The Role And Mechanisms Of ER-phagy Modulation In Intervertebral Disc Degeneration

Part Ⅰ:FAM134B-mediated ER-phagy activation suppresses AGEs-induced senescence and apoptosis via modulating ROS in human nucleus pulposus cellsObjective:To investigate the role of FAM134B-mediated ER-phagy in human NP cells upon AGEs treatment and explore its regulatory mechanisms.Methods:Human primary nucleus pulposus cells were exposed to AGEs(200 μg/mL)for 0,6,12,24 and 36 hours or co-treated with AGEs and NAC,a specific ROS scanvenger,for 36 hours.EdU staining was used to measure cell viability.Annexin V/PI double staining was used to evaluate apoptosis.SA-β-gal assay was performed to detect cell senescence.DCFH-DA ROS detection kit was used to detect the intracellular ROS level.Apoptotic and senescent protein expression were assessed with western blot and immunofluorescence staing.The morphology of ER and lysosome were specificly labeled with ER-tracker and Lyso-tracker,respectively.TEM was used to determine the status of ER positive autophagosomes and autolysosomes formation.Results:AGEs treatment resulted in significantly increased apoptosis,senescence,and ROS accumulation in human NP cells,meanwhile,the enhanced apoptosis and senescence by AGEs treatment could be partially alleviated with the classic ROS scavenger NAC administration.Furthermore,we confirmed that FAM134B-mediated ER-phagy was activated under AGEs stimulation via ROS pathway.Importantly,it was also found that F AM 13 4B overexpres si on could efficiently relieve intracellular ROS accumulation,apoptosis,and senescence upon AGEs treatment.Conclusions:our data demonstrate that FAM134B-mediated ER-phagy plays a vital role in AGEs-induced apoptosis and senescence through modulating cellular ROS accumulation,and targeting FAM 134B-mediated ER-phagy could be a promising therapeutic strategy for IDD treatment.Part Ⅱ:FAM134B-mediated ER-phagy activation suppresses GD-induced senescence and apoptosis via modulating ER stress in human nucleus pulposus cellsObjective:To investigate the role of FAM134B-mediated ER-phagy in human NP cells under glucose-deprivation(GD)treatement and explore the exact underlying mechanisms.Methods:Human primary nucleus pulposus cells were exposed to GD treatment for various times(0,6,12,24 and 48 hours),cell senescence and apoptosis,ER stress,and RETREG-mediated ER-phagy were assessed accordingly.4-PBA,the ER stress specific inhibitor,was employed to explore the effects of ER stress on GD-induced cell damage.Specific overexpressing lentivirus and small interfering RNA(siRNA)of FAM134B were applied to upregulate and downregulate ER-phagy,respectively.The expression of FAM134B and LC3 in human NP tissures were determined with immunohistochemical staining.Results:GD treatment significantly triggered senescence and apoptosis in human nucleus pulposus(NP)cells,which was involved in ER stress activation.Importantly,RETREG1-mediated ER-phagy was also activated by GD treatment,genetical upregulation and downregulation of FAM134B-mediated ER-phagy considerably alleviated and aggravated senescence and apoptosis in human NP cells,respectively.Furthermore,ER stress was suggested an upstream event of FAM134B-mediated ER-pahgy upon GD treatment,while enhancing FAM134B-mediated ER-pahgy could in turn counteract the ER stress response.Additionally,an apparently higher expression pattern of FAM134B and LC3 was observed in degenerated human NP tissues.Conclusions:FAM 134B-mediated ER-phagy activation could protect against GD treatment-induced senescence and apoptosis via modulating ER stress in human NP cells,providing a new therapeutic target for IDD treatment.Part Ⅲ:O-GlcNAcylation regulates GD-induced senescence and apoptosis by targeting FAM134B-mediated ER-phagy in human nucleus pulposus cellsObjective:This study aimed to elucidate the role and underlying mechanisms of O-GlcNAcylation and ER-phagy in IDD.Methods:The expression of O-GlcNAc transferase(OGT)and O-GlcNAc in human nucleus pulposus(NP)tissues and glucose-deprived human NP cells was measured.The O-GlcNAc level was modified by genetic and pharmacological methods,and the effects on FAM13 4B-medi ated ER-phagy,apoptosis,and senescence were assessed.Protein-protein interactions were detected by coimmunoprecipitation and immunofluorescence colocalization assays.Results:The expression profiles of OGT and O-GlcNAc was notably upregulated in degenerated NP tissues and glucose-deprived NP cells.By modulating the O-GlcNAc level by genetic manipulation and specific pharmacological intervention,we revealed that increasing O-GlcNAcylation substantially improved cell function and facilitated cell survival under glucose deprivation.Moreover,FAM134B-mediated ER-phagy activation was regulated by O-GlcNAcylation,and suppression of ER-phagy by FAM134B knockdown considerably counteracted the protective effects of amplified O-GlcNAcylation.Mechanistically,FAM134B was determined to be a possible target of OGT,and O-GlcNAcylation of FAM134B obviously reduced its ubiquitination-mediated degradation.Correspondingly,the protective effect of modulating O-GlcNAcylation homeostasis was further verified in a rat IDD model.Conclusion:Our data demonstrated that OGT directly associates with and stabilizes FAM134B and subsequently enhances FAM134B-mediated ER-phagy to improve the adaptive capability of cells in response to glucose deprivation.These findings may provide new understanding regarding the roles of O-GlcNAcylation,FAM134B-mediated ER-phagy,and NP cell damage in IDD pathophysiology.