13 February 2018 : Laboratory Research
Effects of 17β-Estradiol on Mitophagy in the Murine MC3T3-E1 Osteoblast Cell Line is Mediated via G Protein-Coupled Estrogen Receptor and the ERK1/2 Signaling PathwayXiaoqi Sun1ABCDEFG, Xuhao Yang2ABCDEFG, Yuyan Zhao1ABCDEFG*, Yinan Li2ABCDEFG, Lei Guo2ABCDEFG
Med Sci Monit 2018; 24: LBR903-911
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.
Keywords: Estradiol, MAP Kinase Signaling System, Mitochondrial Degradation, Receptors, G-Protein-Coupled
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