Gastrin activates nuclear factor kappaB (NFkappaB) through a protein kinase C dependent pathway involving NFkappaB inducing kinase, inhibitor kappaB (IkappaB) kinase, and tumour necrosis factor receptor associated factor 6 (TRAF6) in MKN-28 cells transfected with gastrin receptor

Abstract

Background: We previously reported that gastrin induces expression of CXC chemokines through activation of nuclear factor kappaB (NFkappaB) in gastric epithelial cells that express gastrin receptor.

Aims: To clarify gastrin receptor mediated signals leading to activation of NFkappaB.

Methods: MKGR26 cells were created by transfecting gastrin receptor cDNA into MKN-28 cells. Degradation of inhibitor kappaB (IkappaB) and phosphorylation of protein kinase C (PKC)-delta were both detected by western blot analysis. NFkappaB activation was determined by luciferase assay and electrophoretic mobility shift analysis.

Results: Gastrin induced degradation of IkappaB-alpha and activation of NFkappaB, which was abolished by the selective gastrin receptor antagonist L-740,093 and the general PKC inhibitor GF109203X. Gastrin induced phosphorylation of PKC-delta, and its inhibitor rottlerin partially suppressed NFkappaB activation. However, the mitogen activated protein kinase (MAPK) kinase inhibitor PD98059, p38 MAPK inhibitor SB203580, and tyrphostin AG1478 had no effect on NFkappaB activation. Introduction of the dominant negative mutant of IkappaB kinase, of NFkappaB inducing kinase, and of tumour necrosis factor receptor associated factor 6 (TRAF6), but not that of TRAF2, inhibited gastrin induced activation of NFkappaB.

Conclusions: Gastrin activates NFkappaB via a PKC dependent pathway which involves IkappaB kinase, NFkappaB inducing kinase, and TRAF6.

Figure 1

Effects of tyrphostin AG1478, the protein kinase C inhibitor GF109203X, the mitogen activated protein kinase kinase inhibitor PD98059, the p38 mitogen activated protein kinase inhibitor SB203580, and the gastrin receptor antagonist L-740,093 on gastrin induced activation of nuclear factor κB (NFκB), assessed by electrophoretic mobility shift assay (EMSA). (A) After incubation with the inhibitors for one hour, MKGR26 cells were treated with gastrin for 0.5 hours. Nuclear cell extracts of MKGR26 cells were assayed for NFκB DNA binding activity using the labelled oligonucleotide probes. (B) MKGR26 cells were transfected with pNFκB-LUC and pRL-SV40 as an internal standard. After preincubation with the inhibitors for one hour they were treated with gastrin for eight hours and the luciferase assay was performed. The results are expressed as fold induction compared with control cells treated with vehicle alone. Values are means (SEM) from six independent experiments (*p<0.05 v bar 1; †p<0.05 v bar 2).

Figure 2

Effects of protein kinase C inhibitors on gastrin induced activation of nuclear factor κB (NFκB). (A) After incubation with rottlerin or HBDDE for one hour, MKGR26 cells were treated with gastrin for 0.5 hours. Nuclear cell extracts of MKGR26 cells were assayed for NFκB DNA binding activity using the labelled oligonucleotide probe. (B) MKGR26 cells were transfected with pNFκB-LUC and pRL-SV40 as an internal standard. After preincubation with rottlerin or Go6976 for one hour, they were treated with gastrin for eight hours and the luciferase assay was performed. The results are expressed as fold induction compared with control cells treated with vehicle alone. Values are means (SEM) from six independent experiments (*p<0.05 v bar 1; †p<0.05 v bar 2). (C) Guinea pig parietal cells were isolated and incubated with or without GF109203X or rottlerin for two hours. Then, cells were treated with gastrin for 0.5 hours and the nuclear cell extracts were assayed for NFκB DNA binding activity using the labelled oligonucleotide probe.

