Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
Transcriptional regulation of the growth-regulated oncogene α gene by early growth response protein-1 in response to tumor necrosis factor α stimulation
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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