The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway

Abstract

In the present study we investigated a possible role for the p38 mitogen-activated protein (MAP) kinase pathway in mediating nuclear factor-kappa B (NF-kappaB) transcriptional activity in the erythroleukaemic cell line TF-1. TF-1 cells stimulated with the phosphatase inhibitor okadaic acid (OA) demonstrated enhanced NF-kappaB and GAL4p65-regulated transcriptional activity which was associated with elevated p38 phosphorylation. However, pretreatment with the p38 MAPK specific inhibitor SB203580 (1 microM) or overexpression of kinase-deficient mutants of MKK3 or MKK6 did not affect OA-enhanced NF-kappaB transcriptional potency, as determined in transient transfection assays. In fact, 5 and 10 microM SB203580 enhanced rather than inhibited NF-kappaB-mediated promoter activity by 2 fold, which was independent of phosphorylation of the p65 subunit. The SB203580-mediated increase in NF-kappaB transcriptional activity was associated with enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK), but not p38 kinase. Overexpression of kinase-deficient mutants belonging to the ERK1/2, JNK, and p38 pathways showed that only dominant-negative Raf-1 abrogated SB203580-enhanced NF-kappaB activity. This would implicate the involvement of the ERK1/2 pathway in the enhancing effects of SB203580 on NF-kappaB-mediated gene transcription. This study demonstrates that the p38 MAP kinase pathway is not involved in the OA-induced activation of NF-kappaB. SB203580 at higher concentrations activates the ERK pathway, which subsequently enhances NF-kappaB transcriptional activity.

Figure 1

OA enhances NF-κB DNA binding. Electrophoretic mobility shift assay for the transcription factor NF-κB was performed after treatment of TF-1 cells with medium, OA (30 ng ml⁻¹, 6 h), or SB203580 (30 min pretreatment) at 1, 5 and 10 μM plus OA (6 h). Band shift assay was performed as described in ‘Methods' utilizing ³²P-labelled oligonucleotide specific for NF-κB. Excess of free probe is shown.

Figure 2

OA enhances NF-κB transcriptional activity. (A) Schematic representation of the two IL-6 promoter constructs and the pGAL4p65, pGAL4dbd, and pGAL4tkluc plasmids applied in the transfections of TF-1 cell line. Fragments of 122 (pIL6(-122)luc) and 60 (pIL6(-60)luc) basepairs, respectively, of the human IL-6 promoter are fused to the luciferase gene. Binding site for the transcription factors NF-κB is denoted above the largest construct. The Gal4-transactivator fusion proteins, pGal4p65 and pGal4dbd, are exclusively nuclear and are regulated independently of IκB. The reporter gene is under the control of five Gal4-binding sites. (B) Schematic representation of NF-κB- and p65-mediated promoter activity in the transiently transfected TF-1 cells. TF-1 cells were cultured as described in ‘Methods' and subsequently transiently transfected by means of electroporation with the respective plasmids. Six hours after transfection cells were stimulated for 24 h with medium or OA (30 ng ml⁻¹). Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. Basal promoter activity for each construct when treated with medium alone is set at 1. Mean fold induction and standard error of the mean represent six or more identical experiments. ^*P<0.05.

Figure 3

OA enhances phosphorylation of p38 MAP kinase. TF-1 cells were cultured for 16 h in RPMI 1640 containing 0.1% FBS and subsequently stimulated with medium, OA (30 ng ml⁻¹) for 90 min, or SB203580 (30 min pretreatment) at the indicated concentrations. Cell extracts were prepared as described in detail in ‘Methods'. Phosphorylated p38 is depicted in the upper panel. Total p38 protein is shown in the lower panel and represents equal loading. Immunoblotting with anti-phospho-p38 and anti-p38 antibodies was performed by standard procedures and detection was performed using ECL immunodetection kit.

Figure 4

SB203580 at 1 μM and kinase deficient MKK3 and MKK6 do not affect NF-κB transactivation potential. (A) TF-1 cells were transiently transfected as described in ‘Methods' with pIL6luc(-122) or pGAL4p65. Six hours after electroporation cells were stimulated for 24 h with medium, SB203580 (1 μM), OA (30 ng ml⁻¹), or SB203580 (1 μM, 30 min pretreatment) plus OA. Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. Promoter activity after treatment with medium is set at 1. Mean fold induction and standard error of the mean represent six identical experiments. ^*P<0.05. (B) TF-1 cells were transiently transfected as described in ‘Methods' with 15 μg pIL6luc(-122) together with 15 μg (1) pcDNA3 empty vector, (2) pRSV-MKK3(Ala), or (3) pcDNA3-MKK6(K82A) and 2 μg of a CMV-CAT construct. Six hours after electroporation cells were stimulated for 24 h with medium or OA (30 ng ml⁻¹). Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. The IL-6 promoter activity is represented in arbitrary luciferase units corrected for transfection efficiency. Mean and standard error of the mean represent at least three identical experiments.

