The Role And Mechanism Of Stem Cell Transcription Factors In Ectopic Ossification Of Patients With Ankylosing Spondylitis

Ankylosing spondylitis?AS?is a chronic inflammatory disease that affects primarily the spine and pelvis and causes pain and initially reversible stiffness,ultimately leading to joint ankylosis due to ectopic ossification.Ectopic ossification can result in the loss of joint mobility and ankylosis,leading to severe disability.Despite the clinical impact of ankylosis and its consequence of pain and dreadful life style changes in patients,the mechanism?s?of ectopic ossification in AS remains largely unknown.In previous studies,fibroblasts showed the potential to differentiate into osteoblasts under specific circumstances in vitro,which suggested a possibility that fibroblasts may be the potential cells to participate in ossification in AS patients.However,the mechanism of differentiation remains to be explored.The discovery that somatic cells can be reprogrammed to induced pluripotent stem?iPS?cells by merely four transcription factors?OCT4,SOX2,KLF4,and MYC?was a great breakthrough in stem cell research.These four transcription factors play an essential role in maintaining stemness,dedifferentiation,and re-differentiation.Study revealed that combination of some chemicals could induce mouse embryonic fibroblast cells to trans-differentiate into a wide range of somatic lineages and that SOX2 was the important regulator within the induced network.TNF could also dedifferentiate astrocytes by the re-expression of OCT4 through the NF-?B pathway,indicating an association between reprogramming and inflammation.Inflammation is another main characteristics of AS.The treatment strategy is to control inflammatory response to reduce disease activity,thus to relieve the disease.It is a significant milestone in AS treatment that the tumor necrosis factor inhibitors?TNFi?prove to be effective in alleviating symptoms and reducing disease activity.Recent studies have also found that cytokines,including migration inhibitory factor?MIF?and Interleukin?IL?-22,can induce osteogenesis.Since inflammation could regulate stem cell transcription factors and stem cell transcription factors could further participate in cell reprograming,whether they could be the bridge linking inflammation and ossification in patients with AS?Thus,we hypothesized that resident fibroblasts in ligaments could be reprogrammed to osteoblasts via some pivotal transcription factors after a long period of inflammatory stimulation,which would then lead to ectopic ossification in AS.To test this hypothesis,we will verify it in three parts below.Part 1:Profile of stem cell transcription factors in ectopic ossification of patients with ankylosing spondylitisObjectives:To screen stem cell transcription factors that might function during the osteogenic process in osteogenic models in vitro and in ligaments samples.Methods:Firstly,Ligaments were collected from the hip joints of AS patients and osteoarthritis?OA?patients who underwent a hip replacement operation.Hematoxylin-eosin?HE?staining was used to observe its structural characteristics.Secondly,we isolated primary fibroblasts and identified them via immunohistochemistry?IHC?and flow cytometery?FCM?.To simulate the dynamic osteogenic process and assess the changes in stem cell transcription factors,a classic in vitro osteogenic model was adopted to induce fibroblasts to osteoblasts.Based on this model,a Stem Cell TF Activation Profiling Plate Array and quantitative real-time PCR?qPCR?was used to screen for stem cell transcription factors that might function during the osteogenic process.Finally,the potential stem cell transcription factors were further assessed in the ligaments by q-PCR.Results:99%of isolated primary cells were proved to be fibroblasts.Culturing fibroblasts in osteogenic differentiation medium?ODM?could successfully induce them to osteoblasts and MYC and FOXO1 were elevated in this process?p<0.05?.EGR1,ETS1,LEF and NANOG showed the trends of upregulation.Further assessment in ligaments showed that only MYC and EGR1 were upregulated in AS ligaments than OA ligaments?p<0.05?.Conclusions:MYC and EGR1 were involved in the trans-differentiation of fibroblasts into osteoblasts,and thus participate in the occurrence of heterotopic ossification of AS ligament tissue.Part 2:Mechanism of MYC and EGR1 in osteogenesis.Objectives:To explore the molecular mechanism of MYC and EGR1 in ectopic ossification of AS patients.Methods:Firstly,the expression of MYC and EGR1 in fibroblasts was knocked down with lenti-virus,qPCR was used to observe the expression of ossification gene in fibroblasts.ALP staining and mineralization staining were used to assess the level of ossification.Chromatin immunoprecipitation?ChIP?