Evidence that an endothelial cytosolic protein binds to the 3'-untranslated region of endothelial nitric oxide synthase mRNA.
Changes in the endothelial nitric oxide synthase (eNOS) expression could be involved in the endothelium-dependent vasorelaxing dysfunction associated with cardiovascular diseases. We have recently demonstrated the existence of endothelial cytosolic proteins that bind to the 3'-untranslated region (3'-UTR) of eNOS mRNA and could be involved in eNOS mRNA stabilization. In the present work, we have characterized the cytosolic proteins that bind to 3'-UTR eNOS mRNA. An endothelial cytosolic protein (MW 60-kD) specifically bound to 3'-UTR eNOS mRNA as determined by a cross-linking assay followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The endothelial cytosolic protein recognized a cytidine (C)-rich region within 3'-UTR eNOS mRNA. Furthermore, tumor necrosis factor-alpha (TNF-alpha) increased the level of the 60-kD endothelial cytosolic protein. In addition, TNF-alpha reduced eNOS mRNA levels and this was prevented by coincubation with cycloheximide. Cycloheximide also prevented the binding activity of the endothelial cytosolic protein to 3'-UTR eNOS mRNA. In summary, these data suggest that a 60-kD endothelial cytosolic protein binds to 3'-UTR eNOS mRNA. TNF-alpha increased the 60-kD protein levels. Cycloheximide prevented the binding activity of the cytosolic protein to 3'-UTR eNOS mRNA related to TNF-alpha; this effect was associated with greater eNOS mRNA levels. Further specific studies are needed to determine the involvement of this 60-kD endothelial cytosolic protein in the regulation of eNOS mRNA stabilization and in the endothelial dysfunction associated with cardiovascular diseases.
Sánchez de Miguel
L
L
Nephrology, Hypertension and Cardiovascular Research Laboratory, Fundación Jiménez Díaz, Madrid, Spain.
Alonso
J
J
González-Fernández
F
F
de la Osada
J
J
Montón
M
M
Rodríguez-Feo
J A
JA
Guerra
J I
JI
Arriero
M M
MM
Rico
L
L
Casado
S
S
López-Farré
A
A
D020413:3' Untranslated Regions / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D020413:3' Untranslated Regions; D011506:Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III