Effects And Molecular Mechanisms Of 17β-estradiol On Mitophagy In ATDC5 Chondrocytes

Objective:It is well-established that 17β-estradiol is a steroid hormone that plays a critical role in mammalian physiology and pathology.Estrogen receptor（ERs）and17β-estradiol combine to regulate many physiological functions of the reproductive,cardiovascular,nervous,muscular,skeletal and endocrine systems.To date,three main ERs have been identified,i.e.,ERα,ERβand G protein-coupled estrogen receptor-30（GPR30）.The conventional effects of estrogen are mediated by classical nuclear hormone receptors（ERαand ERβ）.Recent studies have identified GPR30 as a critical mediator of rapid signaling in response to estrogen.It has been proven that 17β-estradiol can protect osteoblasts against serum deprivation-induced apoptosis by promoting autophagy through the ER–ERK–mTOR pathway.Recent studies have demonstrated that estrogen exhibits a potentially protective effect on chondrocytes and might alleviate osteoarthritis（OA）.Nevertheless,the molecular mechanisms of the estrogen-mediated protection of chondrocytes against damage remain unclear.Autophagy is a process of the degradation of unnecessary cytoplasmic macromolecules and organelles and is thus a widely present biological phenomena in eukaryotes.Under ordinary conditions,autophagy occurs at a basal rate in cells,but it can be up-regulated in response to environmental stressors and signaling stimuli such as starvation,amino acid depletion,oxidative stress,cell overcrowding,hypoxia and several pharmacological agents.Mitochondria are hardwired to be a major metabolic pathway of the cell.Mitophagy is a type of selective autophagy that is involved in the removal of dysfunctional mitochondria through degradation in the autophagosome-lysosome system.Microtubule-associated protein-1 light chain-3（LC3）is the mammalian orthologue of the yeast autophagy-associated protein ATG8.During the process of autophagy,cytosolic LC3（LC3-I）binds phosphatidylethanolamine to form an LC3-phosphatidylethanolamine conjugate（LC3-II）,which is a good index of the process of autophagy.TOM 20 is a subunit of the translocase of the mitochondrial outer membrane complex that is responsible for recognizing mitochondrial presequences and is considered to be a potential marker of mitophagy.Additionally,some specific antioxidant enzymes that are located in the mitochondria,e.g.,heat shock protein 60（HSP60）,peroxiredoxin 3（Prx3）,and thioredoxin 2（Trx2）,protect mitochondria against ROS-induced damage by catalyzing the reduction of H₂O₂ into water.In a state of stress,cells can activate autophagy through multiple signaling pathways.Among them,the PI3K-Akt-mTOR signaling pathway is considered to be the classic pathway to inhibit autophagy.Recent studies have shown that estradiol can modulate autophagy through the PI3K-Akt-mTOR signaling pathway to reduce the degree of damage to spinal cord injury.However,whether 17β-estradiol regulates mitophagy through the PI3K-Akt-mTOR pathway in ATDC5 chondrocytes remains unclear.Furthermore,the mitogen-activated protein kinases（MAPK）signaling pathway is also closely related to autophagy.MAPK mainly includes p38 kinase,C-Jun N terminal kinase（JNK）,extracellular signal regulated protein kinase（ERK）and so on.In recent years,many studies have shown that the p38 signaling pathway and the JNK signaling pathway play important roles in the process of apoptosis and autophagy in many cells.Among them,p38 can play a dual role in promoting and inhibiting autophagy,and JNK can promote cell autophagy.Whether 17β-estradiol can regulate mitophagy of ATDC5chondrocytes through the p38 signaling pathway and the JNK signaling pathway