| Objective:Ankylosing spondylitis(AS) is the prototypic phenotype of a class of immunemediated spondyloarthropathies that affects spine, sacroiliac joint and hip joint. AS causes spinal rigidity with inflammatory pain even spinal fusion, structural and functional impairment of sacroiliac joint and hip joint. Moreover, the harms of AS also include reactive arthritis, Crohn disease, ulcerative colitis, psoriatic arthritis and acute anterior uveitis. The late stagy of AS may lead to spinal fusion and the development of a "bamboo?like" spine, ultimately result in high risk of disability. Therefore, it is valuable to investigate the pathogenesis and therapeutic strategy of AS.The pathogenesis of AS is complex and not fully understood, involving the change of spinal joints, cartilage, tendons and ligaments. The heterotopic osteogenesis of spinal ligaments is the key risk of AS, however, its pathogenesis remains unclear. Our previous reports indicated the expressions of transforming growth factor β1(TGF-β1), bone morphogenetic proteins(BMP2) and type III transforming growth factor β receptor(TGFβR3) in AS supraspinous ligaments significantly increased compared with control.In the preliminary experiments of this study, AS-derived supraspinous ligament fibroblasts(HSLFs) demonstrated distinct osteogenic potential, suggesting it might participate in the osteogenic process of spinal ligaments. Consistently, the expressions of TGF-β1, BMP2 and TGFβR3 in AS-derived HSLFs also increased compared with control HSLFs. Both of TGF-β1 and BMP2 are the members of TGF superfamily. TGFβR3 recognizes the two factors and thus activates Smad signaling, which is one of the key switches leads to osteogenesis. Therefore, TGF-β1 and BMP2 are presumed to participate in the osteogenesis process of AS-derived HSLFs through activating TGFβR3-Smad signaling.Herein, a hypothesis that TGFβR3 overexpression is associated with osteogenesis of AS-derived HSLFs is proposed based on previous findings. To confirm it, this study aims to further investigate the role of TGFβR3 in osteogenesis of AS-derived HSLFs. Firstly, experiments will be desinged to confirm TGF-β1 combined with BMP-2 induce osteogenesis by acting on TGFβR3. Then, the changes of members involved in TGFβR3/Smad pathway were detected, to find the key downstream effectors. In summary, this study aims to unveil the mechanism of osteogenesis of AS-derived HSLFs mediated by TGFβR3, and thus provide new candidate targets for developing new drugs and treatment strategies.Methods:1. The expression of TGFβR3 in AS supraspinous ligament(1) Hematoxylin-eosin(HE) staining method was applied to compare the pathologic changes of AS supraspinous ligament with control group.(2) Immunohistochemistry(IHC) was carried out to detect the expressions of TGFβR3, TGF-β1 and BMP2 in supraspinous ligaments of AS and control group, to confirm whether the expressions of three molecules of AS are different from control.(3) Real-time PCR was carried out to compare the m RNA expressions of TGFβR3, TGF-β1 and BMP2 in AS supraspinous ligament with control.(4) Western blot was carried out to compare the protein expressions of TGFβR3, TGF-β1 and BMP2 in AS supraspinous ligament with control.(5) Tissue piece method was applied to isolate human supraspinous ligament fibroblasts(HSLFs) from supraspinous ligaments of AS or control.(6) Immunofluorescence(IF) was carried out to compare the protein expressions of TGFβR3, TGF-β1 and BMP2 in AS-derived HSLFs with control HSLFs.2. The role of TGFβR3 in osteogenesis of AS-derived HSLFs(1) Accessment of auto-osteogenesis of AS-derived HSLFsMineralization induction-alizarin red staining method was applied to compare extracellular matrix mineralization of AS-derived HSLFs with control HSLFs.(2) Influence of TGF-β1 combined with BMP2 on osteogenic differentiation of HSLFs overexpressing TGFβR3(1) The overexpression vector of TGFβR3, pc DNA-TGFβR3, was constructed, then verified by double digestion and DNA sequencing.