Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis
2020; Springer Nature; Volume: 12; Issue: 12 Linguagem: Inglês
10.15252/emmm.202012109
ISSN1757-4684
AutoresXiang Li, Siwen Chen, Zaiying Hu, Dongying Chen, Jianru Wang, Zemin Li, Zihao Li, Haowen Cui, Guo Dai, Lei Liu, Haitao Wang, Kuibo Zhang, Zhaomin Zheng, Zhongping Zhan, Hui Liu,
Tópico(s)Bone and Joint Diseases
ResumoArticle1 December 2020Open Access Source DataTransparent process Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis Xiang Li Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Siwen Chen Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Zaiying Hu Department of Rheumatology and Immunology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Dongying Chen Department of Rheumatology and Immunology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Jianru Wang Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Zemin Li Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Zihao Li Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Haowen Cui Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Guo Dai Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Lei Liu Department of Spine Surgery, The Fifth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Haitao Wang Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Kuibo Zhang Department of Spine Surgery, The Fifth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhaomin Zheng Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Zhongping Zhan Department of Rheumatology and Immunology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Hui Liu Corresponding Author [email protected] orcid.org/0000-0003-0754-402X Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Xiang Li Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Siwen Chen Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Zaiying Hu Department of Rheumatology and Immunology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, ChinaThese authors contributed equally to this work Search for more papers by this author Dongying Chen Department of Rheumatology and Immunology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Jianru Wang Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Zemin Li Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Zihao Li Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Haowen Cui Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Guo Dai Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Lei Liu Department of Spine Surgery, The Fifth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Haitao Wang Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Kuibo Zhang Department of Spine Surgery, The Fifth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhaomin Zheng Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Zhongping Zhan Department of Rheumatology and Immunology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Hui Liu Corresponding Author [email protected] orcid.org/0000-0003-0754-402X Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China Search for more papers by this author Author Information Xiang Li1,2, Siwen Chen1,2, Zaiying Hu3, Dongying Chen4, Jianru Wang1,2, Zemin Li1,2, Zihao Li1,2, Haowen Cui1,2, Guo Dai1,2, Lei Liu5, Haitao Wang1,2, Kuibo Zhang5, Zhaomin Zheng1,2, Zhongping Zhan4 and Hui Liu *,1,2 1Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China 2Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, China 3Department of Rheumatology and Immunology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China 4Department of Rheumatology and Immunology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China 5Department of Spine Surgery, The Fifth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China *Corresponding author. Tel: +86 138 2609 6992; Fax: +86 20 87331655; E-mail: [email protected] EMBO Mol Med (2020)12:e12109https://doi.org/10.15252/emmm.202012109 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pathological new bone formation is a typical pathological feature in ankylosing spondylitis (AS), and the underlying molecular mechanism remains elusive. Previous studies have shown that the calcium-sensing receptor (CaSR) is critical for osteogenic differentiation while also being highly involved in many inflammatory diseases. However, whether it plays a role in pathological new bone formation of AS has not been reported. Here, we report the first piece of evidence that expression of CaSR is aberrantly upregulated in entheseal tissues collected from AS patients and animal models with different hypothetical types of pathogenesis. Systemic inhibition of CaSR reduced the incidence of pathological new bone formation and the severity of the ankylosing phenotype in animal models. Activation of PLCγ signalling by CaSR promoted bone formation both in vitro and in vivo. In addition, various inflammatory cytokines induced upregulation of CaSR through NF-κB/p65 and JAK/Stat3 pathways in osteoblasts. These novel findings suggest that inflammation-induced aberrant upregulation of CaSR and activation of CaSR-PLCγ signalling in osteoblasts act as mediators of inflammation, affecting pathological new bone formation in AS. Synopsis This study identifies a critical role of inflammation-induced aberrant upregulation of CaSR in the process of pathological new bone formation in ankylosing spondylitis (AS). Targeting CaSR may be a novel potential therapeutic strategy to slow down the progression of axial structural ankylosis. Increased CaSR+ osteoblasts accumulate at the entheseal sites in AS patients and animal models. Inhibition of CaSR suppresses the ankylosing phenotype in animal models of AS. Activation of CaSR promotes osteogenic differentiation and pathological new bone formation through the PLCγ signalling pathway both in vitro and in vivo. Multiple inflammatory cytokines promote upregulation of CaSR in osteoblast through p65 and Stat3 pathways. CaSR might be a potential target for pathological new bone formation in AS. The paper explained Problem Upregulation of CaSR was found in AS spinal samples and animal models. Whether CaSR plays a role in the process of pathological new bone formation in AS is unknown. Results The expression of CaSR was upregulated in Runx2+ and OCN+ osteoblasts accumulated in the potential bone-forming sites in the tissues from AS patients and animal models. Systemic administration of CaSR antagonist NPS-2143 attenuated pathological new bone formation in animal models. Activation of PLCγsignalling by CaSR promoted osteogenic differentiation and pathological new bone formation. In addition, various inflammatory cytokines including TNFα and interleukin family induced upregulation of CaSR through NF-κB/p65 and JAK/Stat3 signalling pathways in osteoblasts. Impact Inflammation-induced upregulation of CaSR acts as a mediator of inflammation affecting pathological new bone formation in AS. Targeting CaSR may be a novel potential therapeutic strategy to slow the progression of axial structural ankylosis. Introduction Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the axial skeleton that is grouped under the term spondylarthritis (SpA) (Taurog et al, 2016; Sieper & Poddubnyy, 2017). In addition to inflammatory back pain and arthritic destruction, spinal ankylosis resulting from increasing pathological new bone formation is a typical feature of AS, causing disability and tremendous socioeconomic costs (Taurog et al, 2016). Although recent investigations and medications have focused on the aetiology and symptom-controlling therapy, the pathological mechanism of pathological new bone formation is still not clarified. Targeted treatment of axial skeletal ankylosis is demanded because the prognosis so far is unsatisfying (Deodhar, 2018; Molnar et al, 2018). The correlation between inflammation and pathological new bone formation remains enigmatic. