Expression And Function Of Gpr30 In Human Chondrocytes

Purpose: Estrogens play an important role in cartilage homeostasis. However, the role of a non-classical estrogen receptor, the G protein-coupled estrogen receptor (GPER or GPR30), as a mediator of the effects of estrogens in articular tissues is unknown. Thus, the purpose of this work was to evaluate the expression and function of GPR30 in primary adult human articular chondrocytes. Methods: Human cartilage collected from the distal femoral condyles of multi-organ donors at the Bone and Tissue Bank of the University and Hospital Center of Coimbra (CHUC) was used. All procedures were approved by the Ethics Committee of CHUC (authorization number: 0204-8654-DC, date: 12-16-2013). To characterize the expression of GPR30, immunohistochemistry, flow-cytometry, RT-qPCR and western blot were used. The MCF-7 cell line was used as positive control for expression of GPR30. GPR30-dependent signaling was evaluated as the phosphorylation levels of Akt and ERK1/2 induced by known GPR30 agonists (17β-estradiol, G-1 and ICI-182780) by western blot (WB). Results: Immunohistochemistry of articular cartilage sections shows that GPR30 is expressed in human chondrocytes in situ. Expression of GPR30 is also observed in freshly isolated and cultured human chondrocytes, as demonstrated by flow-cytometry and RT-qPCR and in chondrocyte extracts by WB. Nonetheless, the pattern of bands detected by two distinct antibodies does not overlap, suggesting that the proteins detected represent partially degraded forms of the receptor. Treatment with GPR30 agonists did not induce Akt or ERK1/2 phosphorylation, suggesting that GPR30 is not functional in human chondrocytes. Conclusions: The Results obtained suggest that GPR30 is present in human chondrocytes. Nevertheless, these cells probably express a partially degraded form of the receptor, which can explain the lack of functional activity observed. Therefore, the benefit of estrogen-induced responses in human chondrocytes are unlikely mediated by GPR30. However, further studies are required to evaluate the influence of gender, age or degree of cartilage damage on GPR30 expression and function in human chondrocytes.