跨膜蛋白208可影响人软骨细胞的自噬和线粒体功能
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摘要
背景:软骨细胞自噬水平下降是骨关节炎发病的重要机制之一。自噬抑制基因跨膜蛋白208在晚期膝骨关节炎患者软骨组织中表达升高,该基因可能通过抑制软骨细胞自噬促进骨关节炎发展。目的:通过体外骨关节炎模型,观察软骨细胞跨膜蛋白208基因表达对自噬和线粒体功能的影响。方法:软骨组织来源于膝关节表面置换患者术中废弃软骨,非负重面轻度病变软骨(OuterbridgeⅠ-Ⅱ级)作为早期组,负重面重度病变软骨(OuterbridgeⅢ-Ⅳ级)作为晚期组,检测不同软骨组织跨膜蛋白208含量。实验方案符合北京大学第一医院对研究的相关伦理要求,软骨组织供者及其家属在充分了解治疗方案的前提下签署了"知情同意书"。取第4代人正常关节软骨细胞,通过白细胞介素1β刺激人正常软骨细胞诱导骨关节炎体外模型;另外加入自噬激活剂雷帕霉素或自噬抑制剂3-MA预处理软骨细胞后,再加入白细胞介素1β刺激12,24,48h,观察软骨细胞状态。RealTimePCR检测不同病变软骨组织跨膜蛋白208基因的表达;Western Blot检测自噬相关蛋白轻链蛋白3B、P62表达评价自噬水平;AnnexinV-FITC法评估软骨细胞的凋亡情况;MitoSOX Red dye和JC-1染料荧光显色评价线粒体损伤情况。结果与结论:①骨关节炎患者重度退变软骨组织跨膜蛋白208表达升高;②用白细胞介素1β刺激软骨细胞12,24和48 h后,软骨细胞跨膜蛋白208表达水平先降后升,自噬水平先升后降,凋亡水平逐渐升高,线粒体损伤逐渐加重;③雷帕霉素能明显降低跨膜蛋白208的表达量,提高自噬水平,降低白细胞介素1β刺激后软骨细胞的凋亡和线粒体损伤;④结论:跨膜蛋白208可抑制软骨细胞自噬,其表达增高可能导致软骨细胞应激状态下的损伤增加。
BACKGROUND: Decreased level of autophagy in chondrocytes is an important mechanism of osteoarthritis.The autophagy inhibitory gene transmembrane protein 208 is elevated in late-stage osteoarthritis cartilage, and this gene may promote the development of osteoarthritis by inhibiting autophagy of chondrocytes.OBJECTIVE: To investigate the effects of chondrocyte transmembrane protein 208 gene on autophagy and mitochondrial function in osteoarthritis model in vitro.METHODS: The cartilage tissue was from the discarded cartilage of patients undergoing knee arthroplasty. The mild lesion cartilage on non-weight-bearing face(Outerbridge I-II grade) was as early-term group, and the severe lesion cartilage on heavy-weight surface(Outerbridge III-IV grade) was as the late-term group. The content of transmembrane protein 208 in different cartilage tissues was detected.The study was in accordance with the ethical criteria of Peking University First Hospital, and the cartilage donors and their family members signed an informed consent form. Passage 4 normal human chondrocytes were obtained and then stimulated by interleukin-1β to establish the in vitro osteoarthritis. The chondrocytes were pretreated by autophagy activator rapamycin or autophagy inhibitor 3 MA, followed by interleukin-1β added for 12, 24 and 48 hours to observe the morphology of chondrocytes. The content of transmembrane protein 208 gene was detected by real-time PCR. Western blot assay was used to detect autophagy-associated protein light chain proteins 3 B and P62 to evaluate autophagy levels. The apoptosis of chondrocytes was detected by Annexin V-FITC method. Mitochondrial damage was assessed by fluorescent detection with MitoSOX Red dye and JC-1 dye.RESULTS AND CONCLUSION:(1) The expression of transmembrane protein 208 was significantly increased in severe osteoarthritis cartilage.(2) After stimulation with interleukin-1β for 12, 24 and 48 hours, the expression level of transmembrane protein 208 in chondrocytes decreased initially, and then increased. The level of autophagy rose initially and then fell. The apoptotic level gradually increased, and mitochondrial damage gradually became severe.(3) Rapamycin could significantly reduce the expression of transmembrane protein 208,increase the level of autophagy, and reduce the apoptosis and mitochondrial damage in chondrocytes after interleukin-1β stimulation.(4) To conclude, transmembrane protein 208 inhibits autophagy in chondrocytes, and increased expression of this gene may lead to increased damage of chondrocytes under stress.