Figure 5

SB203580 at 1 μM inhibits NF-IL6 DNA binding activity. Electrophoretic mobility shift assay for the transcription factor NF-IL6 was performed after treatment of TF-1 cells with medium, OA (30 ng ml⁻¹, 6 h), and SB203580 at 1 μM (30 min pretreatment) plus OA. Band shift assay was performed as described in ‘Methods' utilizing ³²P-labelled oligonucleotide specific for NF-IL6.

Figure 6

SB203580 at 5 and 10 μM enhances NF-κB activity, but not p65 activity. (A) TF-1 cells were transiently transfected as described in ‘Methods' with pIL6luc(-122). Six hours after electroporation cells were stimulated for 24 h with medium, SB203580 (10 μM), OA (30 ng ml{sF/R}⁻¹{XF/R}), SB203580 5 μM (30 min pretreatment) plus OA, or SB203580 10 μM plus OA. Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. Promoter activity after treatment with medium is set at 1. Mean fold induction and standard error of the mean represent six identical experiments. ^*P<0.05 OA-treated cells versus medium-treated cells. ^**P<0.05 SB203580 plus OA-treated cells versus OA-treated cells. (B) Same as described under (A), but with p(TRE)5xCAT instead of pIL6luc(-122). The amount of produced CAT protein after medium treatment is set at 1. Mean fold induction and standard error of the mean represent three identical experiments. ^*P<0.05. (C) Same as described under (A), but with 5 μg pGAL4p65 together with 5 μg pGAL4tkluc. Promoter activity after treatment with medium is set at 1. Mean fold induction and standard error of the mean represent six identical experiments. ^*P<0.05.

Figure 7

SB203580 at 10 μM enhances phosphorylation of ERK1/2 and JNK1/2. TF-1 cells were cultured for 16 h in RPMI 1640 containing 0.1% FBS and subsequently stimulated with medium, OA (30 ng ml⁻¹) for 90 min, or SB203580 (30 min pretreatment) in the indicated concentrations. Cell extracts were prepared as described in detail in ‘Methods'. Phosphorylated ERK1 and ERK2 (A) and phosphorylated JNK1 and JNK2 (B) are shown in the upper panels. Total ERK and JNK protein are shown in the lower panels and represent equal loading. Immunoblotting with anti-phospho-ERK/JNK and anti-ERK/JNK antibodies was performed by standard procedures and detection was performed using ECL immunodetection kit.

Figure 8

Kinase inactive Raf-1 suppresses SB203580-enhanced NF-κB activity. (A) TF-1 cells were transiently transfected as described in ‘Methods' with 15 μg pIL6luc(-122) together with (1) 15 μg pcDNA3 empty vector, (2) pRSV-NΔRaf1, (3) pcDNA3-MKK4(Ala), (4) pcDNA3-Flag-JNK1, (5) pRSV-MKK3(Ala), or (6) pcDNA3-MKK6(K82A). Six hours after electroporation cells were stimulated for 24 h with medium, OA (30 ng ml⁻¹) or SB203580 at 5 μM (30 min pretreatment) plus OA (30 ng ml⁻¹). Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. The IL-6 promoter activity for pcDNA3 construct when treated with SB203580 at 5 μM plus OA is set at 100%. Mean and standard error of the mean represent three identical experiments. ^*P<0.05. (B) TF-1 cells were transiently transfected as described in ‘Methods' with 15 μg pIL6luc(-122) together with (1) 15 μg pcDNA3 empty vector, (2) 1 μg pRSV-NΔRaf1 and 14 μg pcDNA3 empty vector, (3) 5 μg pRSV-NΔRaf1 and 10 μg pcDNA3 empty vector or (4) 15 μg pRSV-NΔRaf1. Six hours after electroporation cells were stimulated for 24 h with medium or SB203580 at 5 μM (30 min pretreatment) plus OA (30 ng ml⁻¹). Twenty-four hours after stimulation cells were lysed and analysed for the amount of produced luciferase protein. Values depict the luciferase activity in arbitrary units corrected for transfection efficiency. Mean and standard error of the mean represent three identical experiments. ^*P<0.05.