Tumor necrosis factor-alpha induces fibronectin synthesis in coronary artery smooth muscle cells by a nitric oxide-dependent posttranscriptional mechanism

Postcardiac transplant coronary arteriopathy is associated with tumor necro sis factor-alpha (TNF-alpha) induction of fibronectin-dependent smooth musc le cell (SMC) migration into the subendothelium, resulting in occlusive neo intimal formation. Because expression of inducible nitric oxide synthase (i NOS) is elevated in neointimal formation after transplantation and upregula ted in vascular SMCs by TNF-alpha, we investigated whether TNF-alpha induct ion of fibronectin synthesis in coronary artery (CA) SMCs is mediated by ni tric oxide (NO). TNF-alpha caused a dose-dependent increase in reactive oxy gen and nitrogen intermediates in CA SMCs (P <0.05). This correlated with i ncreased NO production (P <0.05) and fibronectin synthesis (P <0.05). TNF-a lpha induction of Fibronectin synthesis was abrogated by the NOS inhibitor N-G-monomethyI-L-arginine (L-NMMA) (P <0.05) or the flavonoid-containing en zyme inhibitor diphenyleneiodonium (DPI) (P <0.05) and reproduced with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (P <0.05). Northern blotti ng showed no effect of TNF-alpha on steady-state fibronectin mRNA levels. T NF-alpha increased expression of light chain 3 (LC-3), a protein shown prev iously to facilitate fibronectin mRNA translation through its interaction w ith an adenosine-uracil rich element (ARE) in the 3 ' -untranslated region of fibronectin mRNA. RNA gel mobility shift and UV cross-linking assays usi ng CA SMC lysates revealed protein binding complexes with radiolabeled olig onucleotide containing the ARE, similar to those generated with recombinant LC-3. One of these complexes increased after TNF-alpha treatment, an effec t inhibited with L-NMMA or DPI. These data demonstrate a novel paradigm whe reby cytokines regulate mRNA translation of extracellular matrix proteins t hrough NO-dependent modulation of RNA binding protein interaction with mRNA .