(2) Liposome method was applied to construct TGFβR3-overexpressing HSLFs by transforming pc DNA-TGFβR3 into normal HSLFs.(3) Overexpression of TGFβR3 in transformed HSLFs was confirmed using western blot method.(4) Influence of TGF-β1 combined with BMP2 on osteogenic differentiation of HSLFs overexpressing TGFβR3i Influence of TGF-β1 combined with BMP2 on cell area of HSLFs overexpressing TGFβR3 was evaluated by staining cytoskeleton using fluorescence-labeled phalloidin.ii Influence of TGF-β1 combined with BMP2 on proliferation of HSLFs overexpressing TGFβR3 was detected using cell counting method.iii Influence of TGF-β1 combined with BMP2 on extracellular matrix mineralization of HSLFs overexpressing TGFβR3 was detected using mineralization induction-alizarin red staining method.iv Influence of TGF-β1 combined with BMP2 on alkaline phosphatase(ALP) activity of HSLFs overexpressing TGFβR3 was detected by developing ALP.(3) Influence of TGF-β1 combined with BMP2 on osteogenic differentiation of HSLFs with low expression of TGFβR3(1) Liposome method was applied to construct HSLFs with TGFβR3 low expression by transforming specific si RNA of TGFβR3 into normal HSLFs.(2) Semi-quantitative PCR was carried out to detect the blocking rate of TGFβR3 using specific si RNA.(3) Influence of TGF-β1 combined with BMP2 on osteogenic differentiation of HSLFs with low expression of TGFβR3.i Influence of TGF-β1 combined with BMP2 on cell area of HSLFs with low expression of TGFβR3 was evaluated by staining cytoskeleton using fluorescence-labeled phalloidin.ii Influence of TGF-β1 combined with BMP2 on extracellular matrix mineralization of HSLFs with low expression of TGFβR3 was detected using mineralization inductionalizarin red staining method.iii Influence of TGF-β1 combined with BMP2 on alkaline phosphatase(ALP) activity of HSLFs with low expression of TGFβR3 was detected by developing ALP.3. Verifying the key molecules involved in TGFβR3/Smad signaling associated with osteogenesis of HSLFs overexpressing TGFβR3(1) Influence of TGF-β1 combined with BMP2 on TGFβR3/Smad signaling of HSLFs overexpressing TGFβR3Influence of TGF-β1 combined with BMP2 on the expressions of TGFβR1, TGFβR2, TGFβR3, Smad2/3, p-Smad2/3, Smad1, p-Smad1, Smad4 and RUNX2 in HSLFs overexpressing TGFβR3 were detected using western blot.(2) Influence of TGF-β1 combined with BMP2 on TGFβR3/Smad signaling of HSLFs with low expression of TGFβR3Influence of TGF-β1 combined with BMP2 on the expressions of TGFβR1, TGFβR2, TGFβR3, Smad2/3, p-Smad2/3, Smad1, p-Smad1, Smad4 and RUNX2 in HSLFs with low expression of TGFβR3 were detected using western blot.4. Accessment of the expressions of key molecules involved in TGFβR3/Smad signaling in AS supraspinous ligament(1) The expressions of p-Smad2/3, p-Smad1, Smad4 and RUNX2 in AS supraspinous ligament were detected using IHC.(2) The expressions of p-Smad2/3, p-Smad1, Smad4 and RUNX2 in AS-derived HSLFs were detected using IF.Results:1. The expression of TGFβR3 in AS supraspinous ligament(1) AS patient demonstrated typical spinal and sacroiliac fusion. The collagen fibrils of AS supraspinous ligament were broken, irregularly organized and vitreously degenerated. Furthermore, fatty infiltration and vascular proliferation were also observed.(2) TGFβR3, TGF-β1 and BMP2 overexpressed in AS supraspinous ligament, especially in perivascular tissue, fibrous tissue and ligament peplos compared with control.(3) Relative m RNA expressions of TGFβR3, TGF-β1 and BMP2 in AS supraspinous ligament respectively increased five-six fold, two-fold and 100-150 fold compared with control.(4) Protein expressions of TGFβR3, TGF-β1 and BMP2 in AS supraspinous ligament also significantly increased compared with control.(5) HSLFs was isolated from the supraspinous ligaments of AS or control.