Phosphoglucose Isomerase/Autocrine Motility Factor Promotes Melanoma Cell Migration through ERK Activation Dependent on Autocrine Production of Interleukin-8

Kenichiro Araki,Tatsuo Shimura,Toshiki Yajima,Soichi Tsutsumi,Hideki Suzuki,Kohji Okada,Tsutomu Kobayashi,Avraham Raz,Hiroyuki Kuwano

doi:10.1074/jbc.m109.008250

Kenichiro Araki, Tatsuo Shimura + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m109.008250

Copy DOI

Abstract

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Highlights

Enhanced expression levels of both autocrine motility factor (AMF) and AMF receptor (AMFR) are correlated with the progression of malignant tumors [5, 6]
The main findings of the present study are as follows: 1) AMF induces a significant increase in IL-8 production in early stage melanoma cells; 2) the increase in IL-8 production is caused by an increase in the IL-8 transcription rate rather than enhanced IL-8 mRNA stability; 3) the ERK1/2 pathway enhances IL-8 transcription in response to AMF; 4) autocrine IL-8 effects are required for AMF-induced melanoma cell migration
For the first time, that AMF induces melanoma cells to produce IL-8, which acts in an autocrine manner to promote the migration of these cells

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Human malignant melanoma SBcl-2 (SB cell line, clone-2) cells were provided by Dr Meenhard Herlyn (Wistar Institute, Philadelphia, PA). SBcl-2 cells were established in culture from primary cutaneous melanoma and are a poorly tumorigenic and nonmetastatic line in nude mice [18, 25]. Melanoma cells were grown in 2% Tu medium comprising MCDB153 medium supplemented with 20% L15 medium, 2% heat-inactivated fetal bovine serum, and 5 ␮g/ml insulin unless otherwise stated. ELISA—The IL-8 protein concentrations in cell culture supernatants were measured using an ELISA kit (BIOSOURCE International, Camarillo, CA), according to the manufacturer’s protocol. Melanoma cells (2 ϫ 103 cells/well) were cultured in 96-well microtiter plates in a total volume of 100 ␮l/well. The standard reaction volume was 20 ␮l and contained 1ϫ SYBR Green PCR Master Mix (Applied Biosystems), 1.0 ␮l of cDNA template, and a 1.0 ␮M concentration of both forward and reverse primers.

RESULTS

We also investigated whether

DISCUSSION