Post-transcriptional Regulation of Vascular Endothelial Growth Factor by Hypoxia

doi:10.1074/jbc.271.5.2746

Post-transcriptional Regulation of Vascular Endothelial Growth Factor by Hypoxia (*)

From the (1)Cardiology and
(2)Hematology-Oncology Divisions, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115

^¶To whom correspondence and reprint requests should be addressed:
Brigham and Women's Hospital, LMRC Rm. 222, 221 Longwood Ave., Boston, MA 02115
. Tel.: 617-732-7646; Fax: 617-739-0748.

↵^§ Current address: Whitaker Cardiovascular Institute, Evans Department of Medicine, Boston University School of Medicine, Boston, MA 02118.

Abstract

The major control point for the hypoxic induction of the vascular endothelial growth factor (VEGF) gene is the regulation of the steady-state level of the mRNA. We previously demonstrated a discrepancy between the transcription rate and the steady-state mRNA level induced by hypoxia. This led us to examine the post-transcriptional regulation of VEGF expression. Actinomycin D experiments revealed that hypoxia increased VEGF mRNA half-life from 43 ± 6 min to 106 ± 9 min. Using an in vitro mRNA degradation assay, the half-life of VEGF mRNA 3′-untranslated region (UTR) transcripts were also found to be increased when incubated with hypoxic versus normoxic extracts. Both cis-regulatory elements involved in VEGF mRNA degradation under normoxic conditions and in increased stabilization under hypoxic conditions were mapped using this degradation assay. A hypoxia-induced protein(s) was found that bound to the sequences in the VEGF 3′-UTR which mediated increased stability in the degradation assay. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the hypoxia-induced stabilization of VEGF 3′-UTR transcripts and inhibited hypoxia-induced protein binding to the VEGF 3′-UTR. These findings demonstrate a significant post-transcriptional component to the regulation of VEGF.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants T32HL07604 and 1KO8HL03405-01 (to A. P. L.), 1F32HL08838-02 (to N. S. L.), and DK45098 (to M. A. G.), an American Heart Association Grant-in-Aid (to M. A. G.), and an American Heart Association Established Investigator Award (to M. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

VEGF

vascular endothelial growth factor

bp

base pair

Epo

erythropoietin

UTR

untranslated region

DMEM

Dulbecco's modified Eagle's medium

kb

kilobase

EMSA

electromobility shift assay

PCR

polymerase chain reaction

TBE

Tris borate EDTA

IRE

iron responsive element.
↵²Sequences for oligonucleotides described under “Materials and Methods” are denoted as follows: VEGF, upper case letters; bacteriophage T7 RNA polymerase promoter core, italicized, upper case letters; appended sequence for PCR, lower case letters; mutated VEGF sequence, underlined letters.
- Received September 5, 1995.
- Revision received November 16, 1995.