Tài liệu Báo cáo khoa học: Gene transcription of fgl2 in endothelial cells is controlled by Ets-1

Gene transcription of fgl2 in endothelial cells is controlled by Ets-1

and Oct-1 and requires the presence of both Sp1 and Sp3

Mingfeng Liu1

, Julian L. Leibowitz2

, David A. Clark1,4, Michael Mendicino1

, Qin Ning1

, Jin Wen Ding1

,

Cheryl D’Abreo3

, Laisum Fung1

, Philip A. Marsden3 and Gary A. Levy1

1

Multi Organ Transplant Program, Toronto General Hospital and The University of Toronto, Canada; 2

Department of Pathology and

Laboratory Medicine, Texas A & M University System College of Medicine, USA; 3

Renal Division and Department of Medicine,

St. Michael’s Hospital and University of Toronto, Canada; 4

McMaster University, Ontario, Canada

The immune coagulant fgl2/fibroleukin has been previously

shown to play a pivotal role in the pathogenesis of murine

and human fulminant hepatitis and fetal loss syndrome.

Constitutive expression of fgl2 transcripts at low levels are

seen in cytotoxic T cells, endothelial, intestinal and tropho￾blast cells, while specific factors (such as virus and cytokines)

are required to induce high levels of fgl2 expression in other

cell types including monocytes/macrophages. To address

the transcriptional mechanisms that regulate constitutive

expression of fgl2, murine genomic clones were characterized

and the transcription start site was defined by 5¢-RACE and

primer extension. A comprehensive assessment of basal

fgl2 promoter activity in murine vascular endothelial cells

defined a minimal 119 bp region responsible for constitutive

fgl2 transcription. A complexpositive regulatory domain

(PRD) spanning a 39-bp sequence from )87 to )49 (relative

to the transcription start site) was identified. Electrophoretic

mobility shift assay studies in vascular endothelial cells

revealed that the nucleoprotein complexes that form on this

positive regulatory domain (PRD) contain Sp1/Sp3 family

members, Oct-1, and Ets-1. Heterologous expression studies

in Drosophila Schneider cells confirmed that the constitutive

expression of this gene is controlled by Ets-1 and requires the

presence both of the Sp1 and Sp3 transcription factors. The

presence of this complexmulticomponent PRD in the fgl2

proximal promoter is consistent with the observation that,

in vivo, fgl2 expression is tightly regulated. Moreover, viral

induced fgl2 expression also requires the presence of this

PRD. These results clearly demonstrate that multiple cis

DNA elements in a clustered region work cooperatively to

regulate constitutive fgl2 expression and interact with indu￾cible elements to regulate viral-induced fgl2 expression in

endothelial cells.

Keywords: fibroleukin; fgl2; hepatitis; immune coagulant;

transcription regulation.

Activation of the coagulation system represents an import￾ant facet of immune and inflammatory reactions, account￾ing for the fibrin deposition that is commonly observed in

these reactions [1]. Cellular procoagulants and the soluble

factors of the coagulation cascade are important partici￾pants in a number of human diseases including allograft

rejection [2,3], glomerulonephritis [4,5], septic shock [6,7],

and bacterial and viral infections [8]. For example, tissue

factor (TF) [9], the transmembrane receptor of factor VII, is

the major procoagulant of the classical extrinsic pathway of

coagulation resulting in thrombin generation, and subse￾quent fibrin deposition. Furthermore, TF has important

roles in the regulation of angiogenesis in cancer, embryonic

blood vessel development, and intracellular signaling

[10–12]. Thrombin is involved in many biological processes.

In addition to its function in blood coagulation and wound

healing, thrombin has diverse functions in many different

cell types. For instance, thrombin induces proliferation of

fibroblasts and smooth muscle cells, and neurite retraction

and synapse reduction in neurons [12]. Moreover, thrombin

is a chemotactic agent for inflammatory cells such as

macrophages and neutrophils [12].

fgl2/fibroleukin was originally described as a fibrinogen￾like protein, constitutively expressed in cytotoxic T cells

[13]. This gene was subsequently determined to encode an

immune coagulant and was localized to the proximal

region of mouse chromosome 5 [14,15]. Fgl2 is a member

of the fibrinogen family, which includes tenascin, cyto￾toxin, and fibrinogen [16–18]. Fibrinogen-like protein

family members all contain fibrinogen-related domains

(FRED), known to represent key regions for protein–

protein interaction. As a procoagulant, fgl2 is involved in

cleaving prothrombin to thrombin endogenously and

when functionally expressed in a heterologous system

[14,19]. Thus, an important biological function of fgl2

might be to regulate the production of thrombin via a

pathway independent of TF, especially in settings where

TF-independent procoagulant activity has been documen￾ted. For instance, fgl2 plays a critical role in the

pathogenesis of both human and mouse fulminant viral

Correspondence to G. A. Levy, Toronto General Hospital, 621

University Ave., NU-10–116, Toronto, Ontario, Canada M5G 2C4.

Fax: 416 340 3378, Tel.: 416 340 5166,

E-mail: [email protected]

Abbreviations: EMSA, electrophoretic mobility shift assays; fgl2,

fibrinogen-like protein; LUC, luciferase; MHV-3, murine hepatitis

virus strain 3; NE, nuclear extracts; PRD, positive regulatory domain;

Stat3, signal transducer and transactivator 3.

(Received 6 December 2002, revised 4 March 2003,

accepted 27 March 2003)

Eur. J. Biochem. 270, 2274–2286 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03595.x