Role Of ERS Mediated By PERK In Regulation Of OPLL Of Cervical Spine By Cx43

Purpose Ossification of the posterior longitudinal ligament(OPLL)occurs mainly in people over 65 years old in East Asia.It is characterized by the hyperplasia,hypertrophy and ossification of the cervical ligament.The progression of ossification could aggravate spinal canal stenosis and compression of spinal cord and nerve roots,gradually.The quality of life would be seriously affected by following neurologic deteriotation.At present,there is no satisfactory conservative treatment.Most patients would eventually choose surgical treatment which is associated with high risks,serious complications and unsure effects for some cases.Therefore,it's urgent to explore the pathogenesis of this disorder.Recently,the theories of heredity,microenvironment,metabolism and mechanical stress have been widely accepted.But it could not explain the complete mechnism,which needs to be further explored.(1)Recent studies have shown that endoplasmic reticulum stress(ERS)mediated by PERK,which is indispensable mechanism for human osteogenesis and bone formation,could regulate the ossification of periodontal ligament induced by mechanical stress.We plan to verify if ERS mediated by PERK influence OPLL,since it's not been reported.(2)In previous studies,we have noticed the expression of Connexin43(Cx43)in ligament fibroblast of patients with OPLL is much higher than that of patients with non-OPLL,and preliminarily confirmed that Cx43 might play an important role in OPLL.On the other hand,researches have found Cx43 could regulate metabolism of osteoblast and chondrocyte through ERK,and Src/MEK/ERK pathway is closely related to the pathogenesis of OPLL.Thus,it is necessary to explore whether Cx43 regulate OPLL through MAPK pathways.(3)It has been reported that Cx43 play a regulatory role through ERS,and ERS can activate MAPK pathways in some pathological processes.We need to verify if Cx43 activate MAPK pathways through ERS mediated by PERK and then regulate osteogenic differentiation.Method From December 2015 to June 2017,16 patients with OPLL and 24 patients without OPLL underwent anterior cervical corpectomy/ discectomy and fusion surgery.Large tissues of the posterior longitudinal ligament were obtained during the operation.The ligament cells were isolated and cultured by enzyme digestion.Vimentin was stained by immunohistochemical technique to identify the fibroblast.Further molecular biological studies were carried out as following:(1)With RT-PCR for mRNA and Western-blotting for protein,the expression levels of ERS related indexes such as PERK,CHOP,GRP78 and osteoblastic markers such as ALP,OCN,COL I were compared between OPLL group andnon-OPLL group.Then,cells in non-OPLL group were stimulated by mechnical stress in Flexercell loading system.The above indexes and reactive oxygen species(ROS)level were detected before and after stimulation.Treated by PERK path-specific inhibitor and mechnical stress,the expressions of osteogenic markers in non-OPLL group were observed.(2)By immunohistochemical technique,the phosphorylation levels of p38 MAPK,ERK1/2and JNK were compared between OPLL group and non-OPLL group.Western-blotting was used to detect if mechnical stress activate MAPK pathways in non-OPLL group.Next,the siRNA sequence of Cx43 was prepared and carried by lentivirus vector to transfect the cells.The knock-down efficiency was tested from expression levels in gene and protein,and the effect of interfering Cx43 on the activation of MAPK pathways by mechnical stress was observed.Handled by specific inhibitors for p38 MAPK,ERK1/2,JNK pathways and stimulated by mechnical stress,the expressions of osteogenic markers were detected,subsequently.(3)After Cx43 was interfered and overexpressed,the expressions of PERK,CHOP,GRP78 and ROS under mechnical stress were detected in non-OPLL group.Then,phosphorylation levels of MAPK stimulated by mechnical stress were observed in cells treated by PERK path-specific inhibitor.Finally,we treated cells in non-OPLL group with siRNA/Cx43 and PERK path-specific activators in turn,then observed the phosphorylation levels of MAPK and expressions of osteogenic markers under mechnical stress.Results Cultured by enzyme digestion,cells of the third generation were in a good state of growth,stable in shape and arranged in an orderly manner,mainly in the form of long spindle and polygon.The cells were abundant in body and radial in shape.The morphology of the cells were not significantly different between OPLL group and non-OPLL group.Immunohistochemical staining of vimentin was positive,which confirmed the nature of fibroblast.(1)The expression levels of ERS related indexes such as PERK,CHOP,GRP78 and osteoblastic markers such as ALP,OCN,COL I in OPLL group were significantly higher than that in non-OPLL group(p < 0.05).Mechnical stress significantly(p < 0.05)upregulated the above indexes and ROS level in non-OPLL group.The expressions of osteoblastic markers under mechnical stress were significantly downregulated by inhibiting ERS mediated by PERK(p < 0.05).(2)The phosphorylation levels of p38 MAPK,ERK1/2 and JNK in OPLL group were significantly higher than that in non-OPLL group(p < 0.05).Mechnical stress significantly activated these pathways in non-OPLL group.The siRNA sequence of Cx43 was successfully constructed,and theknock-down efficiency was higher than 70% in levels of mRNA and protein.The activation of MAPK pathways by mechnical stress was significantly inhibited by interefering Cx43 in non-OPLL group(p < 0.05).The expressions of COL I,OCN,ALP,Runx2 induced by mechnical stress were significantly downregulated by interefering Cx43 and inhibiting p38 MAPK,ERK1/2 pathways.Nonetheless,inhibiting JHK pathway didn't significantly influence the expressions of osteoblastic markers(p >0.05),suggesting OPLL would not be significantly regulated by JNK pathway.(3)In non-OPLL group,interfering Cx43 significantly downregulated the expressions of PERK,CHOP,GRP78 and the level of ROS induced by mechnical stress,while overexpressing Cx43 significantly upregulated these indexes(p < 0.05).Inhibiting ERS mediated by PERK significantly hindered activation of MAPK pathways by mechanical stress(p < 0.05).MAPK phosphorylation and osteogenic differentiation of cells in non-OPLL group were inhibited by interfering Cx43.Reactivation of ERS mediated by PERK restored the activation of MAPK pathways and osteogenic stimulation by mechanical stress.Conclusion In vitro,the cells of posterior longitudinal ligament could be successfully isolated and cultured by enzyme digestion.The nature of fibroblast could be verified by vimentin staining.(1)ERS mediated by PERK could be induced by mechnical stress in posterior longitudinal ligament,and promote the process of OPLL.(2)Cx43 could promote OPLL induced by mechanical stress through p38 MAPK and ERK1/2 pathways.There might be other pathways in this procedure.(3)Besides promoting OPLL via p38 MAPK and ERK1/2 pathways in downstream of Cx43,ERS mediated by PERK might regulate OPLL through other approaches.