| Objective:Osteoporosis is an endocrine disease on bone caused due to reduction of bone tissue per unit volume,and the main clinical features include fracture and bone pain.With aging,the incidence and mortality of osteoporosis along with the associated economic burden has increased every year.Hence,osteoporosis becomes a research hotspot.Primary osteoporosis is four times more prevalent in postmenopausal women than in men.With the decrease of estradiol,bone loss increases and the proportion of osteoporosis significantly increases in peri-menopausal women.Estradiol replacement treatment is the main messure for osteoporosis.Estrogen levels may be the main cause of osteoporosis.Estrogen is a kind of steroid with a wide biological activity.It has the physiological function of promoting and maintaining the female genital and secondary sexual characteristics,as well as in the endocrine system,bone growth and maturation,cardiovascular system,limb metabolism,Skin and other aspects.It was shown estrogen mainly affects the proliferation by binding to osteoblasts directly.However,estrogen binds to osteoclasts in a paracrine manner by acting on Fasl which osteoclasts secrete,and induces osteoclast apoptosis through the Fas/Fasl pathway.Therefore,we chose MC3T3-E1 murine osteoblast cell line as experimental subjects to very the effect of estradiol on bone metabolism.Osteoblast apoptosis has been considered as an important origin of osteoposis,but mitophagy,which also act as a cell terminal regulatory pathway,has been not well studied in osteoporosis.Therefore,it is critical to clarify the relationship between estradiol and mitophagy in osteoblasts.G protein coupled receptor 30?GPR30?is a functional membrane receptor which is different from the classic nuclear receptor of estrogen.It is mainly located in the cell membrane,such as mitochondria and Golgi.It becomes to be the focus of the physiological role of estrogen for its widely expression in the hippocampus,the hypothalamus,the uterus,the ovary,the mammary gland,the bone,and the cardiovascular,and other systemic multiple system organs and tissues.It has been shown that GPR30 is expressed in osteoblasts and that estrogen induces osteoblastic proliferation through GPR30,so we conclude that estrogen expresses its biological effects in osteoblasts by activating GPR30,in turn has an impact on bone mass.Osteoblasts apoptosis has been proved to be an important cause of osteoporosis.However,autophagy,which is also a pathway of cell terminal regulation,has not been studied clearly.Mitophagy is a specific autophagy which selectively clears the damaged mitochondria.It is important to clear damaged mitochondria in time to maintain its quantity and quality.Different mitophagy pathways are involved in mitochondrial degradation processes in different germline and different tissues,and play an important regulatory role in the development and progression of many major diseases such as neurodegenerative diseases,cardiovascular diseases,diabetes and tumors.The relationship between autophagy and bone has always been a hot issue,autophagy is involved in the regulation of osteoblasts and osteoclasts,and is closely related to osteogenesis and bone resorption.Therefore,we speculate that the occurrence of osteoporosis may be related to osteoblast mitophagy.The regulatory mechanism of GPR30 involved in signal transduction gradually clear.At present,many researchers have focused on the signal pathways including EGFR/MAPKs pathway,cAMP-PKA pathway,PI3K/Akt pathway and so on.At present,studies have shown that autophagy can be mediated through the ERK pathway and autophagy can be affected by many factors,such as fip200 and ROS.However,no reports have been reported on how GPR30 regulates osteoblast mitophagy.Therefore,we will put forward our hypothesis and conduct further studies through the molecular mechanism of how estrogen-activated GPR30 regulates mitochondrial autophagy and provide a strong theoretical basis for clinical osteoporosis.Methods:Section 1:the