SIK1 inhibits migration in AGS-G_R cells via suppression of MMP-9.

<p><b>A–B:</b> AGS-G_R cells (<b>A</b>) and MKN45 cells (<b>B</b>) were treated with gastrin, and phospho-LKB1 (Ser-428) protein levels determined by Western blot. The phospho-LKB1 bands from a representative experiment are shown. <b>C–D:</b> AGS-G_R cells (<b>C</b>) and MKN45 (<b>D</b>) were treated with gastrin, and phospho-SIK1 (Thr-182) protein levels determined by Western blot. The phospho-SIK1 bands from a representative experiment are shown. <b>E:</b> AGS-G_R cells transfected with siSIK1 or siCtr and real-time cell migration monitored (0–24 h). Results show one representative of three independent experiments (mean ±SD of three technical replicates). <b>F:</b> MMP-9 mRNA expression in cells transfected with pSIK1 and treated with gastrin. Results show one representative of three independent experiments, (mean ± SD).</p

ICER represses the level of <i>SIK1</i> mRNA and protein.

<p><b>A:</b> AGS-G_R cells were treated with gastrin and mRNA levels of ICER measured by qRT-PCR. Results shown are mean ± SEM of three independent biological experiments. <b>B:</b> AGS-G_R cells were transfected with ICER I, ICER IIγ or control expression plasmids, treated with gastrin (1 h) and mRNA levels of SIK1 measured by qRT-PCR. Results show one representative of three independent experiments; mean ± SD of three technical replicates. <b>C:</b> AGS-G_R cells were transfected with siRNAs, treated with gastrin and mRNA levels measured by qRT-PCR. Results show one representative of three independent experiments; mean ± SD of three technical replicates. <b>D:</b> SIK1 Western blot in cells transfected with siICER. A representative image is shown and quantified.</p

Gastrin-induced activation of SIK1.

<p><b>A:</b> AR42J cells were treated with gastrin and mRNA levels measured by qRT-PCR. Mean expression level relative to untreated cells is shown. Results show one representative of three independent biological experiments; mean ± SD of three technical replicates. <b>B:</b> SIK1 Western blot of gastrin treated AR42J cells. A representative image is shown and quantified <b>C:</b> AGS-G_R cells were treated with gastrin and mRNA levels measured by qRT-PCR. Mean ± SEM of three independent biological experiments is shown. <b>D:</b> SIK1 Western blot of gastrin treated AGS-G_R cells. A representative image is shown and the SIK1 bands from two independent experiments were quantified; results shown are mean intensities ±SD. <b>E:</b> SIK1 Western blot of gastrin treated MKN45 cells. The SIK1 bands from a representative experiment were quantified. <b>F:</b> Intracellular localization of endogenous CRTC2 protein (Red; CRTC2, blue; Draq-5-stained DNA). G: Intracellular localization of SIK1 protein. AGS-G_R cells transfected with pEGFP-SIK1.</p

The role of SIK1 in gastrin responsive cells.

<p>Gastrin binds to the CCK2 receptor (CCK2R) and activates the LKB1–SIK1 signalling pathway in adenocarcinoma cells. SIK1 mediated phosphorylation of HDAC leads to cytosolic translocation and activation of transcription. In the gastric adenocarcinoma cell line AGS-G_R ectopic SIK1 inhibits migration.</p

Immunostaining of NR4A2 in gastric adenocarcinoma.

<p><b>A-B</b>: NR4A2 immunoreactivity in normal oxyntic mucosa showing strong intensity in scattered single cells (neuroendocrine cells) and weaker staining intensity in the other epithelial cells. <b>C-F</b>: NR4A2 immunoreactivity in gastric adenocarcinomas of intestinal (C-D) and diffuse (E-F) type, showing a general staining in tumor cells with mixed nuclear or cytoplasmic localization and variable intensities. (A, C, E at x200 magnification, with boxes representing B, D and F at x400 magnification).</p

Negative regulation of gastrin-induced NR4A2 expression.

<p><b>A</b>: AGS-G_R cells transfected with NR4A2-luc and ICER expression plasmids or empty vector. Cells were treated with gastrin for 6 h prior to measurement of NR4A2 activity. Data shown represent mean ± SEM of five biological replicas (** p<0.03, * p = 0.06). <b>B</b>: AGS-G_R cells transfected with NBRE-luc and ICER expression plasmid or empty vector and treated with gastrin for 4 h prior to measurement of NBRE activity. Data shown represent mean ± SEM of four biological replicas (** p<0.03). <b>C</b>: AGS-G_R cells were transfected with pZfp36l1 expression plasmid or empty vector and treated with gastrin (5 nM) NR4A2 mRNA expression was measured by qRT-PCR. Data shown represent one of three biological replicas; mean ± SD of three technical replicas is shown. <b>D</b>: Cyclin L1 represents one of three control genes examined.</p

NR4A2 suppresses gastrin-induced migration and invasion.

<p><b>A</b>: Real-time cell migration monitored (0-24 h) in AGS-G_R cells transfected with siNR4A2 or siCtr, with or without gastrin treatment (10 nM). Results show one representative of three biological replicas; mean ±SD of three technical replicas. <b>B</b>: Invasion assay with AGS-G_R cells transfected with pCMX-NR4A2 or pCMX (control) was performed in 24-well plates containing 8-µm pore Matrigel-coated inserts (with or without 0.3 nM gastrin). Cells invading the lower surface of the membrane were stained with Reastain Quick-Diff reagents and total numbers of cells in 5 fields per membrane were counted. The mean of three independent experiments is shown.</p

NR4A2 activates NBRE promoter elements.

<p><b>A</b>: Gastrin-induced NBRE-luc activation. Data represent one of two biological replicas. <b>B</b>: The effect of NR4A2 siRNA on gastrin-induced NBRE activation. Data represent mean ± SEM of four biological replicas (** p<0.01, * p=0.1). <b>C-D</b>: Effect of specific inhibitors of PKA (H-89, 10µM), PI3K (LY 294002, 10µM) or PKC (GF 109203x, 3.5µM) on (<b>C</b>) gastrin-induced NR4A2 gene expression and (<b>D</b>) gastrin-induced NBRE activation. Data represent one of three biological replicas; mean ± SD of six technical replicas.</p