphorylation in human IVD cells. DUSP-1 knockdown and untransfected cells were serum starved and then stimulated with IL-1β (10 ng/mL) for 15, 30, 45, and 60 min. The phosphorylation of MAPKs was analyzed by Western blotting. IL-1β-induced MAPK phosphorylation was increased and prolonged in DUSP-1 knockdown cells compared with untransfected cells. Results were reproducible among three individuals and representative blots are shown.

Figure 6

Effects of DUSP-1 knockdown on IL-1β-induced expression of NGF in human IVD cells. Untransfected and DUSP-1 knockdown human IVD cells were serum starved and stimulated with IL-1β (10 ng/mL) for 24 h. Expression levels of NGF were quantified by real-time PCR. Results are expressed as the mean ± SD (n = 4 individuals) after normalization to GAPDH, and are shown as relative values to that of untransfected cells stimulated with IL-1β. IL-1β-induced expression of NGF was significantly enhanced in DUSP-1 knockdown cells. ** p < 0.01 vs. untransfected cells stimulated with IL-1β.