TNF-alpha, inefficient by itself, potentiates IL-1beta-induced PGHS-2 expression in human pulmonary microvascular endothelial cells: requirement of NF-kappaB and p38 MAPK pathways

Abstract

1: Prostaglandin H synthase-2 (PGHS-2), is an inducible enzyme involved in various inflammatory responses. We established here that interleukin-1beta (IL-1beta) but not tumour necrosis factor-alpha (TNF-alpha) increased its expression in human pulmonary microvascular endothelial cells (HPMEC). However, associated with IL-1beta, TNF-alpha greatly potentiated this enzyme induction. 2: Although unable to induce PGHS-2 expression by itself, TNF-alpha promoted a similar transcription nuclear factor-kappaB (NF-kappaB) activation to IL-1beta. This effect was more pronounced when cells were co-exposed to both cytokines. HPMEC pre-treatment with MG-132, a proteasome inhibitor, prevented NF-kappaB activation as well as more distal signalling response, indicating that NF-kappaB activation is required but not sufficient for PGHS-2 expression. 3: Both IL-1beta and TNF-alpha failed to activate c-Jun NH2-terminal kinase (JNK). In addition, PD98059, a p42/44 mitogen-activated protein kinase (MAPK) phosphorylation inhibitor, did not decrease PGHS-2 expression. However, SB 203580, a p38 MAPK inhibitor, suppressed PGHS-2 induction by IL-1beta alone or combined with TNF-alpha, demonstrating that p38 MAPK but not p42/44 MAPK or JNK cascades are required for PGHS-2 up-regulation. 4: Finally, TNF-alpha, unlike IL-1beta, was unable to promote p38 MAPK phosphorylation, indicating that the failure of TNF-alpha to induce PGHS-2 expression is linked, at least in part, to its inability to activate p38 MAPK signalling pathway. Altogether, these data enhanced our understanding of PGHS-2 regulation in HPMEC and emphasize the heterogeneity of cellular responses to proinflammatory cytokines.

Figure 1

Induction of PGHS-2 expression in HPMEC by IL-1β but not TNF-α. Human pulmonary microvascular endothelial cells (HPMEC) were treated with IL-1β and processed for the determination of PGHS-2 expression by immunoblotting according to Methods. Each lane was loaded with 10 μg of protein for both PGHS-2 and standard control β-actin assessments. Adherent HPMEC were incubated for 6 h in the absence and the presence of various concentrations of IL-1β (0–5 ng ml⁻¹ (A)) or TNF-α (0–100 ng ml⁻¹ (B)). Results are representative of three separate experiments.

Figure 2

Potentiation by TNF-α of IL-1β-induced PGHS-2 expression. Cultured HPMEC were cotreated with IL-1β and TNF-α for 6 h and then PGHS-2 and β-actin proteins were analysed as indicated in the legend of Figure 1. (A) Concentration response of TNF-α (0–100 ng ml⁻¹) in the presence of IL-1β (0.3 ng ml⁻¹). (B) Concentration response of IL-1β (0–0.5 ng ml⁻¹) in the presence of TNF-α (30 ng ml⁻¹). Results are representative of three separate experiments.

Figure 3

Potentiation by TNF-α of IL-1β-induced 6 keto-PGF1α generation Adherent HPMEC were co-exposed to TNF-α (30 ng ml⁻¹) or IL-1β (0.3 ng ml⁻¹) alone or in combination for 6 h. Then, cells were washed and exposed to arachidonic acid (30 μM) for 30 min. Amounts of 6 keto-PGF1α released in the incubation media were evaluated as indicated in Methods. Results are expressed as the mean±s.e.mean of three separate experiments (***P<0.001).

