Tài liệu Báo cáo khoa học: Fra-1 targets the AP-1 site/2G single nucleotide polymorphism (ETS site)

Fra-1 targets the AP-1 site/2G single nucleotide polymorphism

(ETS site) in the MMP-1 promoter

Grant B. Tower1,*, Charles I. Coon2

, Karine Belguise3

, Dany Chalbos3 and Constance E. Brinckerhoff1,2

Department of 1

Biochemistry and 2

Medicine at Dartmouth Medical School, Hanover, NH, USA; 3

Institut National

de la Sante´ et de la Recherche´ Me´dicale, Endocrinologie Moleculaire et Cellulaire des Cancers, Montpellier, France

The matrix metalloproteinase (MMP) family degrades the

extracellular matrix. One member of this family, MMP-1,

initiates the breakdown of interstitial collagens. The

expression of MMP-1 is controlled by the mitogen activated

protein kinase (MAPK) pathway(s) via the activity of acti￾vator protein-1 (AP-1) and polyoma enhancing activity-3/

E26 virus (PEA3/ETS) transcription factors through con￾sensus binding sites present in the promoter. Another ETS

site in the MMP-1 promoter is created at )1607 bp by a

single nucleotide polymorphism (SNP), which contains two

guanines (5¢-GGAT-3¢;
2G SNP), rather one guanine

(5¢-GAT-3¢;
1G SNP), adjacent to an AP-1 binding site at

)1602 bp. The 2G SNP displays greater transcriptional

activity than the 1G SNP, and AP-1 and Ets families of

transcription factors cooperate to increase transcription. The

2G SNP has been linked to the incidence and the progression

of several cancers and is also associated with non-neoplastic

diseases; although the underlying mechanism(s) has yet to be

elucidated. In this study we demonstrate that the expression

of Fos-like region antigen (Fra-1), an AP-1 transcription

factor component that also correlates strongly with neo￾plastic disease, is necessary for MMP-1 transcription in

A2058 melanoma cells. The inhibition of Fra-1 expression

preferentially downregulates transcription from the MMP-1

promoter DNA containing the 2G SNP, compared to DNA

containing the 1G SNP. This study provides evidence that,

in cooperation with the 2G DNA polymorphism, the AP-1

family member, Fra-1, contributes to the high constitutive

expression of MMP-1 in melanoma cells.

Keywords: antisense; MAPK; matrix metalloproteinase;

melanoma and metastasis; signal transduction.

The extracellular matrix (ECM) acts as a structural support

network within tissues and as a barrier to cell migration.

Additionally, the destruction of the ECM can regulate

disease progression and severity in a variety of pathological

situations [1–3]. The matrix metalloproteinase (MMP)

family is responsible for degradation of the ECM, and the

MMP subfamily of collagenases specifically cleaves the

interstitial collagens (types I, II and III). MMP-1 is a

collagenase that is expressed at very low levels in normal

physiological situations, and expression can be increased

transiently when remodeling of the ECM is required (e.g.

wound healing, uterine resorption and development).

However, in many pathological states, MMPs are expressed

at high levels. This is due to continuous induction by

external stimuli, as seen in rheumatoid arthritis and

athlerosclerosis, or to constitutive expression because of

an activating mutation in a regulatory pathway [4–7]. High

constitutive levels of MMP-1 correlate with a poor prog￾nosis in many types of cancer [8–10].

The mitogen activated protein kinase (MAPK) signaling

pathway is composed of four main cascades, three of which

are known to control the expression of MMP-1. They are

the extracellular response kinase (ERK 1/2), the p38 and the

Jun N-terminal kinase, and they activate factors that target

multiple activator protein-1 (AP-1) and polyoma enhancing

activity-3/E26 virus (PEA3/ETS) factor binding sites within

the MMP-1 promoter [11–16]. These families of transcrip￾tion factors act synergistically when AP-1 and ETS

consensus sites are located in proximity to one another

[17,18], such as the AP-1/ETS site located at )73/)88 bp in

the MMP-1 promoter [19].

We have described a single nucleotide polymorphism

(SNP) at )1607 bp that generates an additional ETS site

when two guanines (5¢-GGAT-3¢;
2G SNP) are present

instead of one guanine (5¢-GAT-3¢;
1G SNP) (Fig. 1), and

this SNP results in increased transcription from the

2G-containing promoter relative to the 1G-containing

promoter [20,21]. We have verified this ETS site by

demonstrating that it binds recombinant Ets-1 protein,

and that the adjacent AP-1 site at )1602 bp binds recom￾binant c-Jun. Further, we carried out gel shift analyses

with nuclear extracts from A2058 melanoma cells and

Correspondence to C. E. Brinckerhoff, Department of Biochemistry,

505 Vail Building, Hanover, NH 03755, USA.

Fax: + 1 603 650 1128, Tel.: + 1 603 650 1609,

E-mail: [email protected]

Abbreviations: MMP, matrix metalloproteinase; MAPK, mitogen

activated protein kinase; AP-1, activating protein; PEA3/ETS, poly￾oma enhancing activity-3/E26 virus; FGF, fibroblast growth factor;

SNP, single nucleotide polymorphism; ERK, extracellular response

kinase; Fra, Fos-related antigen; Elf, ETS-like factor; DMEM,

Dulbecco’s Modified Eagles Medium; CMV, cytomegalovirus;

GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

*Present address: Schering-Plough, 2015 Galloping Hill Rd., K-15,

D-129C Kenilworth, NJ 07033, USA.

(Received 11 June 2003, revised 4 September 2003,

accepted 8 September 2003)

Eur. J. Biochem. 270, 4216–4225 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03821.x