remains unclear.Recent studies have demonstrated that 17β-estradiol is not only associated with oxidative stress,the inflammatory response and apoptosis but is also associated with autophagy.The neuroprotective effects of 17β-estradiol have been found to be partly related to the suppression of excessive autophagy in a rat spinal cord injury model.Osteoarthritis（OA）is a form of progressive joint inflammation that is characterized by articular cartilage damage.Recent studies have demonstrated that 17β-estradiol exhibits a potential protective function against OA.Nevertheless,the mechanisms by which 17β-estradiol influences chondrocytes remain unclear.The following paper investigates whether17β-estradiol protects ATDC5 chondrocytes by inhibiting mitophagy and whether17β-estradiol can inhibit mitophagy via the PI3K-Akt pathway through the G protein-coupled estrogen receptor-30（GPR30）.Methods:1.Effects of 17β-estradiol and GPR30 on mitophagy in ATDC5chondrocytes（1）Effects of 17β-estradiol on GPR30 mRNA expression in ATDC5 chondrocytesATDC5 chondrocytes were treated with 17β-estradiol（0,10^-99 M,10^-88 M,10^-77 M）.The expressions of GPR30 mRNA were detected by Real-time PCR.（2）Effects of 17β-estradiol on GPR30 protein activity in ATDC5 chondrocytesATDC5 chondrocytes were treated with 17β-estradiol（0,10^-99 M,10^-88 M,10^-77 M）and/or the GPR30 inhibitor（G15）.The activities of the GPR30 protein were detected by western blot.（3）Observation of the distribution of GPR30 protein in ATDC5 cells through immunofluorescence staining.（4）Observation of the effects of 17β-estradiol on the mitophagosomes in ATDC5 cells by a transmission electron microscopy.ATDC5 chondrocytes were treated with 17β-estradiol and/or the GPR30 inhibitor（G15）.The mitophagosomes in ATDC5 chondrocytes were analyzed by a transmission electron microscope.（5）Observation of the effects of 17β-estradiol on the number of autophagic lysosomes in ATDC5 cells through immunofluorescence double staining.2.Effects of 17β-estradiol on the activation of autophagy-related protein in ATDC5chondrocytes via GPR30ATDC5 chondrocytes were treated with 17β-estradiol（0,10^-77 M）.The protein activation of LC3,TOM20 and Hsp60 were detected by western blot.Furthermore,ATDC5 chondrocytes were treated with 17β-estradiol（0,10^-77 M）and/or the GPR30 inhibitor（G15,15μM）and the PI3K inhibitor（LY294002,20μM）.The protein activation of LC3,TOM20 and Hsp60 were detected by western blot.3.The mechanism of 17β-estradiol and GPR30 on mitophagy in ATDC5chondrocytes（1）Detection of the effect of 17β-estradiol on the activation of Akt and mTOR in ATDC5chondrocytesATDC5 chondrocytes were treated with 17β-estradiol(0,10^-7 M)for 24 h.The protein activation of Akt and mTOR were detected by western blot.Furthermore,ATDC5 chondrocytes were treated with 17β-estradiol(0,10^-7 M)and/or the GPR30 inhibitor（G15,15μM）and the PI3K inhibitor（LY294002,20μM）.The protein activation of Akt and mTOR were detected by western blot.（2）Detection of the effect of 17β-estradiol on the activation of p38 in ATDC5chondrocytesATDC5 chondrocytes were treated with 17β-estradiol(0,10^-7 M)for 24 h.The protein activation of p38 was detected by western blot.Furthermore,ATDC5 chondrocytes were treated with 17β-estradiol(0,10^-7 M)and/or the GPR30 inhibitor（G15,15μM）and the p38 inhibitor（10μM）.The protein activation of p38 was detected by western blot.