The Role And Mechanism Of The Canonical Hedgehog Pathway In Cervical Ossification Of The Posterior Longitudinal Ligament

ObjectiveCervical ossification of the posterior longitudinal ligament(OPLL)is a degenerative disease where ligament tissue is replaced by ectopic bone,predominantly affecting the cervical spine.Severe cases can result in compression of the spinal cord and nerve roots,leading to varying degrees of cervical myelopathy and/or cervical radiculopathy,including sensory and motor impairments,alongside sphincter dysfunction.More importantly,OPLL can make patients intolerant to trauma,where even mild trauma may lead to spinal cord injury resulting in paralysis and other serious consequences,imposing a significant burden on families and society.From an epidemiological perspective,cervical OPLL predominantly affects males,accounting for approximately 66% of all cases.The occurrence of OPLL is regional,primarily affecting East Asian populations.Currently,surgical intervention remains the sole treatment approach for symptomatic OPLL,despite its inherent complexity and substantial risks,potentially leading to severe complications.The unclear molecular mechanisms hinder the development of pharmacological and preventative intervention strategies.In order to delve deeper into the etiology of OPLL,particularly its molecular mechanisms,this study conducted transcriptome sequencing analysis on primary ligament cells extracted from patients with and without cervical OPLL.With this as a foundation,we aim to elucidate the molecular mechanisms underlying osteogenic differentiation of posterior longitudinal ligament cells in OPLL,potentially offering avenues to halt promising avenues to impede disease progression and offer therapeutic benefits.MethodsPart One:(1)We collected posterior longitudinal ligaments(PLL)from patients undergoing anterior cervical spine surgery without OPLL(PLL,control group)and from patients with OPLL(OPLL,case group)for primary cell culture;(2)Surface marker detection was performed using flow cytometry,and cells were induced to differentiate towards osteogenic and adipogenic lineages to identify the stemness of cultured primary cells.Part Two:(1)Total RNA was extracted from PLL and OPLL cells for highthroughput sequencing to identify differential expression genes(DEGs);(2)DEGs showing statistical and functional significance were selected through GO enrichment analysis and enrichment analyses of KEGG,Reactome,and Wiki Pathways;(3)To validate sequencing findings,m RNA and protein expression of the promising DEGs were evaluated,providing evidence for the altered expression of GLI1 in OPLL;(4)Osteogenic induction was performed on posterior longitudinal ligament cells to detect changes in GLI1 protein expression and cellular localization;(5)Functional experiments involving knockdown/overexpression of GLI1 were conducted to further validate its role in osteogenic differentiation of posterior longitudinal ligament cells;(6)Animal experiments were carried out by subcutaneously implanting cell-seeded collagen scaffolds into nude mice to further investigate the role of GLI1 in osteogenic differentiation of posterior longitudinal ligament cells in vivo.Part Three:(1)The role of the Hedgehog pathway in osteogenic differentiation of posterior longitudinal ligament cells was explored by introducing Hedgehog pathway antagonist(Cyclopamine,CPN)/ activator(Purmorphamine,PM)into OPLL and PLL cells,respectively;(2)Overexpression of GLI1 in PLL cells was accompanied by Hedgehog pathway antagonism,while in OPLL cells,GLI1 knockdown was coupled with Hedgehog pathway activation,elucidating the regulatory role of the HedgehogGLI1 pathway in osteogenic differentiation of posterior longitudinal ligament cells.Part Four:(1)Database predictions were utilized to identify the target genes of GLI1;(2)Activation of the Hedgehog pathway / overexpression of GLI1 and antagonism of the Hedgehog pathway / GLI1 knockdown were conducted in PLL and OPLL cells,respectively,to validate the regulatory effect of the Hedgehog-GLI1 pathway on target genes BMP2,BMP4,and their associated BMP pathway;(3)Proteinprotein interactions between GLI1 and BMP2,BMP4 were explored through immunoprecipitation;(4)Rescue assay was performed.In GLI1-knockdown OPLL cells followed by rh-BMP2 introduction,changes in osteogenic differentiation capability were assessed at different exposure times.Similarly,in PLL cells with GLI1 overexpression,BMP pathway inhibitor Noggin was added to examine changes in osteogenic differentiation capability,further clarifying the regulatory role of Hedgehog-GLI1-BMP on