(6) Protein expressions of TGFβR3, TGF-β1 and BMP2 in AS-derived HSLFs also increased compared with control HSLFs.2. The role of TGFβR3 in osteogenesis of AS-derived HSLFs(1) AS-derived HSLFs produced more extracellular matrix mineralization compared with control HSLFs.(2) TGF-β1 combined with BMP2 induced osteogenesis of HSLFs overexpressing TGFβR3 compared with TGF-β1 or BMP2 alone(1) pc DNA-TGFβR3 was successfully constructed.(2) TGFβR3 expression of HSLFs overexpressing TGFβR3 was much higher than normal HSLFs used as control.(3) TGF-β1 combined with BMP2 increased cell area of HSLFs overexpressing TGFβR3 compared with TGF-β1 or BMP2 alone.(4) TGF-β1 combined with BMP2 could not affect the proliferation of HSLFs overexpressing TGFβR3.(5) TGF-β1 combined with BMP2 induced more extracellular matrix mineralization of HSLFs overexpressing TGFβR3 compared with TGF-β1 or BMP2 alone.(6) TGF-β1 combined with BMP2 increased ALP activity of HSLFs overexpressing TGFβR3 compared with TGF-β1 or BMP2 alone.(3) TGF-β1 combined with BMP2 lost osteogenic induction effect on HSLFs with TGFβR3 lower expression(1) TGFβR3 expression of HSLFs transformed with TGFβR3 si RNA was only 30% of which transformed with control si RNA.(2) TGF-β1 combined with BMP2 could not increase cell area of HSLFs with low expression of TGFβR3 compared with TGF-β1 or BMP2 alone.(3) TGF-β1 combined with BMP2 could not increase extracellular matrix mineralization of HSLFs with low expression of TGFβR3 compared with TGF-β1 or BMP2 alone.(4) TGF-β1 combined with BMP2 could not increase ALP activity of HSLFs with low expression of TGFβR3 compared with TGF-β1 or BMP2 alone.3. Verifying the key molecules involved in TGFβR3/Smad signaling associated with osteogenesis of HSLFs overexpressing TGFβR3(1) TGF-β1 combined with BMP2 increased the expressions of TGFβR3, p-Smad2/3, p-Smad1, Smad4 and RUNX2 but not TGFβR1, TGFβR2, Smad2/3 or Smad1 in HSLFs overexpressing TGFβR3.(2) In HSLFs with lower expression of TGFβR3, the synergistic effect of TGF-β1 combined with BMP2 on increasing the expressions of TGFβR3, p-Smad2/3 and p-Smad1 lost. Meanwhile, the synergistic effect on increasing Smad4 and RUNX2 expression was reversed as antagonistic effect.4. Accessment of the expressions of key molecules involved in TGFβR3/Smad signaling in AS supraspinous ligament(1) The expressions of p-Smad2/3, p-Smad1, Smad4 and RUNX2 increased in AS supraspinous ligament compared with control ligament.(2) The expressions of p-Smad2/3, p-Smad1, Smad4 and RUNX2 increased in AS-derived HSLFs compared with normal HSLFs.Conclusions:1. Histopathological changes were observed in AS supraspinous ligament compared with normal control.2. The expressions of TGFβR3, TGF-β1 and BMP2 increased in AS-derived supraspinous ligament and HSLFs.3. AS-derived HSLFs demonstrated auto-osteogenetic potential.4. TGF-β1 synergy with BMP2 induced osteogenesis of HSLFs overexpressing TGFβR3.5. TGF-β1 combined with BMP2 lost osteogenic induction effect on HSLFs with low expression of TGFβR3, suggesting the synergic effect of TGF-β1 and BMP2 depends on TGFβR3 overexpression.6. TGF-β1 combined with BMP2 increased TGFβR3 expression and thus up-regulated phosphorylations of Smad2/3 and Smad1, expressions of Smad4 and RUNX2.7. TGF-β1 combined with BMP2 lost the effect on TGFβR3/Smad signaling in HSLFs with low expression of TGFβR3, suggesting the synergic effect of TGF-β1 and BMP2 depends on TGFβR3 overexpression.8. Smad2/3 and Smad1 phosphorylations, Smad4 and RUNX2 expressions increased in AS-derived supraspinous ligament and HSLFs.9. In summary, our results demonstrated that TGF-β1 combined with BMP2 induced osteogenesis of AS-derived HSLFs via acting on overexpressed TGFβR3, and thus resulted in excessively activating both TGF-β/Smad and BMP/Smad signaling, including increased phosphorylation of Smad2/3 and Smad1, Smad4 expression and culminating in the induction of RUNX2. |
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