Ets1 transcription factor mediates gastrin-releasing peptide-induced IL-8 regulation in neuroblastoma cells

Abstract

Angiogenesis plays a critical role in tumor progression in various cancers, including neuroblastoma. We have previously shown that gastrin-releasing peptide (GRP) stimulates neuroblastoma growth and that its cell surface receptors, gastrin-releasing peptide receptors (GRP-R), are overexpressed in advanced-stage human neuroblastomas; however, the effects of GRP on angiogenesis are not clearly elucidated. Interleukin (IL) 8, a proinflammatory chemokine, plays an important role during tumor angiogenesis. Ets transcription factors, such as oncoproteins, cause tumor development and are also known to induce IL-8 expression. In the present study, we found an increased expression of Ets1 in more undifferentiated human neuroblastomas. Stable transfection of SK-N-SH human neuroblastoma cells with Ets1 plasmid resulted in increased IL-8 luciferase activity and IL-8 secretion into cell culture media. Conversely, silencing of Ets1 resulted in a significant decrease in IL-8 secretion in SK-N-SH cells. Moreover, exogenous GRP treatment increased Ets1 (T38) phosphorylation and Ets1 nuclear accumulation, and enhanced Ets1 binding to its DNA consensus sequence, resulting in the stimulation of IL-8 mRNA expression and protein secretion. Our findings demonstrate that GRP upregulates proangiogenic IL-8 expression in an Ets1-dependent manner, suggesting a critical role of this process during GRP-induced neuroblastoma angiogenesis and metastasis.

Figure 1

Ets1 expression in human neuroblastomas. (A) Paraffin-embedded human neuroblastoma sections were stained with an anti-Ets1 antibody visualized by diaminobenzidine staining. Ets1 was strongly expressed in undifferentiated neuroblastoma cells (panels b and d), whereas weakly stained Ets1 was found in well-differentiated neuroblastoma sections (panels a and c). Original magnification: x200 for panels (a) and (b); x400 for panels (c) and (d). (B) Ets1 protein was expressed in 18 of 21 (86%) undifferentiated tumor sections, and in 7 of 16 (44%) differentiated tumor sections.

Figure 2

Ets1 regulates IL-8 expression and secretion in SK-N-SH cells. (A) Cells were transfected with the Ets1 expression plasmid, and then Western blot analysis was performed. (B) IL-8 promoter transcriptional activity was enhanced by cotransfection with Ets1 expression plasmid (mean ± SEM; *P < .05 vs control vector). (C) IL-8 secretion into a culture supernatant was increased by Ets1 overexpression (mean ± SEM; *P < .05 vs control vector).

Figure 3

Ets1 phosphorylation and nuclear localization after GRP treatment. (A) SK-N-SH cells were serum-starved overnight and then treated with GRP (10^-7 M). Induction of Ets1 phosphorylation was detected after GRP treatment in both SK-N-SH and BE(2)-C cells by Western blot analysis. (B) For immunofluorescent staining, SK-N-SH cells were seeded on coverslips and serum-starved overnight. Ets1 protein was localized predominantly in the nuclei at 1 hour (bottom panels) after GRP treatment (10^-7 M) when compared to controls (top panels). DAPI specifically stained for the DNA of nuclei.

Figure 4

GRP increases Ets1 DNA binding and transcription activity. (A) Nuclear protein was extracted from cells treated with GRP (10^-7 M) for 4 hours. Nuclear protein (2.5 µg/lane) was added in binding reactions, and protein/DNA complexes were resolved in a 6% DNA retardation gel. (B) SK-N-SH cells were cotransfected with 5x GAL4-Luc plus GAL4-Ets1 (WT) or GAL4-Ets1 (T38A) (dominant-negative mutant vector). Cells were treated with GRP (10^-7 M) for 24 hours after serum starvation. Ets luciferase activity was significantly increased after GRP treatment (data represent triplicate determinations; mean ± SEM; *P < .05 vs control vector).

Figure 5

GRP increases IL-8 luciferase activity, and Ets1 regulates GRP-induced IL-8 secretion in SK-N-SH cells. (A) For IL-8 promoter activity assays, cells were seeded in 24-well plates, transfected with IL-8 luciferase plasmid DNA, and then treated with GRP (10^-7 M). (B) For IL-8 secretion assay, cells in 12-well plates (1 x 10⁵ cells/well) were treated with GRP (10^-7 M) for 24 hours. The culture supernatants were collected, and IL-8 in the culture supernatant was measured by ELISA (data represent triplicate determinations; mean ± SEM; *P < .05 vs control). (C) Ets1 siRNA transfection yielded a significantly decreased Ets1 protein expression by Western blot analysis. (D) Ets1 knockdown using siRNA significantly decreased IL-8 secretion. In addition, GRP-stimulated increase in IL-8 secretion was markedly attenuated in SK-N-SH cells transfected with Ets1 siRNA vector when compared to control cells transfected with control siRNA vector (mean ± SEM; *P < .05 vs control siRNA vector; †P < .05 vs no GRP). All data represent triplicate determinations.