osteogenic differentiation of posterior longitudinal ligament cells.ResultsPart One: Flow cytometry revealed the expression of typical mesenchymal stem cell(MSC)surface markers,CD105,CD73,and CD90,in cells derived from both PLL and OPLL,while CD45,CD34,and HLA-DR were negative,meeting the criteria for mesenchymal stem cell identification.Moreover,osteogenic and adipogenic differentiation capabilities were shared by both cell types.Specifically,Oil Red O staining verified their ability for adipogenesis.After inducing osteogenic differentiation in PLL and OPLL cells,Western blot was employed to assess the expression of osteogenic-related genes,while ALP staining and Alizarin Red staining were utilized for a visual evaluation of their osteogenic differentiation capabilities.The results revealed that compared to PLL cells,OPLL cells exhibited a stronger osteogenic potential.Part Two: High-throughput sequencing identified a total of 233 DEGs,comprising128 upregulated genes and 105 downregulated genes.We sifted through DEGs associated with bone formation and ectopic bone formation processes within top 30 enrichment GO terms and took the intersection of these genes with the DEGs derived from the top 20 upregulated pathways across the three databases,in an attempt to unveil key DEGs relevant to OPLL.GLI1 emerged as a compelling candidate,emphasizing both statistical and biological significance.We gauged the m RNA and protein expression levels of GLI1 in both cell types using q RT-PCR and WB.In comparison to the control group,OPLL cells exhibited a significant increase in GLI1 expression at both the m RNA and protein levels,aligning with the heightened in vivo GLI1 levels observed via IHC.As osteogenic differentiation is induced,a pattern of initial elevation followed by subsequent downregulation in GLI1 expression was observed,with its localization gradually shifting towards nuclear aggregation in OPLL cells.Following GLI1 knockdown,there was a significant decrease in the protein levels of osteogenicrelated genes,accompanied by diminished ALP-secretion and mineralization capacity in OPLL cells.Conversely,GLI1 overexpression led to a substantial upregulation of these genes and an enhanced ALP-secretion and mineralization ability in PLL cells.Moreover,knocking down GLI1 can inhibit ectopic bone formation subcutaneously in nude mice,reducing the bone mass of heterotopic ossification.Part Three: When PM was introduced to PLL cells to activate the Hedgehog pathway,WB confirmed the upregulation of osteogenic-related genes expression,while ALP staining and Alizarin Red staining demonstrated a stronger osteogenic ability compared to PLL cells.Correspondingly,adding CPN to OPLL cells to antagonize the Hedgehog pathway led to a decrease in osteogenic capability.Furthermore,antagonizing the Hedgehog pathway rescued the enhanced osteogenic ability caused by GLI1 overexpression.Conversely,the elimination of GLI1,even under Hedgehog pathway activation,sustained osteogenic-related genes at diminished levelsPart Four,A protein-protein interaction(PPI)network analysis based on transcriptomic data identified GLI1 as a pivotal hub gene associated with the BMP pathway.Co-immunoprecipitation experiments confirmed the interaction between GLI1 and BMP2.Overexpression of GLI1 in PLL cells led to upregulation of BMP2,BMP4,smad1,and phosphorylated smad 1/5/9(P-smad1/5/9)protein expression.Conversely,knockdown of GLI1 suppressed the BMP pathway in OPLL cells.Similarly,activation of the Hedgehog pathway in PLL cells resulted in upregulation of BMP2,BMP4,smad1,and P-smad1/5/9 expression,while antagonism of the Hedgehog pathway in OPLL cells led to a decrease in the expression of BMP2,BMP4,smad1,and P-smad1/5/9.Introduction of rh-BMP2 rescued the decreased levels of GLI1 and RUNX2 caused by GLI1 knockdown,whereas it didn’t exert the same influence on ALP,OPN and OCN.ALP secretion and mineralization capacity were also restored.Likewise,Noggin reversed the osteogenic ability of PLL cells overexpressing GLI1 and reduced it.ConclusionIn our study,we have validated GLI1 as a pivotal gene in OPLL pathogenesis and proposed its potential regulatory mechanism.In OPLL,the canonical Hedgehog signaling pathway is activated,leading to the upregulation and nuclear translocation of its downstream transcription factor GLI1.GLI1 further induces the expression of BMP2,activating the BMP signaling pathway,mediating crosstalk between the canonical Hedgehog and BMP signaling pathways.This process promotes osteogenic differentiation of posterior longitudinal ligament cells,ultimately leading to ectopic ossification.