Estrogen induced inhibitory effect on urothelial cell proliferation is mediated by GPR30

Previous studies demonstrated that estrogen induces urothelial cell proliferation through ERα and ERβ but some putative plasma membrane‐associatedestrogen binding sites may mediate an inhibitory effect on urothelial proliferation. GPR30, an orphan G protein‐coupled receptor, has been identified as a candidate for non‐genomic estrogen signaling and is expressed at high levels in urothelial cells. Primary cultures of normal human bladder urothelial cells and T24 bladder carcinoma cells were used in these studies. Increased GPR30 expression was induced by transfection with a pcDNA3‐GPR30‐GFP construct and inhibited by 27mer siRNA against GPR30. Expression of GPR30, c‐fos, c‐jun, and cyclin D1 was determined by real‐time PCR and immunoblotting. Cell proliferation was determined by 3 H‐thymidine incorporation. Primary urothelial and T24 cells constitutively expressed high levels of GPR30, and this was selectively inhibited by siRNA. In both cell types, E2 induced a 70–120 % increase, but membrane‐impermeable E2‐BSA induced a 30–50 % decrease, in DNA synthesis. Expression of c‐fos, c‐jun, and cyclin D1 mRNA and protein was significantly up‐regulated by E2. In cells overexpressing GPR30, E2‐induced DNA synthesis was significantly decreased, and E2 failed to induce c‐fos, c‐jun, and cyclin D1 expression. Inhibition of GPR30 by siRNA caused a higher proliferative response to E2 treatment and reversed the inhibition caused by E2‐BSA. The inhibitory effect on urothelial cell proliferation induced by E2 treatment is at least partially mediated by GPR30. Supported by NIH R01 DK066349