Nitric oxide mediates LC-3-dependent regulation of fibronectin in ductus arteriosus intimal cushion formation

Ductus arteriosus intimal cushion formation is characterized by fibronectin -dependent smooth muscle cell (SMC) migration. Enhanced fibronectin synthes is in ductus SMC is regulated by the interaction of LC-3, a microtubule-ass ociated protein, with an AU-rich element (ARE) in the 3'-untranslated regio n of fibronectin mRNA, facilitating its recruitment to polyribosomes for tr anslation. Since nitric oxide (NO) is implicated in posttranscriptional gen e regulation and is produced in the ductus, we investigated its mechanistic role in LC-3-mediated fibronectin synthesis, NO production was sevenfold h igher in ductus vs. aortic SMC (P<0.005) associated with increased neuronal NO synthase (nNOS) expression. The NOS inhibitor L-NMMA decreased fibronec tin synthesis by similar to 45-50% (P<0.05), whereas the NO donor, SNAP, in creased ductus fibronectin synthesis similar to onefold (P<0.05); neither a gent altered fibronectin mRNA levels. Immunoblotting revealed that SNAP inc reased and GNMMA reduced a membrane-associated phosphorylated form of LC-3. RNA gel mobility shift assays confirmed that NO enhanced LC-3 binding to t he fibronectin mRNA ARE. Our studies indicate a tissue-specific program in the ductus arteriosus whereby elevated nNOS expression and NO production re gulate the posttranscriptional increase in fibronectin synthesis required f or SMC motility.