The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Abstract

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB.

Figure 1

Interleukin (IL)-1β stimulation of intestinal epithelial cells results in activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38 (a), and p38 mitogen-activated protein kinase (p38 MAPK) signalling can be inhibited using the specific pharmacological inhibitor SB 203580 (10 µm). (a) Caco-2 cells were stimulated with IL-1β (2 ng/ml) for the indicated times, before being harvested. Western immunoblotting was carried out as described in the Materials and methods. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-Tyr, tyrosine phosphorylation. (b) Caco-2 cells were preincubated with SB 203580 for 2 hr before being stimulated with IL-1β (2 ng/ml) for 30 min and harvested. Ten microlitres of cell lysate was then used in a kinase assay, with the heat shock protein 27 (hsp 27) as substrate. Samples were boiled in 5× sample buffer and resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). After transfer to nitrocellulose membranes, the substrate bands were visualized using autoradiography. The panel on the left is an autoradiogram and the one on the right is a Ponceau stain of the same membrane, showing co-localization of the hsp 27 band. The results shown are representative of at least three independent experiments. (c) Cells were pretreated with SB 203580 or PD 98059 (25 µm) for 2 hr, stimulation with IL-1β (2 ng/ml) for 36 hr. Supernatants were collected and analysed for the presence of IL-8 (c) or monocyte chemotactic protein 1 (MCP-1) (d) by enzyme-linked immunosorbent assay (ELISA). (Lane 1, control; lane 2, IL-1β; lane 3, IL-1β with SB 203580; lane 4, IL-1β with PD 98059.) The results shown are representative of at least three independent experiments. The results are expressed as mean ± standard error of the mean (SEM) (*P <0·01).

Figure 2

(a) Interleukin (IL)-1β-induced degradation of IκB is independent of p38 mitogen-activated protein kinase (p38 MAPK) regulation in Caco-2 cells. Cells were starved overnight before preincubation with the appropriate inhibitor for 2 hr. Cells were stimulated with IL-1β (2 ng/ml) for 30 min, then harvested. Cells were lysed, resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotted with the appropriate antibody. The results are representative of at least four independent experiments. DRB, a specific inhibitor of protein kinase CK2; SB, SB 203580. (b) IL-1β-induced translocation and DNA binding of nuclear factor (NF)-κB is independent of p38 MAPK regulation in Caco-2 cells. Cells were preincubated overnight for 2 hr before being stimulated with IL-1β (2 ng/ml) for 30 min. Nuclear extracts were prepared and assayed for κB binding by using electromobility shift assays (EMSAs). (c) Specificity of κB binding was determined by antibody supershifting or competition with cold probe. Nuclear extracts were preincubated with an anti-Rel A antibody or 100-fold excess of cold unlabelled probe, before adding radiolabelled probe. The results are representative of at least three independent experiments.

Figure 3

Interleukin (IL)-1β-induced trans-activation of nuclear factor (NF)-κB is independent of p38 mitogen-activated protein kinase (p38 MAPK) regulation in Caco-2 cells. Cells were co-transfected with the NF-κB reporter 4x κB-luc and LacZ for 12 hr before the medium was replaced with fresh serum-containing medium for 24 hr. (a) Cells were preincubated with the appropriate inhibitor for 2 hr, before being stimulated with IL-1β (2 ng/ml) for 6 hr. Cells were harvested and the luciferase activity was determined. Results were normalized for transfection efficiency using LacZ activity. Results are representative of at least five independent experiments. (b) Caco-2 cells were transfected with the indicated plasmids using Effectene and then stimulated with IL-1β (p38wt = p38 wild-type, p38kd = p38 kinase-dead). (c) DNA binding was carried out as described in the Materials and methods. (d) Expression of both the native and transfected p38 MAPK is shown. (e) Activator protein-1 DNA binding in the presence or absence of IL-1β and SB 203580 is shown.

Figure 4

The p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, SB 203580, inhibits interleukin (IL)-1β-induced IL-8 and monocyte chemotactic protein 1 (MCP-1) mRNA expression. (a) Cells were preincubated with the appropriate inhibitor for 2 hr, before being stimulated with IL-1β for 3 hr. Total RNA was isolated, and semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR) was performed using 1 µg of RNA. PCR products were run on a 2% agarose gel containing ethidium bromide. (b) Densitometry was performed using Bio-Rad Quantity One Software. The results depicted are representative of three independent experiments. (c) Reduction in IL-8 message is independent of message stability. Cells were stimulated with IL-1β for 3 hr and then cultures were adjusted so that they contained 10 µg/ml of actinomycin D (Act D), either alone or together with 10 µm SB 203580 (SB). The samples were then harvested at the indicated time-points, total RNA extracted and semiquantitative PCR performed as described above. The data shown are representative of three independent experiments.

Figure5

Interleukin (IL)-1β-induced trans-activation of the IL-8 promoter is dependent on p38 mitogen-activated protein kinase (p38 MAPK) regulation in Caco-2 cells. (a) Cells were co-transfected with the IL-8 promoter and LacZ for 12 hr before the medium was replaced with fresh serum-containing medium for 24 hr. Cells were preincubated with the appropriate inhibitor for 2 hr, before being stimulated with IL-1β (2 ng/ml) for 6 hr. (b) Alternatively, the IL-8 promoter was co-transfected with the p38 MAPK constructs (p38WT = p38 wild-type, p38KD = p38 kinase-dead) and then stimulated with IL-1β. Cells were harvested and the luciferase activity determined. Results were normalized for transfection efficiency using LacZ activity. Results are representative of at least three independent experiments.