Transcriptional regulation of the growth-regulated oncogene α gene by early growth response protein-1 in response to tumor necrosis factor α stimulation

https://doi.org/10.1016/j.bbagrm.2013.07.005Get rights and content

Highlights

  • GROα is a CXC chemokine which plays an important role in tumor progression, including tumor invasion.

  • Regulatory mechanism underlying TNFα-induced GROα expression is not fully characterized.

  • Egr-1 is induced by TNFα through the Erk and JNK MAPK pathways.

  • TNFα-induced Egr-1 directly binds to the GROα gene promoter and transactivates the GROα gene.

  • Egr-1-induced GROα participates in TNFα-induced invasion of HeLa cells.

Abstract

Growth-regulated oncogene α (GROα) plays an important role in a wide range of normal and pathological conditions, including inflammation, angiogenesis, wound healing, tumor invasion, and metastasis. Egr-1 is a member of the zinc-finger transcription factor family induced by diverse stimuli, including TNFα. However, the role of Egr-1 in GROα expression was previously unknown. This study shows that Egr-1 directly binds to the GROα promoter and transactivates the GROα gene. Silencing of Egr-1 by expression of Egr-1 siRNA abrogated TNFα-induced GROα transcription. We also found that Egr-1 mediates ERK and JNK MAPK-dependent GROα transcription upon TNFα stimulation. Our findings suggest that Egr-1 may play an important role in tumor development through transactivation of the GROα gene in response to TNFα within the tumor microenvironment.

Introduction

Chronic inflammation is thought to facilitate carcinogenesis by providing an ideal microenvironment for tumor cell development [1], [2], [3], [4]. Tumor necrosis factor α (TNFα) is a potent inflammatory cytokine produced by many cell types, including immune cells and fibroblasts, in response to inflammation and tissue injury. TNFα induces diverse inflammatory cytokines and chemokines, modulating a broad range of inflammatory and immunological processes. In the tumor microenvironment, TNFα is produced by tumor cells and tumor-associated fibroblasts and plays crucial roles in tumor progression, including tumor invasion and metastasis [1], [2], [5].

The melanoma growth stimulatory activity/growth-regulated oncogene α (MGSA/GROα), which is designated as a CXC chemokine ligand 1 (CXCL1), was first identified as an autocrine growth factor in human malignant melanoma [6]. GROα plays an important role in the regulation of inflammation through attraction of neutrophils and in the progression of tumor development through stimulation of angiogenesis and metastasis [7], [8], [9]. GROα is produced from diverse cell types, including fibroblasts, airway muscle cells, and melanoma and epithelial ovarian cancer cells in response to pro-inflammatory agents such as TNFα, interleukin-1 (IL-1), and lipopolysaccharide (LPS) [10]. Transcription of the GROα gene is controlled by multiple cis-acting response elements, including the TATA box (− 25 to − 30 nt), NF-κB element (− 67 to − 77 nt), AT-rich HMGI(Y) binding elements within the NF-κB sites, the immediate upstream region (IUR; − 78 to − 93 nt), and SP1 binding sites (− 117 to − 128 nt), which are involved in basal and induced transcriptional responses [11], [12], [13].

In an attempt to discover an additional cis-element responsible for the regulation of GROα transcription in tumor cells, we analyzed the regulatory elements located within the 5′-flanking regulatory region using a Genomatix Web-based database, and found that two putative Egr-1-binding sequences (EBSs) are located at − 367 (5′-AGGAGCTggggcgGATC-3′) and − 134 (5′-CACCCTGggggcgGGGC-3′) within the GROα genes (the lowercased letters are core sequences). Because Egr-1 can be induced by TNFα in many cell types [14], [15], [16], [17] and regulate matrix metalloproteinase-9 (MMP-9) expression in response to TNFα stimulation [18], it is of interest to investigate whether Egr-1 is involved in GROα expression. Here, we demonstrate that in addition to the NF-κB element, two EBSs are crucial for maximal induction of GROα transcription by TNFα. Furthermore, exogenous Egr-1 transactivated GROα promoter activity and stable silencing of Egr-1 by siRNA strongly attenuated TNFα-induced GROα mRBA expression in HeLa cells. We also found that Egr-1 mediates ERK and JNK, but not p38, in mitogen-activated protein kinase (MAPK)-dependent GROα transcription in response to TNFα stimulation.

Section snippets

Cell culture and reagents

The human cervix carcinoma HeLa cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA). HeLa cells expressing control scrambled siRNA (siCont) or Egr-1 siRNA (siEgr-1) are described elsewhere [18]. Human recombinant TNFα, U0126, SB203580, and SP600125 were purchased from Calbiochem (San Diego, CA, USA) and JNK-interacting protein

Upregulation of GROα transcription by TNFα in HeLa cells

We first verified the transcriptional activation of the GROα gene following TNFα stimulation in HeLa cells. RT-PCR (Fig. 1A), Northern blot (Fig. 1B), and quantitative real-time PCR (Fig. 1C) analyses showed that the levels of the GROα transcript increased as a function of time of exposure to TNFα.

Identification of the Egr-1-binding sequences (EBSs) within the human GROα gene promoter

Transcription of the GROα gene is regulated by multiple cis-acting elements, including NF-κB, CDP, and IUR [13]. In an attempt to discover an additional cis-element responsible for the regulation of

Acknowledgement

This work was supported by the Bio & Medical Technology Development Program (No. 2007-2004337) and the Basic Research Promotion Fund (No. 2012R1A2A2A01013311) of the National Research Foundation (NRF) funded by Ministry of Science, ICT & Future Planning, and by a grant of the Korean Healthcare Technology R&D Project, the Ministry for Health, Welfare & Family Affairs (No. A111778). This paper was supported by the KU Research Professor Program of Konkuk University.

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