Cyclooxygenase (Cox)-2 regulating transcriptional and post-transcriptional activation of aromatase is involved in Leydig cell tumor proliferation

Our recent studies have revealed that estrogens stimulate an autocrine mechanism determining Leydig tumor cell proliferation. Estrogen over-production is due to an elevated SF1 expression and CREB phosphorylation, both inducing aromatase overexpression. In this study, we investigated the role of cyclooxygenase (COX)-2 in CREB dependent-aromatase expression in Leydig tumor cells. We found that COX-2 is expressed in rat and human Leydigiomas as well as in rat Leydig tumor cell line R2C, but not in normal testis. In R2C cells the use of a specific inhibitor for COX-2 (NS398) as well as COX-2 siRNA were able to reduce aromatase expression as a consequence of a decreased CREB activation and recruitment to the PII promoter of the aromatase gene. COX-2 is responsible for prostaglandin E2 (PGE2) synthesis, that binds the PGE2 receptor EP4 activating PKA and ultimately CREB. In fact, the use of inhibitors for EP4 (AH23848), and PKA (H89) reproduced the same effects observed in the presence of NS398, decreasing CREB activation and consequently aromatase expression and activity. Immunoprecipitation experiments demonstrated also the ability of these inhibitors to decrease enzymatic activity interfering with aromatase phosphorylation on tyrosine residues. Indeed, this study demonstrated that the altered expression of COX-2 induces a PGE2/PKA/CREB pathway responsible for transcriptional and post-transcriptional activation of aromatase determining an increase in estrogen production, estrogen-dependent cell cycle regulator cyclin E and Leydig cell tumor proliferation. The observation that COX-2 signalling inhibitors decrease R2C cell proliferation suggests their potential application as new adjuvant therapies for Leydig tumor cell treatment.