Phosphorylation Of Keratin 8 On Ser43 Contributes To Excessive Mechanical Load Induced Intervertebral Disc Degeneration

BackgroundIntervertebral disc degeneration（IDD）represents the pathological foundation of degenerative spinal diseases,which could result in neck and low back pain,spinal stenosis and finally lead to disability,bringing severer medical burden to individuals and society.Three distinct yet interrelated structures constitute the intervertebral disc（IVD）:the peripheral anulus fibrosus（AF）;the cartilaginous endplates（CEP）;and the central nucleus pulposus（NP）.It is widely acknowledged that the degeneration of the NP marks the degeneration of IVD.Excessive mechanical load is an independent risk factor of the IDD.Excessive mechanical load promotes the catabolism,and reduces the anabolism of the NP cells,and finally results in apoptosis of NP cells and consequently the degradation of NP matrix and structural failure of the IVD.The elucidation of the mechanisms of excessive mechanical load induced degeneration and apoptosis and NP cells is of great significance to the development of new therapeutic approaches of the IDD.Previous studies from our research group showed than Keratin 8（KRT8）was specifically expressed within the human NP cells.Also,the protein level of KRT8 decreases when the human disc degeneration aggravated.In vitro studies also showed that the protein level of KRT8 is mechanical sensitive.Yet whether Krt8 was involved in the IDD and the underlying mechanism remains elusive.ObjectivesTo clarify the role of Krt8 during disc degeneration and to investigate the mechanisms by which Krt8 influences the fate and function of NP cells under excessive mechanical load,and to evaluate the therapeutic effects targeting Krt8 or mechano transduction pathways.Methods1.NP conditional Krt8 knockout mice was constructed and load induced IDD models were employed to challenge the discs.2.Rat tail load induced IDD model was employed,human healthy primary NP cells and rat primary NP cells were isolated,and in vitro compression model was established,to explore the expression tendency of Krt8 under excessive mechanical load.3.Knockdown or overexpression of Krt8 was performed under in vitro compression,to clarify the role of Krt8 within NP cells under excessive mechanical load.4.Proteomics,m RNA sequencing and bioinformatic analysis were employed to investigate the mechanisms by which Krt8 influences the fate and function of NP cells under excessive mechanical load.5.The therapeutic effects for load induced IDD,targeting Krt8 or mechano transduction pathways,were evaluated in vitro and in vivo.Results1.Conditional knockout of Krt8 within NP aggravated load-induced disc degeneration in vivo.A Krt8 NP conditional knockout mice line(Lepr-Cre;krt8^fl/fl)was established by Cre-Lox P system.Load induced IDD models,lumbar spine instability model（LSI）for lumbar discs and tail compressive suture model（TCS）for coccygeal discs,were employed to challenge the discs.Results from radiological and histological studies showed that conditional knockout of Krt8 within NP aggravated load-induced disc degeneration in vivo.2.The expression tendency of Krt8 under excessive mechanical load.Rat tail load induced IDD model was employed,human healthy primary NP cells and rat primary NP cells were isolated,and in vitro compression model was established.Results from molecular biology studies showed a promotion of the protein level of KRT8 within NP cells under excessive mechanical load,followed by a reduction of it when the compression continues.And such pattern of the KRT8 protein level within NP cells under in vitro compression was a result of joint efforts from an elevated transcription,translation of the Krt8 as well as an increased proteasome activity.3.Loss of Krt8 predisposed NP cells to,and overexpression of Krt8 endowed NP cells with greater resistance to,excessive mechanical load-induced apoptosis and degeneration in vitro.Rat primary NP cells were isolated,and in vitro compression model was established.Loss of Krt8 predisposed NP cells to and overexpression of Krt8 endowed NP cells greater resistance to excessive mechanical load-induced apoptosis and degeneration in vitro.Only the newly expressed KRT8 protein,rather than the MG132 restored KRT8 protein level,was capable of rescuing the deleterious phenotype under compression.4.A damped