JNK/AP-1 pathway is involved in tumor necrosis factor-α induced expression of vascular endothelial growth factor in MCF7 cells

Abstract

Vascular endothelial growth factor (VEGF) has been implicated in breast tumor angiogenesis. And tumor necrosis factor-α (TNF-α) is a positive regulator of VEGF. This study was aimed to identify the signalling pathway of TNF-α in VEGF expression regulation in breast cancer cell line MCF7. Using luciferase reporter assays, we demonstrated that TNF-α significantly increased activator protein-1 (AP-1) transcriptional activity in the MCF7 cells. The expression of the AP-1 family members c-Jun, c-Fos and JunB and phosphorylation levels of c-Jun were upregulated by TNF-α, whereas other AP-1 family members Fra-1, Fra-2, and JunD were unaffected. The activation of AP-1 was associated with the formation of p-c-Jun-c-Jun and p-c-Jun-JunB homodimers. Furthermore, the phosphorylation levels of c-Jun N-terminal kinase (JNK) but not P38 and ERK were elevated by TNF-α in MCF7 cells. TNF-α potently upregulated the mRNA and protein levels of VEGF, which were significantly reversed by JNK inhibitor SP600125. Finally using chromatin immunoprecipitation (CHIP) assays, we found that p-c-Jun bound to the VEGF promoter and regulated VEGF transcription directly. These data suggest that the pro-inflammatory cytokine TNF-α is a critical regulator of VEGF expression in breast cancer cells, at least partially via a JNK and AP-1 dependent pathway.

Introduction

Vascular endothelial growth factor (VEGF) is a potent endothelial mitogen that is upregulated in a number of tumor types, including breast cancer [1], [2]. Because it can stimulate macrophage migration [3], VEGF appears to be important for breast cancer growth and neovascularization. The VEGF promoter region includes several potential binding sites for the transcription factors SP-1, AP-1 and AP-2 [4]. Hypoxia up-regulates VEGF via the binding of hypoxia inducible factor-1 (HIF-1) to a hypoxia response element in the VEGF promoter [5]. VEGF gene expression is also enhanced by cytokines such as TNF-α, EGF, PDGF and TGF-β [6].

The cytokine TNF-α appears to have different roles depending upon whether it is studied during breast cancer pathogenesis or when used as a therapeutic agent. High-dose local administration of TNF-α selectively destroys tumor blood vessels, and has powerful anti-cancer actions [7]. However, when TNF-α is chronically elevated endogenously during breast cancer development, it appears to act as a tumor enhancer, contributing to the tissue remodeling and stromal development necessary for tumor growth and spread [8]. The effects of TNF-α are mediated by several signal transduction pathways, including the NF-κB pathway and mitogen-activated protein kinase (MAPK) pathways. TNF-α has also been shown to acts as positive regulator of VEGF in human glioma cells [9], although the details of their relationship in breast cancer pathogenesis are not yet clear.

AP-1 is known to be involved in the TNF-α receptor signalling pathway, enabling TNF-α to influence the expression of many genes [10], some of which may be important in tumor invasion [8]. The AP-1 complex is composed of heterodimers of members of the Jun (c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) families, or Jun-Jun homodimers [11], [12], [13]. In particular, c-Jun has been implicated in events leading to tumor development [14], whereas c-Fos appears to be involved in the malignant conversion of benign papillomas [15].

The activation of AP-1 is mediated by at least two regulatory events [16], [17]. First, some AP-1 proteins (e.g. c-Jun) are encoded by immediate early genes that are transcriptionally induced following treatment. Second, AP-1 activity can also be regulated by post-translational modification, including phosphorylation by MAPKs. The MAPK family comprises the extracellular signal regulated kinase (ERK), p38 MAPK and c-Jun NH₂-terminal kinase (JNK). However, the exact mechanism of specific condition or treatment on AP-1 activation and the relative role of different MAPKs in these processes are diverse. TNF-α regulates AP-1 activity, in part, by increasing the expression of members of the Jun and Fos families. An important role for the JNK signalling pathway in AP-1 activation by TNF-α has also been proposed [18]. Although some links have been made between TNF-α and the activation of JNK and AP-1, it remains unclear whether these events are linked to TNF-α-mediated VEGF expression in breast cancer.

This study was designed to identify the signalling pathway of VEGF expression regulation in the MCF7 breast cancer cell line. Our results demonstrate, for the first time, that the inflammatory cytokine TNF-α is a critical regulator of VEGF expression in breast cancer cells through the JNK/AP-1 dependent pathway.