Identification and characterization of the promoter of cancer-related gene LOXL2

Abstract

Lysyl oxidase like 2, LOXL2, as a member of the lysyl oxidase (LOX) family, has been shown to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 is also engaged to transcription regulation, cell signaling transduction and cell adhesion regulation. It has been reported that LOXL2 is highly expressed in several types of tumors and promotes cell proliferation and migration in various cancer cells. However, the regulatory mechanism of LOXL2 expression remains largely unknown. To further investigate its transcriptional regulatory mechanism, LOXL2 promoter region has been cloned and identified in the present study. Chromatin state analysis revealed that LOXL2 gene locus contained an active promoter near its first exon. We then constructed five different LOXL2 gene promoter luciferase reporter constructs covering 1.7 kb upstream of LOXL2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all the five constructs showed notable promoter activity, and LOXL2 core promoter was located in a region of 185 bp near the transcription initiation site. Transcriptional factor binding analysis indicated that, LOXL2 promoter lacked classical TATA box, but contained putative binding sites for classic transcriptional factors such as Sp1 and NF-κB. Ectopic overexpression of Sp1 significantly enhanced LOXL2 promoter activity as well as its endogenous expression in cells. In contrast, mithramycin A (a selective Sp1 inhibitor) treatment repressed LOXL2 promoter as well as its endogenous transcription. Site directed mutagenesis assay further confirmed that the Sp1 binding sites were essential for proximal prompter activity of LOXL2 gene. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 bound LOXL2 promoter in vivo. Of note, the expression of Sp1 and LOXL2 are positively correlated, and the higher expression of LOXL2 is associated with poor prognosis in colorectal cancer, strongly suggesting the implication of Sp1-mediated LOXL2 transactivation in the pathogenesis of colorectal cancer.

Introduction

Lysyl oxidase like 2 (LOXL2) is a member of the lysyl oxidase (LOX) family which possess a conserved carboxy-terminal (C-terminal) amine oxidase catalytic domain including a His-X-His-X-His copper binding motif and a lysine tyrosylquinone (LTQ) cofactor [[1], [2], [3], [4], [5]]. Researches have revealed that LOX family could catalyze the oxidative deamination of the e-amino group of lysines and hydroxylysines in collagen and elastin to promote crosslinking of these molecules, which is essential for the tensile strength of ECM [6,7]. In addition, LOX family is proposed to participate in the transcription regulation, cell signaling transduction, and cell adhesion processes which are pivotal to cell survival [8,9]. Based on the primary structure of their N-termini, LOX family can be divided into two subgroups [10]. LOX and LOXL1 both contain a highly basic peptide at their N-termini, while LOXL2, LOXL3 and LOXL4 respectively possess four scavenger receptor cysteine-rich (SRCR) domains at their N-termini [3,10,11].

Among them, LOXL2 has got more and more attention in these years because of its significant role in cancer progression. It has been demonstrated that LOXL2 is associated with various cancer, including breast cancer, colon cancer, esophagus cancer, gastric cancer etc. It has been proved that the higher expression of LOXL2 is associated with poor prognosis of patients [12] and promoted the cancer cells metastasis [[13], [14], [15]]. As to the mechanism through which LOXL2 promote the progression of cancer, there are quite a few studies revealed that LOXL2 could induce the epithelial to mesenchymal transition (EMT) in cancer cells [[16], [17], [18], [19]] which was well-known as an important cellular process in the progression of localized tumors.

Previous studies have showed that LOXL2 is highly expressed and promotes cell proliferation and migration in several types of cancers. In addition, LOXL2 gene expression is induced by extracellular stimuli such as TGF-beta and hypoxia (Saito et al., 1997; Borczuk et al., 2005; Higgins et al., 2007; Salnikow et al., 2008) [2,[20], [21], [22]]. However, the regulatory mechanism of LOXL2 expression remains unknown. Fong et al. (2007) have previously performed predictive analysis of the potential promoter region of human LOXL2 [12]. However, the promoter region of human LOXL2 remains elusive and has not been experimentally identified and characterized till now. In the present study, we identify and characterize its promoter region for the first time, finding that Sp1 is a critical regulator for LOXL2 and Sp1-mediated LOXL2 transactivation is implicated in the pathogenesis of colorectal cancer.