Gastrin-induced proliferation involves MEK partner 1 (MP1)

Abstract

The peptide hormone gastrin is an important factor for the maintenance and homeostasis of the gastric mucosa. We show that gastrin stimulates proliferation in a dose-dependent manner in the human gastric adenocarcinoma cell line AGS-GR. Furthermore, we demonstrate that the MAPK scaffold protein MEK partner 1 (MP1) is important for gastrin-induced phosphorylation of ERK1 and ERK2 and that MP1 promotes gastrin-induced proliferation of AGS-GR cells. Our results suggest a role of MP1 in gastrin-induced cellular responses involved in proliferation and homeostasis of the gastric mucosa.

Figure 1.

xCELLigence proliferation assay of wild-type AGS and AGS-G_R cells in response to gastrin. AGS and AGS-G_R cells were seeded in 96-well E-plates for xCELLigence assay monitoring impedance (cell index). The cells were treated with either 10% FCS or 100 ng/ml EGF as controls or gastrin at different concentrations (0.1, 1, 10, or 100 nM) in either absence or presence of serum. (a) Complete growth curves of AGS and AGS-G_R cells from 0 to 70 h after seeding. The cells were treated with gastrin and EGF in absence of serum. Panel to the right shows bar graph of cell index at 24 h growth. (b) Complete growth curves of AGS and AGS-G_R cells from 0 to 70 h after seeding. The cells were treated with gastrin and EGF in presence of serum. Panel to the right shows bar graph of cell index at 24 h growth. Data is represented as mean ± SD of three independent experiments.

Figure 2.

Gastrin-mediated activation of MP1 in AR42J and AGS-G_R cells. (a) Verification of gene expression microarray results with quantitative RT-PCR in AR42J. Cells were seeded in 6-well plates and serum starved for 24 h the following day prior to treatment with gastrin (10 nM) as indicated in figure. (b) Quantitative RT-PCR analysis of MP1 expression in AGS-G_R. Cells were seeded in 6-well plates and cultivated 24 h prior to gastrin treatment (10 nM) as indicated in figure. Fold induction levels were calculated using the ΔΔCt-method (Livak and Schmittgen 2001), where the expression levels were normalized to the level of β-actin (AR42J) or GAPDH (AGS-G_R) expression, and gastrin-treated cells were compared to untreated cells. One representative experiment is shown and data presented as mean ± SD of three technical replicates. (c) Western blot of AGS-G_R lysates from cells treated with 10 nM of gastrin 2–10 h before harvesting. The blot was treated with non-commercial antibodies against MP1, and β-actin was used as loading control. Lower panels show relative quantification of band intensities from western blot analysis.

Figure 3.

MP1 depletion reduces gastrin-induced phosphorylation of ERK1 and ERK2 (p-ERK1/2). (a) AGS-G_R cells were transfected with siMP1 and siCtr for 24 h. Then, the cells were serum starved for 24 h and treated with 10 nM gastrin for 5 to 30 min before harvesting. Protein extracts were subjected to SDS-PAGE and analyzed for p-ERK1 and p-ERK2 in addition to GAPDH (loading control). (b, c) Densiometric analysis of relative amounts of p-ERK1 and p-ERK2, respectively. One representative experiment is shown. Similar results were obtained three times.

Figure 4.

MP1 enhances cell cycle progression. AGS-G_R cells were grown and treated with siCtr, siMP1, or siCycD1. Cells were treated with 10 nM gastrin without medium replacement 24 h after transfection and then grown for another 24 h. Finally, the cells were harvested in citrate buffer and treated with buffers A, B, and C according to a slightly modified version of the Vindelov protocol (Vindelov et al. 1983). The proportion of cells in S-phase of the cell cycle in untreated (black) and gastrin-treated (red) bars. DNA histograms were created from 20,000 propidium iodide stained nuclei. Representative histograms are shown, and data is presented as percent cells in S-phase mean ± SD (n = 3). Using qRT-PCR, MP1 depletion was found to be approximately 90% (data not shown).