Accumulation of hypoxia-inducible factor-1alpha through a novel electrophilic, thiol antioxidant-sensitive mechanism.

15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a peroxisome-activated proliferator receptor-gamma (PPARgamma) agonist which contains an alpha,beta-unsaturated electrophilic ketone involved in nucleophilic addition reactions to thiols. Here we studied its effect on hypoxia-inducible factor-1alpha (HIF-1alpha) in human proximal tubular cells HK-2. 15d-PGJ(2) induced stabilization of HIF-1alpha protein, without affecting HIF-1alpha mRNA levels or proteasome activity, leading to its nuclear accumulation and activation of HIF-induced transcription. Accumulation of HIF-1alpha was unaffected by selective PPARgamma blockade nor mimicked by the PPARgamma agonists ciglitazone and 9,10-dihydro-15d-PGJ(2). N-acetylcysteine, reduced glutathione (GSH) or dithiothreitol (i.e. agents that act as thiol reducing agents and/or increase the GSH content), but not reactive oxygen species (ROS) scavengers, prevented 15d-PGJ(2)-induced HIF-1alpha accumulation whereas the inhibitor of GSH synthesis buthionine sulfoximine cooperated with 15d-PGJ(2) to accumulate HIF-1alpha. Finally, HIF-1alpha expression was increased by the electrophilic alpha,beta-unsaturated compounds acrolein and PGA(2), but not by 9,10-dihydro-15d-PGJ(2), which lacks the electrophilic cyclopentenone moiety. Taken together, these results point out to a new mechanism to increase pharmacologically the cell levels of HIF-1alpha through the electrophilic reaction of alpha,beta-unsaturated ketones with thiol groups.