The involvement of estrogen receptor (ER) and G protein-coupled estrogen receptor (GPR30) in rapid cellular signaling of phytoestrogens in osteoblasts

Abstract

Naringin, Icariin and Genistein are phytoestrogen found in citrus fruits, Herba Epimedii, and legumes, respectively. In vivo studies reported that these three phytoestrogens mimicked estrogen in exerting anabolic effects on bone, making them potential alternatives for the prevention and treatment of osteoporosis. Recently, estrogen was found to be not only acting on genomic signalling by classical nuclear estrogen receptors, ERα and ERβ, but also acting on rapid cellular signalling that involved both ERs and a novel membrane-bound estrogen receptor, GPR30. This study was designed to investigate the anabolic effects of each phytoestrogen on osteoblastic functions and the underlying signaling pathways by which they exert their effects. Rat osteoblastic UMR106 cells were treated with naringin, icariin and genistein for 24hrs or 48 hrs to study their effects on cell proliferation (MTS assay), differentiation (ALP assay) and modulation of osteoclastogenesis (OPG/RANKL mRNA expression ratio). The results indicated that naringin and icariin significantly increased cell proliferation rate, stimulated alkaline phosphatase (ALP) activity, and up-regulated OPG/RANKL ratio in UMR106 cells. Genistein exerted stimulatory effect at a lower concentration (10⁻⁹M to 10⁻¹²M) and inhibitory effect at a higher concentration (10⁻⁵M) on UMR106 cell proliferation and differentiation, but did not alter the OPG/RANKL ratio. The promoting effects of all three phytoestrogens on cell proliferation and differentiation could be abolished by co-treatment with ER antagonist ICI182780 as well as with GPR30 specific antagonist G15, indicated that both ER-mediated genomic pathway and GPR30-mediated cellular signaling might be involved in regulating the action of phytoestrogens in osteoblasts. However, G15 did not affect the modulation of OPG/RANKL ratio by phytoestrogens, suggesting that GPR30 has limited role in modulation of osteoclastogenesis by osteoblasts. As the effects of these compounds in UMR106 cells were similar to that of 17β-estradiol, the molecular actions of each compound via estrogenic signalling pathways in UMR106 cells were investigated. To explore the action of phytoestrogens via ER-mediated signalling pathway, the binding ability of each compound with classical ERs, their abilities to induce ERE-dependent transcription and ligand-independent phosphorylation of ERα at Ser118 in UMR106 cells were examined. Our results indicated that naringin and icariin showed no binding affinity to both ERα and ERβ, while genistein bound both ER isoforms but showed stronger affinity to ERβ. Genistein, but not naringin and icariin, was able to induce ERE-luciferase activity via both ERα and ERβ in UMR106 cells, with stronger induction by ERβ. However, all three compounds were able to significantly induce ERα phosphorylation at Ser118, suggesting that their actions might involve ligand-independent activation of ER in UMR106 cells instead of direct binding with ER to induce ERE transcription. Since the ligand-independent activation of ER is mediated by rapid estrogenic signalling cascades, the molecular actions of phytoestrogens in UMR106 cells via MAPK signalling pathway, PI3K/Akt pathway and intracellular cAMP-dependent signaling were examined. Our results demonstrated that all three compounds were able to activate MAPK signalling through stimulating phosphorylation of ERK-1/2 as rapid as 5 minutes without altering MEK phosphorylation. Whereas 17β-estradiol and genistein inhibited the basal phosphorylation of Akt and downregulated protein expression of Akt and PI3K, naringin and icariin were able to activate Akt in the PI3K/Akt signaling. On the other hand, instead of stimulating cAMP production, treatment with 17β-estradiol and all compounds for 10 minutes demonstrated no effect or even weakly inhibit the intracellular cAMP content and unable to induce CRE-dependent gene transcription in UMR106 cells. To investigate the role of GPR30 as an estrogen receptor in the actions of phytoestrogens in UMR106 cells, G15 were pre-incubated for 20 minutes before treatment with the compounds. While pretreatment with ICI182780 completely abolish the stimulation of ERE-dependent transcription by genistein via both ERα and ERβ, pretreatment with G15 only partially inhibited ERE-dependent transcription via ERα but not ERβ, indicating the presence of interrelationship between ERα and GPR30. Surprisingly, pretreatment with G15 not only failed to abolish the stimulating effects of phytoestrogens on ERK-1/2 phosphorylation, ERα phosphorylation at Ser118, and Akt phosphorylation, but elevated their basal phosphorylation as well as promoted the effects of phytoestrogens treatment. The results suggest that the estrogenic signaling of phytoestrogens in UMR106 cells does not require GPR30 activation and that GPR30 might play a different role in regulating the molecular actions of estrogenic compounds in osteoblasts. In conclusion, naringin, icariin and genistein demonstrated anabolic effects on osteoblast functions, and their estrogenic actions are ER-dependent and regulated by rapid cellular signalling via MEK/ERK and PI3K/Akt activation. GPR30 is involved in the effects of phytoestrogens but has a distinct role in the estrogenic signalling mechanism in osteoblast.