Figure 3

Effects of protein kinase C (PKC) isozyme mutants on gastrin induced nuclear factor κB (NFκB) activation. MKGR26 cells were cotransfected with 0.5 μg of pNFκB-LUC and a construct encoding the dominant negative (dn) mutant of PKC-δ or PKC-θ. After 24 hours, cells were treated with gastrin for eight hours and the luciferase assay was performed. The results are expressed as fold induction compared with control cells treated with vehicle alone. Values are means (SEM) from six independent experiments (*p<0.05 v bar 1; †p<0.05 v bar 2).

Figure 4

Gastrin induced phosphorylation of protein kinase C (PKC)-δ. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, 30, or 60 minutes and cell lysates were then obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to membranes. Membranes were reacted with rabbit antihuman-phospho-PKC-δ (Thr505) polyclonal antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system.

Figure 5

Gastrin induced degradation of inhibitor κB (IκB)-α. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, or 30 minutes and cell lysates were obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidine difluoride membranes. Membranes were reacted with rabbit antihuman-IκB-α antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system. Quantitated signals were determined by densitometry.

Figure 6

Effects of inhibition of inhibitor κB kinase (IKK) or NFκB inducing kinase (NIK) on gastrin induced nuclear factor κB (NFκB) activation. (A) MKGR26 cells were transfected with 1 μg of pRK5 or a construct encoding the dominant negative (dn) mutant of IKKα, IKKβ, or NIK. After 24 hours, MKGR26 cells were treated with gastrin for 0.5 hours. Nuclear cell extracts of MKGR26 cells were assayed for NFκB DNA binding activity using the labelled oligonucleotide probe. (B) MKGR26 cells were transfected with pNFκB-LUC and pRK5 or a construct encoding the dominant negative (dn) mutant of IKKα, IKKβ, or NIK. pRL-SV40 was cotransfected as an internal standard. After 24 hours they were treated with gastrin for eight hours and the luciferase assay was performed. Results are expressed as fold induction compared with control cells treated with vehicle alone. Values are means (SEM) from six independent (*p<0.05 v bar 1; †p<0.05 v bar 2).

Figure 7

Effects of inhibition of tumour necrosis factor receptor associated factor (TRAF) 2 or TRAF6 on gastrin induced nuclear factor κB (NFκB) activation. (A) MKGR26 cells were transfected with 1 μg of pRK5 or a construct encoding the dominant negative (dn) mutant of TRAF2 or TRAF6. After 24 hours cells were treated with gastrin for 0.5 hours. Nuclear cell extracts of MKGR26 cells were assayed for NFκB DNA binding activity using the labelled oligonucleotide probe. (B) MKGR26 cells were transfected with pNFκB-LUC and pRK5 or a construct encoding the dominant negative (dn) mutant of TRAF2 or TRAF6. pRL-SV40 was cotransfected as an internal standard. After 24 hours they were treated with gastrin for eight hours and the luciferase assay was performed. Results are expressed as fold induction compared with control cells treated with vehicle alone. Values are means (SEM) from six independent (*p<0.05 v bar 1; †p<0.05 v bar 2).

Figure 8

Effect of the dominant negative transforming growth factor β activated kinase 1 (dnTAK1) on gastrin induced nuclear factor κB (NFκB) activation. MKGR26 cells were transfected with pNFκB-LUC and control vector or a construct encoding the dominant negative mutant of TAK1. pRL-SV40 was cotransfected as an internal standard. After 24 hours they were treated with gastrin for eight hours and the luciferase assay was performed. The results are expressed as fold induction compared with control cells treated with vehicle alone. Values are means (SEM) from six independent (*p<0.05 v bar 1; †p<0.05 v bar 2).

Figure 9

Effects of inhibition of inhibitor κB kinase (IKK), NFκB inducing kinase (NIK), or tumour necrosis factor receptor associated factor (TRAF) 6 on nuclear factor κB (NFκB) activation induced by forced expression of protein kinase C (PKC)-δ. MKGR26 cells were cotransfected with 0.5 μg of pNFκB-LUC, pTB701HA/PKC-δ, a construct encoding wild-type PKC-δ, and the dominant negative (dn) mutant of IKKα, IKKβ, NIK, or TRAF6. After 24 hours, the luciferase assay was performed. Values are means (SEM) from six independent (*p<0.05 v bar 1; †p<0.05 v bar 2).