BACKGROUND: Decreased level of autophagy in chondrocytes is an important mechanism of osteoarthritis.The autophagy inhibitory gene transmembrane protein 208 is elevated in late-stage osteoarthritis cartilage, and this gene may promote the development of osteoarthritis by inhibiting autophagy of chondrocytes.OBJECTIVE: To investigate the effects of chondrocyte transmembrane protein 208 gene on autophagy and mitochondrial function in osteoarthritis model in vitro.METHODS: The cartilage tissue was from the discarded cartilage of patients undergoing knee arthroplasty. The mild lesion cartilage on non-weight-bearing face(Outerbridge I-II grade) was as early-term group, and the severe lesion cartilage on heavy-weight surface(Outerbridge III-IV grade) was as the late-term group. The content of transmembrane protein 208 in different cartilage tissues was detected.The study was in accordance with the ethical criteria of Peking University First Hospital, and the cartilage donors and their family members signed an informed consent form. Passage 4 normal human chondrocytes were obtained and then stimulated by interleukin-1β to establish the in vitro osteoarthritis. The chondrocytes were pretreated by autophagy activator rapamycin or autophagy inhibitor 3 MA, followed by interleukin-1β added for 12, 24 and 48 hours to observe the morphology of chondrocytes. The content of transmembrane protein 208 gene was detected by real-time PCR. Western blot assay was used to detect autophagy-associated protein light chain proteins 3 B and P62 to evaluate autophagy levels. The apoptosis of chondrocytes was detected by Annexin V-FITC method. Mitochondrial damage was assessed by fluorescent detection with MitoSOX Red dye and JC-1 dye.RESULTS AND CONCLUSION:(1) The expression of transmembrane protein 208 was significantly increased in severe osteoarthritis cartilage.(2) After stimulation with interleukin-1β for 12, 24 and 48 hours, the expression level of transmembrane protein 208 in chondrocytes decreased initially, and then increased. The level of autophagy rose initially and then fell. The apoptotic level gradually increased, and mitochondrial damage gradually became severe.(3) Rapamycin could significantly reduce the expression of transmembrane protein 208,increase the level of autophagy, and reduce the apoptosis and mitochondrial damage in chondrocytes after interleukin-1β stimulation.(4) To conclude, transmembrane protein 208 inhibits autophagy in chondrocytes, and increased expression of this gene may lead to increased damage of chondrocytes under stress.
引文
[1]Loeser RF,Collins JA,Diekman BO.Ageing and the pathogenesis of osteoarthritis.Nat Rev Rheumatol. 2016;12(7):412-420.
[2]Carames B,Olmer M,Kiosses WB,et al.The relationship of autophagy defects to cartilage damage during joint aging in a mouse model.Arthritis Rheumatol.2015;67(6):1568-1576.
[3]López de Figueroa P, Lotz MK, Blanco FJ, Caramés B.Autophagy activation and protection from mitochondrial dysfunction in human chondrocytes. Arthritis Rheumatol. 2015;67(4):966-976.
[4]Loeser RF.Aging and osteoarthritis:the role of chondrocyte senescence and aging changes in the cartilage matrix.Osteoarthritis Cartilage.2009;17(8):971-979.
[5]Meckes JK,Carames B,Olmer M,et al.Compromised autophagy precedes meniscus degeneration and cartilage damage in mice.Osteoarthritis Cartilage. 2017;25(11):1880-1889.
[6]Almonte-Becerril M,Navarro-Garcia F,Gonzalez-Robles A,et al.Cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of Osteoarthritis within an experimental model.Apoptosis. 2010;15(5):631-638.
[7]Huang ZM, Du SH, Huang LG, et al. Leptin promotes apoptosis and inhibits autophagy of chondrocytes through upregulating lysyl oxidase-like 3 during osteoarthritis pathogenesis.Osteoarthritis Cartilage.2016;24(7):1246-1253.