autophagosome initiation was responsible for the detrimental effect of loss of Krt8 on NP cells under excessive mechanical load.Mechanism study revealed that loss of Krt8 impeded the trafficking of Golgi resident small GTPase RAB33B,further blocked the interaction of RAB33B and ATG16L1,leading to a damped autophagosome initiation.Only newly expressed KRT8 protein,rather than the MG132 restored KRT8 protein level,could facilitate the trafficking of RABB33B.5.Phosphorylation of Keratin 8 on Ser43 contributed to excessive mechanical load induced intervertebral disc degeneration.The paradoxical results that only the newly expressed KRT8,rather than MG132 restored KRT8 protein expression,could rescue the trafficking of RAB33B,warranted the investigation of an underlying mechanism,rather than the whole protein level of KRT8,for example,mechanisms involving the phosphorylation of KRT8.Results showed a strong positive correlation between p-KRT8 and KRT8 bound RAB33B,accompanied by a corresponding reduction of ATG16L1 bound RAB33B.To further elucidate the role of KRT8 phosphorylation during excessive mechanical load-induced disc degeneration,we employed quantitative phosphoproteomic analysis to evaluate the phosphorylation status of KRT8 comprehensively and accurately,under in vivo compression-induced rat tail disc degeneration model.Total 27 phosphorylated sites of KRT8 were identified,and the top 6sites（>1.75 fold change）were included for further study.Mutations of each included phosphorylated sites to aspartic acid（D）were constructed respectively,and adenovirus-mediated overexpression of each mutated KRT8 protein were performed.Moreover,5 mutations among these tested mutations showed a comparable rescuing effect with wild-type KRT8,except for S43D,whose overexpression did not facilitate the trafficking of RAB33B and showed no significant rescuing effect.These results clearly demonstrated that phosphorylation of KRT8 on Ser43 blocked the autophagosome initiation by impeding the trafficking of RAB33B from Golgi to autophagosome through trapping RAB33B with p-Ser43 KRT8.6.Mechanical load activated RHOA-PKN1/2 phosphorylated Ser43 of Keratin 8.Results from multiple sequence comparison showed that serine residue 43（Ser43）of rat KRT8 protein and the surrounding sequence was highly conserved in other species,which further warrants the investigation of a detailed mechanism,by which Ser43 of KRT8 was phosphorylated under excessive mechanical load.To this end,a polyclonal phosphorylation site–specific antibody targeting rat p-Ser43 KRT8 was generated.To identify the protein kinases responsible for the phosphorylation of Ser43 of KRT8 under excessive mechanical load,bioinformatic prediction was employed to predict the protein kinase based on the motifs surrounding the Ser43 of rat KRT8.Top 5 predicted kinases based on the prediction score was included for further study.Results from si RNA mediated knockdown showed that both PKN1 and PKN2 are responsible for the phosphorylation of Ser43 of KRT8.The activation of PKN1 and PKN2 was also validated by quantitative phosphoproteomic analysis,GESA,and in vitro western blot analysis.Further studies revealed that mechanical load activated PKN through RHOA.Knockdown of Pkn1/2 rescued the trafficking of RAB33B,ameliorated the degenerative phenotype and reduced the apoptosis of NP cells under in vitro compression.7.Evaluation of the therapeutic effects for load induced IDD,targeting Krt8 or mechano transduction pathways.Rat tail compression induced IDD model was established and AAV5 mediated overexpression of Krt8 and sh RNA mediated knockdown of Pkn1/2 were performed.Results showed that both overexpression of Krt8 as well as knockdown of Pkn1 and Pkn2,when treated at early stage of IDD model,ameliorated disc degeneration in vivo.However,only knockdown of Pkn1 and Pkn2,when treated at late stage of IDD model,ameliorated disc degeneration in vivo.ConclusionThe current study validated a protective role of Krt8 during overloading-induced IDD,showed that phosphorylation on Ser43 of Keratin 8 by mechanical load activated RHOA-PKN1/2 contributes to disc degeneration,and demonstrated that targeting Krt8 or mechano transduction pathways could be novel and effective approaches to mechano stress-induced IDD,and targeting overloading activated of PKNs could be an approach with a wider window of therapeutic opportunity.