（3）Detection of the effect of 17β-estradiol on the activation of JNK in ATDC5chondrocytesATDC5 chondrocytes were treated with 17β-estradiol（0,10^-77 M）for 24 h.The protein activation of JNK was detected by western blot.Furthermore,ATDC5 chondrocytes were treated with 17β-estradiol（0,10^-77 M）and/or the GPR30 inhibitor（G15,15μM）and the JNK inhibitor（10μM）.The protein activation of JNK was detected by western blot.Results:1.Effects of 17β-estradiol and GPR30 on mitophagy in ATDC5chondrocytesReal-time PCR assays demonstrated that GPR30 mRNA was expressed in ATDC5chondrocytes.17β-estradiol induced a significant increase of GPR30 mRNA levels and GPR30 protein levels in a dose-dependent manner.10^-88 M and 10^-77 M 17β-estradiol induced a significant increase of GPR30 mRNA levels,with the highest level at 10^-77 M.17β-estradiol（10^-77 M）induced a significant increase of GPR30 protein levels.The GPR30 inhibitor（G15,15μM）can inhibit these effects of 17β-estradiol.Compared with the control group,the 17β-estradiol could increase the number of GPR30labeled red fluorescent particles in ATDC5 cytoplasm and increase the number of GPR30protein in the cytoplasm.Compared with the control group,the 17β-estradiol(10^-7 M)significantly reduced the number of autophagosomes in the cytoplasm of ATDC5.Compared with the control group,the 17β-estradiol could reduce the number of fluorescent particles double stained with TOM20 cytoplasmic red fluorescence and LAMP2 green fluorescence in ATDC5 cytoplasm,and reduce the number of mitochondria autophagic lysosomes in the cytoplasm.2.Effects of 17β-estradiol on the activation of autophagy-related protein in ATDC5chondrocytes via GPR30We further examined the activation of LC3,TOM20 and Hsp60 with western blot analysis.When the cells were treated with 17β-estradiol(10^-7 M),the activation of LC3-II was significantly decreased,while the activation of TOM20 and Hsp60 were significantly increased.Furthermore,the GPR30 inhibitor（G15,15μM）and the PI3K inhibitor（LY294002,20μM）can inhibit these effects of 17β-estradiol.3.The mechanism of 17β-estradiol and GPR30 on mitophagy in ATDC5chondrocytesWhen ATDC5 chondrocytes were treated with 17β-estradiol(10^-7 M),the activation of Akt was up-regulated.The GPR30 inhibitor（G15,15μM）and the PI3K inhibitor（LY294002,20μM）can inhibit these effects of 17β-estradiol.When ATDC5 chondrocytes were treated with 17β-estradiol(10^-7 M),the activation of mTOR was up-regulated.The GPR30 inhibitor（G15,15μM）and the mTOR inhibitor（0.1μM）can inhibit these effects of 17β-estradiol.These results indicate that 17β-estradiol could inhibit mitophagy in ATDC5 chondrocytes through the PI3K-Akt-mTOR signaling pathway.When ATDC5 chondrocytes were treated with 17β-estradiol(10^-7 M),the activation of p38 and JNK were not significantly changed,demonstrating that 17β-estradiol could not inhibit mitophagy in ATDC5 chondrocytes through the p38 or the JNK signaling pathways.Conclusions:1.GPR30 mRNA and protein exist in ATDC5 chondrocytes.17β-estradiol improved the mRNA levels of GPR30,and increased the expression level of GPR30protein,and the role is related to 17β-estradiol dose.2.17β-estradiol and the GPR30 receptor decreased the formation of mitophagosomes in the cytoplasm of ATDC5.Therefore,17β-estradiol could inhibite mitophagy of ATDC5chondrocyte through GPR30.3.When ATDC5 chondrocyte were treated with 17β-estradiol,the activation of LC3-II was significantly decreased,while the activation of TOM20 and Hsp60 were significantly increased.Furthermore,the GPR30 inhibitor and the PI3K inhibitor can inhibit these effects of 17β-estradiol.Therefore,17β-estradiol could inhibite mitophagy of ATDC5chondrocyte through GPR30 and PI3K signaling pathway.4.17β-estradiol could up-regulated the activation of Akt and mTOR,while have no significant effects on the the activation of p38 and JNK,demonstrating that 17β-estradiol may inhibit mitophagy of ATDC5 chondrocyte through GPR30 via the PI3K-Akt-mTOR signaling pathway.