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BACKGROUND Osteoarthritis is a progressive inflammatory joint disease resulting in damage to articular cartilage. G-protein coupled estrogen receptor (GPER/GPR30) activates cell signaling in response to 17β-estradiol, which can be blocked by the GPR30 agonist, G15, an analog of G-1. The aims of this study were to investigate the effects of 17β-estradiol on the expression of G-protein coupled estrogen receptor (GPER/GPR30) on mitophagy and the PI3K/Akt signaling pathway in ATDC5 chondrocytes in vitro. MATERIAL AND METHODS Cultured ATDC5 chondrocytes were treated with increasing concentrations of 17β-estradiol with and without G15, p38 inhibitor (SB203580), JNK inhibitor (SP600125), PI3K inhibitor (LY294002, S1737), and mTOR inhibitor (S1842). Expression of GPER/GPR30 and components of the PI3K/Akt pathway in cultured ATDC5 chondrocytes were detected by immunofluorescence (IF) staining, Western blot, and real-time polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) and IF were used to detect mitophagosomes. Expression of LC-3, LAMP2, TOM20, Hsp60, p-Akt, p-mTOR, p-p38, and p-JNK was investigated by Western blot. Proliferation and viability of the ATDC5 chondrocytes were determined using BrdU and MTT assays. RESULTS In 17β-estradiol-treated ATDC5 chondrocytes, increased expression of GPER/GPR30 was found, but fewer mitophagosomes were observed, and decreased numbers of TOM20-positive granules were co-localized with decreased LAMP2 and increased expression levels of TOM20, Hsp60, p-Akt, and p-mTOR, and reduced expression of LC3-II, were found. In 17β-estradiol-treated ATDC5 chondrocytes, the proliferation and viability of the 17β-estradiol-treated ATDC5 chondrocytes were significantly elevated. CONCLUSIONS Treatment with 17β-estradiol protected ATDC5 chondrocytes against mitophagy via the GPER/GPR30 and the PI3K/Akt signaling pathway.

BACKGROUND Osteoporosis is associated with 17β-estradiol deficiency. The G protein-coupled receptor 30 (GPR30) is known to be an estrogen-responsive receptor, but its role in the degradation of mitochondria in osteoblasts by autophagy, or mitophagy, remains unclear. The aim of this in vitro study was to evaluate the effects of 17β-estradiol, GPR30, and its signaling pathway, on mitophagy in the murine MC3T3-E1 osteoblast cell line. MATERIAL AND METHODS In the murine MC3T3-E1 osteoblast cell line, cells were treated with 17β-estradiol, or G15, a selective GPR30 antagonist, or U0126, a mitogen-activated protein (MAP) kinase (ERK1/2) inhibitor, or with vehicle as control. The expression of GPR30 was determined by Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and confocal immunofluorescence imaging. Cell morphology and mitochondrial autophagosomes were identified using transmission electron microscopy (TEM). Phosphorylation of the mitophagy markers, heat shock protein 60 (Hsp60), translocase of outer membrane (Tom)20, and microtubule-associated protein 1A/1B-light chain 3 (LC3) were determined by Western blot, and cell proliferation was determined using the bromodeoxyuridine (BrdU) assay. RESULTS The optimum concentration of 17β-estradiol that resulted in GPR30 expression in MC3T3-E1 cells was 10^-7 M, which led to the accumulation of mitochondrial autophagosomes and increased protein phosphorylation levels of Hsp60, Tom20, and LC3. In cells pretreated with G15 or U0126, 17b-estradiol treatment did not increase mitophagy in MC3T3-E1 cells. CONCLUSIONS In murine osteoblasts cultured in vitro, treatment with 17β-estradiol resulted in the expression of GPR30 and enhanced mitophagy through the GPR30 and ERK1/2 signaling pathway.