[8]Ribeiro M, López de Figueroa P, Blanco FJ, et al.Insulin decreases autophagy and leads to cartilage degradation.Osteoarthritis Cartilage,2016,24(4):731-739.
[9]Martinez-Lopez N,Athonvarangkul D,Singh R.Autophagy and aging.Adv Exp Med Biol.2015;847:73-87.
[10] Loeser RF,Collins JA,Diekman BO.Ageing and the pathogenesis of osteoarthritis.Nat Rev Rheumatol.2016;12(7):412-420.
[11] Lotz MK,Carames B.Autophagy and cartilage homeostasis mechanisms in joint health, aging and OA. Nat Rev Rheumatol.2011;7(10):579-587.
[12] Ansari MY,Khan NM,Ahmad I,et al.Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes. Osteoarthritis Cartilage. 2018;26(8):1087-1097.
[13] Zhang Y,Vasheghani F,Li YH, et al.Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis.Ann Rheum Dis.2015;74(7):1432-1440.
[14] Carames B,Hasegawa A,Taniguchi N,et al.Autophagy activation by rapamycin reduces severity of experimental osteoarthritis.Ann Rheum Dis.2012;71(4):575-581.
[15] Vasheghani F,Zhang Y,Li YH,et al.PPARgamma deficiency results in severe, accelerated osteoarthritis associated with aberrant mTOR signalling in the articular cartilage.Ann Rheum Dis.2015;74(3):569-578.
[16] Zhang H,Wang H,Zeng C,et al.mTORC1 activation downregulates FGFR3 and PTH/PTHrP receptor in articular chondrocytes to initiate osteoarthritis.Osteoarthritis Cartilage.2017;25(6):952-963.
[17] Ito M,Yurube T,Kakutani K,et al.Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis,senescence, and extracellular matrix catabolism with Akt and autophagy induction. Osteoarthritis Cartilage.2017;25(12):2134-2146.
[18] Cravedi P,Ruggenenti P,Remuzzi G.Sirolimus to replace calcineurin inhibitors? Too early yet. Lancet. 2009;373(9671):1235-1236.
[19] Zhao Y, Hu J, Miao G, et al.Transmembrane protein 208:a novel ER-localized protein that regulates autophagy and ER stress.PLoS One.2013;8(5):e64228.
[20] Hosseinzadeh A, Kamrava SK,Joghataei MT,et al.Apoptosis signaling pathways in osteoarthritis and possible protective role of melatonin.J Pineal Res.2016;61(4):411-425.
[21] Yorimitsu T,Nair U,Yang Z,et al.Endoplasmic reticulum stress triggers autophagy.J Biol Chem,2006,281(40):30299-30304.
[22] Deegan S,Saveljeva S,Gorman AM,et al.Stress-induced self-cannibalism:on the regulation of autophagy by endoplasmic reticulum stress. Cell Mol Life Sci.2013;70(14):2425-2441.
[23] Levine B,Kroemer G.Autophagy in the pathogenesis of disease.Cell.2008;132(1):27-42.
[24] Shapiro IM, Layfield R,Lotz M,et al.Boning up on autophagy:the role of autophagy in skeletal biology.Autophagy.2014;10(1):7-19.
[25] Sasaki H,Takayama K,Matsushita T,et al.Autophagy modulates osteoarthritis-related gene expression in human chondrocytes.Arthritis Rheum.2012;64(6):1920-1928.
[26] Barranco C.Osteoarthritis:Activate autophagy to prevent cartilage degeneration? Nat Rev Rheumatol.2015;11(3):127.
[27] Harrison DE, Strong R,Sharp ZD,et al.Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.Nature.2009;460(7253):392-395.
[2]Carames B,Olmer M,Kiosses WB,et al.The relationship of autophagy defects to cartilage damage during joint aging in a mouse model.Arthritis Rheumatol.2015;67(6):1568-1576.
[3]López de Figueroa P, Lotz MK, Blanco FJ, Caramés B.Autophagy activation and protection from mitochondrial dysfunction in human chondrocytes. Arthritis Rheumatol. 2015;67(4):966-976.
[4]Loeser RF.Aging and osteoarthritis:the role of chondrocyte senescence and aging changes in the cartilage matrix.Osteoarthritis Cartilage.2009;17(8):971-979.