Figure 4

Induction by IL-1β of PGHS-2 expression and its potentiation by TNF-α: inhibition by actinomycin D. (A) Concentration-dependent potentiation by TNF-α of IL-1β-induced PGHS-2 mRNA expression. Adherent cells were treated as indicated on the figure. Then, total cellular RNA in each sample was extracted. After reverse transcription reaction, cDNA of PGHS-2 and that of β-actin were amplified and hybridized with specific [γ-³²P]-labelled probes. (B) Inhibition by actinomycin D of PGHS-2 expression. HPMEC were pretreated with actinomycin D (1 μM) for 1 h and then exposed to IL-1β (0.3 ng ml⁻¹) alone or in combination with TNF-α (30 ng ml⁻¹) for 6 h. The samples were analysed by immunoblotting. Results are representative of two to three separate experiments.

Figure 5

Inhibition by MG-132 of NF-κB activation and PGHS-2 expression. Adherent cells were treated as indicated on the figure and processed for the PGHS-2 protein determination (A) or for NF-κB-DNA binding determination (B) according to Methods. (C) Supershift analysis. Nuclear extracts from MG-132 and TNF-α treated cells as indicated in B were incubated for (20 min) with antibodies against p50, p65 or c-Rel of NF-κB subunits and then submitted to EMSA analysis. Results are representative of two to three separate experiments.

Figure 6

Activation of AP-1 and STAT3 by IL-1β and TNF-α. Nuclear proteins were isolated from IL-1β- (0.3 ng ml⁻¹) and TNF-α- (30 ng ml⁻¹) treated HPMEC (2 h and 30 min for AP-1 and STAT3, respectively) and then analysed by EMSA, using a radiolabelled DNA probe containing AP-1 or STAT3 consensus sequences. Results are representative of two separate experiments.

Figure 7

Activation of p42/44 MAPK by IL-1β and TNF-α and its inability to regulate PGHS-2 expression. Adherent HPMEC were pretreated with or without PD98059, a specific inhibitor of p42/44 MAPK phosphorylation and then stimulated with IL-1β (0.3 ng ml⁻¹) and TNF-α (30 ng ml⁻¹). (A and B) IL-1β and TNF-α-induced p42/44 MAPK phosphorylation. HPMEC were exposed to IL-1β (0.3 ng ml⁻¹, A) or TNF-α (30 ng ml⁻¹, B) for the times indicated and harvested for immunoblotting using p42/44 MAPK antibodies. (C) Effect of PD98059 on PGHS-2 expression. Cells were pre-incubated with or without PD98059 (10 μM) for 30 min and then exposed to IL-1β (0.3 ng ml⁻¹) alone or in association with TNF-α (30 ng ml⁻¹) for 6 h, and processed for the PGHS-2 protein determination. (D) Inhibition by PD98059 of IL-1β- and TNF-α-induced p42/44 MAPK phosphorylation. Cells treatment with or without PD98059 (10 μM) before IL-1β (0.3 ng ml⁻¹, 5 min) or TNF-α (30 ng ml⁻¹, 5 min) addition, were processed for immunoblotting using p42/44 MAPK antibodies. Results are representative of two separate experiments.

Figure 8

Activation of p38 MAPK by IL-1β but not by TNF-α and its involvement in PGHS-2 up-regulation. HPMEC were preincubated for 1 h with or without SB 203580 (10 μM) and then exposed to IL-1β (0.3 ng ml⁻¹) alone or combined with TNF-α (30 ng ml⁻¹). PGHS-2 and p38 MAPK proteins were determined by immunoblotting. (A) Inhibition by SB 203580 of PGHS-2 expression. HPMEC were pre-incubated with or without SB 203580 (10 μM) for 1 h and then exposed to IL-1β (0.3 ng ml⁻¹) alone or combined with TNF-α (30 ng ml⁻¹) for 6 h and PGHS-2 expression was analysed by Western blot. (B) Ineffectiveness of TNF-α to induce p38 MAPK activation. HPMEC were treated as described in A, and samples were analysed by immunoblotting using p38 MAPK antibodies. Results are representative of two separate experiments.