TNF-α and LPA promote synergistic expression of COX-2 in human colonic myofibroblasts: role of LPA-mediated transactivation of upregulated EGFR

Abstract

Background: Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α.

Methods: 18Co cells, a model of human colonic myofibroblasts, were grown to confluence on 35 × 10 mm cell culture dishes and were used from passages 10-14. 18Co cells were treated with TNF-α (8.3 ng/ml) and LPA (10 μM). EGFR and COX-2 protein expression, Y1068 phosphorylation, and p42/44 MAPK phosphorylation were assessed by Western Blot, in the presence and absence of various inhibitors.

Results: Exposure of 18Co cells to either TNF-α or LPA alone had no effect on EGFR autophosphorylation at Y1068. However, chronic exposure to TNF-α led to upregulation of EGFR in association with sustained LPA-mediated EGFR phosphorylation at Y1068. TNF-α and LPA also led to sustained p42/44 MAPK phosphorylation and synergistic COX-2 expression, effects that were partially inhibited by the EGFR tyrosine kinase inhibitor AG1478. p42/44 MAPK phosphorylation and COX-2 expression were inhibited to the same degree by the MMP inhibitors GM6001 and BB-94, suggesting that LPA-mediated EGFR transactivation involved MMP-mediated release of EGFR ligands from the cell surface. The Src inhibitor SU6556 inhibited TNF-α/LPA-mediated EGFR phosphorylation at Y1068, p42/44 MAPK phosphorylation, and COX-2 expression in a dose-dependent fashion, suggesting an upstream role of Src in the transactivation of EGFR.

Conclusion: Synergistic COX-2 expression induced by TNF-α and LPA involves Src/MMP-mediated transactivation of EGFR and downstream p42/44 MAPK activation in human colonic myofibroblasts. Enhanced EGFR expression induced by TNF-α promotes GPCR-mediated EGFR transactivation in colonic myofibroblasts, providing an important mechanism for stromal COX-2 over-expression that may predispose to the development of colitis-associated cancer.

Figure 1

TNF-α Potentiates LPA-mediated EGFR Phosphorylation at Y1068. Panel A: Confluent 18Co cells were exposed to 10 μM LPA for various times (broken lines), with or without pre-incubation with 10 ng/ml TNF-α for 18 h (indicated by solid line). Western blot analysis was used with antibodies that detect EGFR and EGFR phosphorylation at Y1068. Densitometry analysis shows the mean ± S.E. (n ≥ 3), expressed as percentage of the maximal level of EGFR Y1068 phosphorylation. In all experiments, equal protein loading was verified using α-SMA antibody. Panel B: 18Co cells were incubated with EGF (5 ng/ml) for 5 or 60 min (broken lines), with or without pre-incubation with 10 ng/ml TNF-α for 18 h (indicated by solid line). Western blot analysis used an antibody detecting EGFR phosphorylation at Y1068. The results are representative of three separate experiments. Panel C: 18Co cells were exposed to 10 μM LPA for 8 h (broken lines), without or with pre-incubation with 10 ng/ml TNF-α for 18 h (indicated by solid line). Western blot analysis detected COX-2 and p42/44 MAPK phosphorylation. The results shown are the mean ± S.E. (n ≥ 3), expressed as a percentage of the maximal level of p42/44 MAPK phosphorylation, displayed graphically on the right.

Figure 2

Enhanced p42/44 MAPK phosphorylation and COX-2 expression induced by TNF-α and LPA are partially inhibited by AG1478 and MMP inhibitors. Panel A: 18Co cells were pre-treated with 1 μM AG1478 for 1 h and then 5 ng/ml EGF or 10 μM LPA with or without pre-incubation with 10 ng/ml TNF-α for 18 h. Western blot analysis detected COX-2 and p42/44 MAPK phosphorylation. The results shown are the mean ± S.E. (n ≥ 3), expressed as a percentage of the maximal level of COX-2 expression and p42/44 MAPK phosphorylation, displayed graphically on the right. For all experiments, * denotes statistical significance, p < 0.05. Panel B: 18Co cells were incubated with or without 10 ng/ml TNF-α for 18 h, followed by 10 μM LPA for 4 h with or without pre-treatment with 1 μM AG1478, 10 μM GM6001, or 5 μM BB-94 for 1 h. The results shown are the mean ± S.E. (n ≥ 3), expressed as a percentage of the maximal level of COX-2 expression and p42/44 MAPK phosphorylation, displayed graphically on the right. Panel C: 18Co cells were incubated with or without 10 ng/ml TNF-α for 18 h, followed by exposure to 5 ng/ml EGF for 4 h with or without pre-treatment with 1 μM AG1478, 10 μM GM6001, or 5 μM BB-94 for 1 h. The results are representative of three separate experiments.

Figure 3

The Src inhibitor SU6556 partially inhibits EGFR phosphorylation at Y1068, p42/44 MAPK activation, and COX-2 Expression. Panel A: 18Co cells were incubated with or without 10 ng/ml TNF-α for 18 h, then were pre-treated with SU6556 for 1 h at the indicated concentrations, followed by 10 μM LPA for 4 h. The results are representative of three separate experiments. Panel B: 18Co cells were incubated for 18 h with or without 10 ng/ml TNF-α, then 250 nM SU6556 for 1 h followed by LPA for 4 h. Densitometry analysis is presented as mean ± S.E., (n ≥ 3), expressed as a percentage of the maximal level of COX-2 and p42/44 MAPK phosphorylation, depicted graphically on the right.

Figure 4

Synergistic COX-2 expression induced by TNF-α and LPA involves EGFR Transactivation in Primary Human Colonic Myofibroblasts. Panel A: Cell lysates of primary colonic myofibroblasts isolated from human colon tissue were incubated with 10 ng/ml TNF-α, 10 μM LPA, or both for 24 h. Western blot analysis used an antibody that detects COX-2. Densitometry analysis is presented as mean ± S.E. (n ≥ 3). Panel B: Primary colonic myofibroblasts were incubated with or without 10 ng/ml TNF-α for 18 h, then with AG1478 for 1 h followed by 10 μM LPA for 4 h. Western blot analysis detected COX-2. Densitometry analysis is presented as mean ± S.E., n ≥ 3.

Figure 5

LPA-mediated transactivation of upregulated EGFR enhances COX-2 expression in colonic myofibroblasts. We have previously demonstrated that TNF-α potentiates LPA-mediated COX-2 expression via PKD [8], and that TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts [11]. Here, we demonstrate cross talk between these two signaling pathways through LPA-mediated transactivation of upregulated EGFR, leading to enhanced COX-2 expression in colonic myofibroblasts. A summary of the signaling interactions is illustrated in graphical form.