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 o f endothelial cytosolic proteins that bind to the 3'-untranslated region (3 '-UTR) of eNOS mRNA and could be involved in eNOS mRNA stabilization. In th e present work, we have characterized the cytosolic proteins that bind to 3 '-UTR eNOS mRNA. An endothelial cytosolic protein (MW 60-kD) specifically b ound 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 l evel of the 60-kD endothelial cytosolic protein. In addition, TNF-a reduced eNOS mRNA levels and this was prevented by coincubation with cycloheximide . Cycloheximide also prevented the binding activity of the endothelial cyto solic 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 incr eased the 60-kD protein levels. Cycloheximide prevented the binding activit y of the cytosolic protein to 3'-UTR eNOS mRNA related to TNF-alpha this ef fect was associated with greater eNOS mRNA levels. Further specific studies are needed to determine the involvement of this 60-kD endothelial cytosoli c protein in the regulation of eNOS mRNA stabilization and in the endotheli al dysfunction associated with cardiovascular diseases.