Nitric oxide increases lysine 48-linked ubiquitination following arterial injury.

Background: Proteins are targeted for degradation by the addition of a polyubiquitin chain. Chains of ubiquitin linked via lysine 48 (K48) are associated with protein degradation while chains linked via lysine 63 (K63) are associated with intracellular signaling. We have previously shown that nitric oxide (NO) inhibits neointimal hyperplasia in association with increasing the ubiquitination and degradation of UbcH10. The aim of this study is to characterize the effect of arterial injury and NO on K48- or K63-linked ubiquitination of cellular proteins.

Methods: The rat carotid artery balloon injury model was performed. Treatment groups included uninjured, injury alone, injury + proline NONOate (PROLI/NO), and PROLI/NO alone. Arteries were harvested at designated time points and sectioned for immunohistochemical analysis of K48- and K63-linked ubiquitination or homogenized for protein analysis. Vascular smooth muscle cells (VSMC) harvested from rat aortae were exposed to the NO donor diethylenetriamine NONOate (DETA/NO). Protein expression was determined by Western blot analysis, or immunoprecipitation and Western blot analysis.

Results: Arterial injury increased K48-linked ubiquitination in vivo. The addition of PROLI/NO following injury caused a further increase in K48-linked ubiquitination at 1 and 3 d, however, levels returned to that of injury alone by 2 wk. Interestingly, treatment with PROLI/NO alone increased K48-linked ubiquitination in vivo to levels similar to injury alone. There were lesser or opposite changes in K63-linked ubiquitination in all three treatment groups. DETA/NO increased K48-linked ubiquitination in VSMC in vitro but had minimal effects on K63-linked ubiquitination. Low doses of DETA/NO decreased K48-linked ubiquitination of cyclin A and B, while high doses of DETA/NO increased K48-linked ubiquitination of cyclin A and B. Minimal changes were seen in K63-linked ubiquitination of cyclin A and B in vitro.

Conclusions: Arterial injury and NO increased K48-linked ubiquitination in vivo and in vitro. Remarkably, minimal changes were seen in K63-linked ubiquitination. These novel findings provide important insights into the vascular biology of arterial injury and suggest that one mechanism by which NO may prevent neointimal hyperplasia is through regulation of protein ubiquitination.