-qPCR was applied to explore the target genes of transcription factors.Results:The primary fibroblasts infected with negative control?NC?or MYC knockdown lentivirus?MYC-KD?were cultured in ODM.When induced for 10 days,the expression of alkaline phosphatase?ALP?and bone morphogenetic protein 2?BMP2?were lower in the MYC-KD group than in the NC group?p<0.05?.At the 20^th day,only ALP expression in the MYC-KD group was lower than that in the NC group?p<0.01?.The ALP staining of MYC-KD group was significantly lower than that of the NC group by direct observation or microscopic observation in the culture plate.Both bright field and fluoroscopy showed that the mineralized nodules in the MYC-KD group were less than those in the NC group.The results of ChIP-qPCR showed that compared with the IgG negative control,MYC could bind to the promoters of ALP and BMP?p<0.05?,respectively.This indicates that MYC can promote osteogenesis by regulating the transcription of ALP and BMP2.The primary fibroblasts infected with NC or EGR1 knockdown lentivirus?EGR1-KD?were cultured in ODM.When induced for 10 days,the expression level of BMP2 and Runt related transcription factor 2?RUNX2?in the EGR1-KD group was higher than NC group?p<0.05?.At the 20^th day,only BMP2 was up-regulated in EGR1-KD group?p<0.01?.Both brightfield and fluoroscopy showed that the mineralized nodules of the EGR1-KD group were significantly higher than those of the NC group.Knocking down EGR1could promote ossification,which suggested that EGR1 itself could inhibit osteogenetic process.Next,the relationship between MYC and EGR1 was explored.At 20 days after ossification induction,the expression level of EGR1 was significantly decreased in the MYC-KD group?p<0.01?,suggesting that EGR1 may be the downstream of MYC.The ChIP-qPCR results showed that MYC binds to the EGR1 promoter?p<0.05?.However,in the EGR1-KD group,the expression of MYC increased compared with NC group,with no statistical significance.Conclusions:In osteogenic model,MYC can directly regulate ALP and BMP2transcription to promote osteogenesis.EGR1 is downstream of MYC.It can inhibit the expression of BMP2 and RUNX2,thus hindering the ossification of fibroblasts.Part 3:Effect of inflammation on stem cell transcription factors.Objectives:Given that the above experiments to induce ossification were all performed by using non-in-vivo chemical substances,we attempted to use recombinant human cytokines,including TNF-?,IL-17,IL-23,IL-22 and interferon-??IFN-??,to mimic the environment of the inflammatory response to further explore the relationship between inflammation and MYC expression in fibroblasts.Methods:Recombinant cytokines were used for short-term?24-hour?or long-term?10 or 20 days?stimulation of primary fibroblasts,qPCR was applied to detect the expression of stem cell transcription factors and ostogenetic genes.IHC method was applied for detecting inflammatory cytokine expression in ligament tissues of patients with AS and OA.Results:Recombinant human cytokines?including TNF-?,IL-17,IL-23,IL-22,IFN-??stimulated primary fibroblasts for 24 hours and results showed that TNF-?inhibits the expression of MYC?p<0.05?,IFN-?and IL-23 can promote MYC expression?p<0.05?.At the same time,we also detected the expression level of ALP,and found that both IL23and IFN-?can up-regulate ALP expression?p<0.05?.Therefore,we used IL-23 and IFN-?to stimulate primary fibroblasts for 10 or 20 days.The results showed that IL-23stimulation only up-regulated the expression of ALP in the ODM group?p<0.05?.After stimulated by IFN-?,both of MYC?p<0.01?and BMP2?p<0.05?were up-regulated.IHC results showed that the number of IL-23 and IFN-?positive cells in AS ligament tissue was higher than that of OA ligament tissue?p<0.05?.Conclusions:IL-23 and IFN-?can promote the expression of osteogenetic genes to some extent.Since IFN-?can promote MYC and BMP2 at the same time,IFN-?may promote ossification through MYC-BMP2 pathway,IHC results confirmed that local inflammation in the AS ligament tissue was also more severe than OA patients.In conclusion,our hypothesis has been validated:under the stimulation of chronic inflammation?possibly IFN-??,MYC of fibroblasts in ligaments of AS patients was upregulated,which further up-regulated the expression of ALP and BMP2,so that fibroblasts could trans-differentiate to osteoblasts and participate in the ectopic ossification.