VEGF gene expression is regulated post-transcriptionally in macrophages.
The macrophage is critical to the innate immune response and contributes to human diseases, including inflammatory arthritis and plaque formation in atherosclerosis. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that is produced by macrophages. To study the regulation of VEGF production in macrophages we show that stimulation of monocyte-macrophage-like RAW-264.7 cells by lipopolysaccharide (LPS) increases expression of VEGF mRNA and protein. Three alternative splicing VEGF mRNA isoforms are produced, and the stability of VEGF mRNA increases following cellular activation. To study post-transcriptional regulation of the VEGF gene the 3'-untranslated region (3' UTR) was introduced into the 3' UTR of the luciferase gene in a reporter construct. In both RAW-264.7 cells and thioglycollate-elicited macrophages, the 3' UTR sequence dramatically reduces reporter expression. Treatment with activators of macrophages, including LPS, lipoteichoic acid, and VEGF protein, stimulates expression of 3' UTR reporters. Finally, mapping studies of the 3' UTR of VEGF mRNA show that deletion of the heterogeneous nuclear ribonucleoprotein l binding site affects basal reporter expression in RAW-264.7 cells, but does not affect reporter activation with LPS. Together these results demonstrate that a post-transcriptional mechanism contributes to VEGF gene expression in activated macrophage cells.
Du
Min
M
Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH, and Veterans Administration Research Service, White River Junction, VT 05009-0001, USA.
Roy
Kristen M
KM
Zhong
Lihui
L
Shen
Zheng
Z
Meyers
Hannah E
HE
Nichols
Ralph C
RC
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008070:Lipopolysaccharides; D020033:Protein Isoforms; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A