expression of GPR30 in MC3T3-E1 cellsThe MC3T3-E1 cells were trained with different concentrations of estradiol(10-9-10-7)for 48h with or without GPR30 specific inhibitor G15.Western blot?Real Time-PCR together with immunofluorescence were taken to messure GPR30 in MC3T3-E1osteoblasts.Section2:the effects of estradiol and GPR30 on mitophagy in MC3T3-E1 cellsThe MC3T3-E1 cells were trained with different concentrations of estradiol(10-9-10-7)for 48h with or without GPR30 specific inhibitor G15.The mitophagosomes of MC3T3-E1 cells were detected by TEM.The co-location of mitophagy related protein Tom20 and lysosome protein Lamp2 were detected by Fluorescence microscope.Western blot was used to detect the expression of mitochondrial autophagy-related proteins LC3,Tom20 and Hsp60.Section 3:the role of ERK1/2 signaling pathway in the regulation of mitophagy by estrogen in INS-1 cellsThe MC3T3-E1 cells were trained with different concentrations of estradiol(10-9-10-7)for 48h with or without GPR30 specific inhibitor G15/ERK signal pathway inhibitor U0126.The co-location of mitophagy related protein Tom20 and lysosome protein Lamp2 were detected by Fluorescence microscope.Western blot was used to detect the expression of mitochondrial autophagy-related proteins LC3,Tom20 and Hsp60.The proliferation were detected by CCK-8 assay.Results:Section 1:the expression of GPR30 in MC3T3-E1 cells?1?The presence of GPR30 was detected in MC3T3-E1 cells.?2?Different concentrations of estradiol(10-9-10-77 M)could up-regulate the activity of GPR30 protein in MC3T3-E1 cells in a dose-dependent way.The effect of 10-77 M estradiol group was statistically significant.The GPR30 specific antagonist of G15(15*10-6 M)could block the effects of estradiol.Section2:the effects of estradiol and GPR30 on mitophagy in MC3T3-E1 cells?1?10-77 M estradiol upregulated the formation of mitochondrial autophagosomes in MC3T3-E1 cells.After the addition of GPR30 specific antagonist,the mitochondrial autophagosome decreased significantly.?2?After 10-77 M estradiol acted on MC3T3-E1 cells,the positive particles co-localization of TOM20 and Lamp2 were increased.The effect of estradiol was blocked by the addition of GPR30 inhibitor.?3?The band of LC3-II?TOM20 and Hsp60 increased in cells of 10-77 M estradiol comparing to control group.After adding G15 the transformation of LC3 from type I to type II was significantly decreased,as well as the expression of TOM20 and Hsp60Section 3:the role of ERK1/2 signaling pathway in the regulation of mitophagy by estrogen in INS-1 cells?1?Estradiol?10-77 M?could regulate the phosphorylation of ERK1/2 in MC3T3-E1cells and the t-ERK1/2 had no obvious change.ERK1/2 inhibitor?U0126,20*10-66 M?and GPR30 antagonist(G15,15*10-6 M)both could block this activation of p-ERK1/2mediated with estradiol.?2?After 10-77 M estradiol acted on MC3T3-E1 cells,the positive particles co-localization of TOM20 and Lamp2 were increased.The effect of estradiol was blocked by the addition of ERK1/2 inhibitor.?3?Estradiol?10-77 M?increased the expression of LC3-II?Tom20 and Hsp60 in MC3T3-E1 cells comparing to control group.The treatment with ERK1/2 inhibitor?U0126,20*10-66 M?decreased the expression of LC3-II protein,as well as the expressions of Tom20 and Hsp60.?4?Estradiol?10-77 M?increased the MC3T3-E1 cells viability comparing to control group.The addition of ERK1/2 inhibitor U0126 and GPR30 antagonist(G15,15*10-6M)decreased the proliferation in those corresponding groups.Conclusions:?1?Osteoblast cells had GPR30 expression.Estradiol regulated the activition of GPR30 protein in a dose dependent way in osteoblast cells.?2?GPR30 was binded with estradiol,and activated GPR30 up-regulated the occurrence of mitophagy in osteoblast cells.?3?Estradiol activated ERK1/2 signaling pathway and the pathway was involved in the process of up-regulating the biological effects of mitophagy.?4?Estradiol protected osteoblast cells from osteoposis by inducing mitophagy. |
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