Down-Regulation of COX-2 by Beta2-Adrenergic Receptor-Based Pepducins

Cyclooxygenase 2 (COX-2) is one of two main isoforms that highly abundant in various pathologies, such as inflammation and cancer. Overexpression of COX-2 is characteristic of many chronic inflammatory diseases therefore reducing its levels may be a new strategy to fight these diseases. Previous results in our lab showed that certain G protein-coupled receptors (GPCRs) down-regulate the levels of COX-2 in a pathway that increases its levels of ubiquitination and accelerates its degradation via the proteasome. Here we show that co-expression of COX-2 and the β₂ adrenergic receptor (β₂AR) in HEK293 causes a significant reduction in COX-2 levels. This effect is specific to COX-2 because co expression of the ubiquitously expressed COX-1 with β₂AR is not affected.To further investigate which parts of the receptor are involved in the effect on COX-2 we screened 32 lipidated peptides (termed `pepducins`) derived from the three intracellular loops of the receptor for their ability to affect COX-2 expression. To this end, HEK293 were transfected with YFP-COX-2 in the presence of each pepducin, and the effect was evaluated 24 h later by flow cytometry.Of the 32 pepducins, most did not affect COX-2 (N=19). Three pepducins in ICL-2 elevated COX-2 by 1.5 fold or more. Three pepducins in ICL-1, two in ICL-2 and five in ICL-3 lowered COX-2 expression by 20% or more. Using western blot, we confirmed that the pepducins derived from ICL-3 lower COX-2 expression in the nanomolar range.

In conclusion, our findings indicated that there are a specific parts in the intracellular loops of the receptor that are involved in the regulation of COX-2, which may be good candidates for development of next generation therapeutics.