Inhibition of Cyclooxygenase-2 Down-regulates Aromatase Activity and Decreases Proliferation of Leydig Tumor Cells

Abstract

Our recent studies have revealed that estrogens stimulate an autocrine mechanism determining Leydig tumor cell proliferation. Estrogen overproduction is due to an elevated steroidogenic factor-1 (SF-1) expression and cAMP-response element-binding protein (CREB) phosphorylation, both inducing aromatase overexpression. Although we have shown that increased SF-1 expression depends mainly on higher local insulin-like growth factor I production, the mechanisms and factors determining increased CREB activation in Leydig tumor cells are not completely understood. In this study, we investigated the role of cyclooxygenase-2 (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 the rat Leydig tumor cell line R2C, but not in normal testis. Our data indicate that in R2C cells the COX-2-derived prostaglandin E2 (PGE2) binds the PGE2 receptor EP4 and activates protein kinase A (PKA) and ultimately CREB. Inhibitors for COX-2 (NS398), EP4 (AH23848), and PKA (H89) decreased aromatase expression and activity as a consequence of a decreased phosphorylated CREB recruitment to the PII promoter of the aromatase gene. The COX-2/PGE2/PKA pathway also seems to be involved in aromatase post-translational activation, an observation that requires further studies. The reduction in aromatase activity was responsible for a drop in estrogen production and subsequent reduction in cyclin E expression resulting in a decrease in tumor Leydig cell proliferation. Furthermore, COX-2 silencing caused a significant decrease in CREB phosphorylation, aromatase expression, and R2C cell proliferation. These novel findings clarify the mechanisms involved in the growth of Leydig cell tumors and should be taken into account in determining new therapeutic approaches.

Cyclooxygenase and prostaglandins in somatic cell populations of the testis

“…Furthermore, production of testosterone by decapsulated mouse testes is significantly inhibited by adding some PGs (PGA 1 , PGA 2 , and PGE 1 ) to the incubation medium (Bartke et al 1976). On the other hand, COX2 seems to be involved in aromatase post-translational activation and increased cell proliferation in the rat Leydig tumor cell line R2C (Sirianni et al 2009).…”

The Prostanoid EP4 Receptor and Its Signaling Pathway

“…Among them, cAMP-response element-binding protein (CREB), a transcription factor, is one of the major downstream targets of PKA, which controls cellular functions via synthesis of a wide variety of proteins (Shaywitz and Greenberg, 1999). EP4-mediated CREB activation was reported in colon epithelial cells (Srivastava et al, 2012), dorsal root ganglion neurons (Cruz Duarte et al, 2012), Leydig tumor cells (Sirianni et al, 2009), and breast cancer cells (Subbaramaiah et al, 2008). Other signaling pathways in addition to CREB are activated via PKA.…”

Teasing out the role of aromatase in the healthy and diseased testis

“…Suffice to say, deciphering the specific roles of aromatase and estrogens and their underlying cellular and molecular mechanisms in the healthy testis, would be extremely valuable in advancing our understanding of how these mechanisms become compromised in the diseased testis, to ultimately reveal potential therapies for aromatase driven testicular disease. the CRE: (1) as a result of overexpression of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) stimulates the cAMP/PKA dependent pathway in mouse Leydig cell tumors 121 and (2) in a similar manner, the xenoestrogen BPA increases rat Leydig cell aromatase expression by upregulating COX-2. 122 Furthermore, a recent study has shown elegantly that aromatase expression is regulated by 1α,25(OH) 2 vitamin D 3 (1,25D) in day 30 rat Sertoli cells cultures with the data suggesting the existence of a genomic and non-genomic activation of the vitamin D receptor involving at least the PKA pathway.…”