[5]Meckes JK,Carames B,Olmer M,et al.Compromised autophagy precedes meniscus degeneration and cartilage damage in mice.Osteoarthritis Cartilage. 2017;25(11):1880-1889.
[6]Almonte-Becerril M,Navarro-Garcia F,Gonzalez-Robles A,et al.Cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of Osteoarthritis within an experimental model.Apoptosis. 2010;15(5):631-638.
[7]Huang ZM, Du SH, Huang LG, et al. Leptin promotes apoptosis and inhibits autophagy of chondrocytes through upregulating lysyl oxidase-like 3 during osteoarthritis pathogenesis.Osteoarthritis Cartilage.2016;24(7):1246-1253.
[8]Ribeiro M, López de Figueroa P, Blanco FJ, et al.Insulin decreases autophagy and leads to cartilage degradation.Osteoarthritis Cartilage,2016,24(4):731-739.
[9]Martinez-Lopez N,Athonvarangkul D,Singh R.Autophagy and aging.Adv Exp Med Biol.2015;847:73-87.
[10] Loeser RF,Collins JA,Diekman BO.Ageing and the pathogenesis of osteoarthritis.Nat Rev Rheumatol.2016;12(7):412-420.
[11] Lotz MK,Carames B.Autophagy and cartilage homeostasis mechanisms in joint health, aging and OA. Nat Rev Rheumatol.2011;7(10):579-587.
[12] Ansari MY,Khan NM,Ahmad I,et al.Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes. Osteoarthritis Cartilage. 2018;26(8):1087-1097.
[13] Zhang Y,Vasheghani F,Li YH, et al.Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis.Ann Rheum Dis.2015;74(7):1432-1440.
[14] Carames B,Hasegawa A,Taniguchi N,et al.Autophagy activation by rapamycin reduces severity of experimental osteoarthritis.Ann Rheum Dis.2012;71(4):575-581.
[15] Vasheghani F,Zhang Y,Li YH,et al.PPARgamma deficiency results in severe, accelerated osteoarthritis associated with aberrant mTOR signalling in the articular cartilage.Ann Rheum Dis.2015;74(3):569-578.
[16] Zhang H,Wang H,Zeng C,et al.mTORC1 activation downregulates FGFR3 and PTH/PTHrP receptor in articular chondrocytes to initiate osteoarthritis.Osteoarthritis Cartilage.2017;25(6):952-963.
[17] Ito M,Yurube T,Kakutani K,et al.Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis,senescence, and extracellular matrix catabolism with Akt and autophagy induction. Osteoarthritis Cartilage.2017;25(12):2134-2146.
[18] Cravedi P,Ruggenenti P,Remuzzi G.Sirolimus to replace calcineurin inhibitors? Too early yet. Lancet. 2009;373(9671):1235-1236.
[19] Zhao Y, Hu J, Miao G, et al.Transmembrane protein 208:a novel ER-localized protein that regulates autophagy and ER stress.PLoS One.2013;8(5):e64228.
[20] Hosseinzadeh A, Kamrava SK,Joghataei MT,et al.Apoptosis signaling pathways in osteoarthritis and possible protective role of melatonin.J Pineal Res.2016;61(4):411-425.
[21] Yorimitsu T,Nair U,Yang Z,et al.Endoplasmic reticulum stress triggers autophagy.J Biol Chem,2006,281(40):30299-30304.
[22] Deegan S,Saveljeva S,Gorman AM,et al.Stress-induced self-cannibalism:on the regulation of autophagy by endoplasmic reticulum stress. Cell Mol Life Sci.2013;70(14):2425-2441.
[23] Levine B,Kroemer G.Autophagy in the pathogenesis of disease.Cell.2008;132(1):27-42.
[24] Shapiro IM, Layfield R,Lotz M,et al.Boning up on autophagy:the role of autophagy in skeletal biology.Autophagy.2014;10(1):7-19.
[25] Sasaki H,Takayama K,Matsushita T,et al.Autophagy modulates osteoarthritis-related gene expression in human chondrocytes.Arthritis Rheum.2012;64(6):1920-1928.
[26] Barranco C.Osteoarthritis:Activate autophagy to prevent cartilage degeneration? Nat Rev Rheumatol.2015;11(3):127.
[27] Harrison DE, Strong R,Sharp ZD,et al.Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.Nature.2009;460(7253):392-395.