Interferon-gamma suppresses S100A4 transcription independently of apoptosis or cell cycle arrest

Abstract

The S100A4 protein has been associated with increased metastatic capacity of cancer cells, and recent studies have suggested a correlation between the expression level of S100A4 and the prognostic outcome for patients with various types of cancer. The knowledge about the mechanisms underlying the metastasis-promoting effects is still limited, and the aim of the present study was to elucidate signal transduction pathways involved in the regulation of S100A4. After treatment of human carcinoma cells with interferon-gamma (IFN-gamma), we observed downregulation of S100A4 both at mRNA and protein levels. The effect was not dependent on IFN-gamma-induced apoptosis or IFN-gamma-mediated cell cycle arrest. Moreover, IFN-gamma-mediated decrease in mRNA stability could not account for the observed decrease in S100A4 transcript level. Finally, microarray analysis suggests ISGF3G, ETV5, ZNF133 and CEBPG as possible candidate genes involved in IFN-gamma-mediated repression of S100A4.

Figure 1

Representative Northern blots showing effects of different mitogens on the expression of S100A4. The OHS, PM1 and WiDr cell lines were incubated with 20 μM PD98059 for 24 h, 0.2 nM TGF-β for 24 h, 0.5 ng ml⁻¹ thapsigargin for 24 h, 10 ng ml⁻¹ TNF-α for 48 h or 200 U ml⁻¹ IFN-γ for 48 h.

Figure 2

Optimisation of the IFN-γ response. (A) Northern blots showing transcriptional regulation of S100A4 in time–response experiments. OHS and II-11b cells were incubated with 10 U ml⁻¹ IFN-γ, and WiDr and PM1 cells were incubated with 200 U ml⁻¹ IFN-γ. Expression of 18S rRNA serves as loading control. (B) Western blots showing regulation of S100A4 and STAT1 by IFN-γ in time–response experiments. Incubation conditions as in (A) Immunostaining of α-tubulin serves as loading control.

Figure 3

Confirmation of active Jak/STAT1 signalling pathway. Western blot stained with antiphospho-STAT1 (Tyr-701). OHS cells were treated with 1000 U ml⁻¹ IFN-γ as indicated. Cells were harvested at indicated time points and analysed by Western blotting as described in Materials and Methods.

Figure 4

Effect of IFN-γ on S100A4 mRNA stability in OHS cells. Northern blot analysis demonstrating S100A4 mRNA stability in OHS control cultures and cultures treated with 10 U ml⁻¹ IFN-γ. The cultures were incubated for 24 h before the addition of 5 μM actinomycin D. The cells were then harvested at different time points as indicated. Isolation of total RNA and Northern blotting were performed as described in Materials and Methods. The chart is a graphical presentation of the band intensities as measured by means of densitometric scanning. The figure shows one of three independent experiments with similar results.