Loss of the EP₂ prostaglandin E₂ receptor in immortalized human keratinocytes results in increased invasiveness and decreased paxillin expression

ostaglandin E₂ (PGE₂) receptor subtype EP₂, which is coupled to cAMP metabolism, is known to mediate proliferation of primary human keratinocytes in vitro. The effect of gain or loss of EP₂ receptors in immortalized human keratinocytes (HaCat cells) was examined. HaCat keratinocytes were transfected with sense or anti-sense constructs of the EP₂ receptor. Loss or gain of EP₂ expression was documented by immunoblot and associated changes in agonist-stimulated cAMP production. Loss or gain of EP₂ receptor expression correlated with alterations in plating efficiencies but with modest affects on growth. When cell lines were studied in an organ culture model, antisense clones were highly invasive compared with vector controls and sense transfectants. A marked increase in prostaglandin production is commonly seen in malignant lesions. Because prostaglandin receptors are known to undergo ligand-induced receptor down-regulation, we sought to determine whether EP₂ receptor down-regulation results in increased invasiveness. In vector controls, invasiveness was reproduced by ligand-dependent EP₂ receptor down-regulation as assessed by immunohistochemistry. In addition, loss of EP₂ receptor expression was associated with decreased paxillin expression, a critical component of focal adhesion assembly. Thus, downregulation of EP₂ receptors represents a potential mechanism for neoplastic progression to an invasive phenotype.