Tài liệu Báo cáo khóa học: A multi-protein complex containing cold shock domain (Y-box) and

A multi-protein complex containing cold shock domain (Y-box)

and polypyrimidine tract binding proteins forms on the vascular

endothelial growth factor mRNA

Potential role in mRNA stabilization

Leeanne S. Coles1,*, M. Antonetta Bartley1,*, Andrew Bert1

, Julie Hunter1

, Steven Polyak2

, Peter Diamond1

,

Mathew A. Vadas1,3 and Gregory J. Goodall1,3

1

Division of Human Immunology, The Hanson Institute, Institute of Medical and Veterinary Science; 2

Division of Biochemistry,

Department of Molecular Biosciences, The University of Adelaide; 3

Department of Medicine, The University of Adelaide,

North Terrace, Adelaide, South Australia, Australia

Vascular endothelial growth factor (VEGF) is a key regu￾lator of angiogenesis and post-transcriptional regulation

plays a major role in VEGF expression. Both the 5¢- and

3¢-UTR are required for VEGF post-transcriptional regu￾lation but factors binding to functional sequences within

the 5¢-UTR have not been fully characterized. We report

here the identification of complexes, binding to the VEGF

mRNA 5¢- and 3¢-UTR, that contain cold shock domain

(CSD) and polypyrimidine tract binding (PTB) RNA

binding proteins. Analysis of the CSD/PTB binding sites

revealed a potential role in VEGF mRNA stability, in both

noninduced and induced conditions, demonstrating a gen￾eral stabilizing function. Such a stabilizing mechanism had

not been reported previously for the VEGF gene. We further

found that the CSD/PTB-containing complexes are large

multiprotein complexes that are most likely preformed in

solution and we demonstrate that PTB is associated with the

VEGF mRNA in vivo. Complexformation between CSD

proteins and PTB has not been reported previously. Analysis

of the CSD/PTB RNA binding sites revealed a novel CSD

protein RNA recognition site and also demonstrated that

CSD proteins may direct the binding of CSD/PTB com￾plexes. We found the same complexes binding to an RNA￾stabilizing element of another growth factor gene, suggesting

a broader functional role for the CSD/PTB complexes.

Finally, as the VEGF gene is also regulated at the tran￾scriptional level by CSD proteins, we propose a combined

transcriptional/post-transcriptional role for these proteins in

VEGF and other growth factor gene regulation.

Keywords: cold shock domain proteins; Y-boxprotein;

polypyrimidine tract binding protein; mRNA stabilization;

vascular endothelial growth factor.

VEGF is an essential regulator of angiogenesis that acts on

vascular endothelial cells to induce proliferation and

promote cell migration [1–3]. Disregulated VEGF expres￾sion is implicated in a number of diseases that are

characterized by abnormal angiogenesis [1–6]. In the case

of solid tumors, the overexpression of VEGF, produced in

response to activated oncogenes, growth factors or low

oxygen conditions (hypoxia), plays a major role in promo￾ting tumor angiogenesis and progression [1–3,7]. Both the

cancer cells themselves and nontumor support cells, such as

fibroblasts, are sources of VEGF [8]. In contrast, in the case

of coronary artery disease, inadequate VEGF expression

rather than VEGF overexpression, plays a role in disease

progression. A number of cell types, including cardiac

myocytes, fibroblasts and endothelial cells produce VEGF

in response to hypoxia, but this natural response is not

sufficient to prevent the further progression of heart disease

[9–11]. It is therefore important to understand the mecha￾nisms of VEGF regulation to develop means to control

VEGF expression.

Post-transcriptional regulation plays a major role in

VEGF expression, with regulation occurring at the level of

splicing, mRNA stability and translation [2,7]. The VEGF

mRNA is normally unstable and its stability is increased in

response to cytokines and stress conditions such as hypoxia

[7,11–14]. Regions in both the 5¢- and 3¢-UTR have been

shown to be involved in VEGF mRNA stabilization

[7,12,13,15–18]. The presence of an internal ribosome entry

site (IRES) in the VEGF 5¢-UTR ensures continual

translation of the VEGF mRNA in stress conditions that

normally decrease cap-dependent translation [19–21]. Little

is known about the factors involved in VEGF post￾transcriptional regulation. Factors such as HuR and

hnRNPL have been implicated in hypoxic stability via their

Correspondence to L. S. Coles, Division of Human Immunology,

The Hanson Institute, Institute of Medical and Veterinary Science,

Frome Road., Adelaide, South Australia, 5000, Australia.

Fax: + 61 88 2324092, Tel.: + 61 88 2223432,

E-mail: [email protected]

Abbreviations: CSD, cold shock domain; IRES, internal ribosome

entry site; VEGF, vascular endothelial growth factor.

*These authors contributed equally to this work.

(Received 16 October 2003, revised 14 December 2003,

accepted 16 December 2003)

Eur. J. Biochem. 271, 648–660 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2003.03968.x