Mechanism of Caveolin-1 Degradation

In the present study, we tested the hypothesis that oxidative/nitrosative stress promotes caveolin-1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction in patients with idiopathic pulmonary artery hypertension (IPAH). It has been shown that there is a decrease in Cav-1 in IPAH patient samples, but the mechanism of this loss has not been elucidated. Our work aimed to reveal the mechanism of Cav-1 loss that is seen in several diseases, including IPAH. When we analyzed the human pulmonary artery endothelial cells (PAECs) from patients with IPAH, we observed reduced Cav-1 expression and endothelial nitric oxide synthase (eNOS) hyperphosphorylation. This is consistent with the theory that Cav-1 is a regulator of eNOS. In the IPAH samples, Cav-1 protein levels were decreased despite increased expression of Cav-1 messenger ribonucleic acid (mRNA). In control human lung endothelial cells, tumor necrosis factor alpha (TNF-α)-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 cysteine-156 (C156) was associated within 5 minutes with Src displacement and activation, Cav-1 tyrosine-14 (Y14) phosphorylation, and destabilization of Cav-1 oligomers, which was blocked by inhibiting either eNOS with the NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) or Src with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Prolonged (72 hours) stimulation with the NO donor DETA-NONOate reduced oligomerized and total Cav-1 levels by 40-80%, similar to that observed in IPAH patient samples. Inhibition of the proteasome and Src prevented Cav-1 degradation in cells treated with DETA-NONOate, suggesting that Cav-1 SNO and sustained phosphorylation promote ubiquitination and degradation by the proteasome. In transduced human embryonic kidney (HEK) cells, we observed reduced oligomerization of cysteine 156 to serine (C156S) Cav-1 mutant, indicating this mutation might mimic the effect of nitrosation. Mass spectrometry analysis revealed ubiquitination of Cav-1 on lysine-86 (K86). Thus, reduced Cav-1 expression due to oxidative stress-induced Cav-1 SNO of C156, Src activation, and phosphorylation of Cav-1 Y14, ubiquitination of Cav-1 K86, and ultimately proteosomal degradation of Cav-1 is associated with eNOS hyperactivation and loss of caveolae in PAECs from patients with IPAH. These studies indicate chronic inflammation and sustained oxidative stress may promote endothelial cell dysfunction via degradation of Cav-1.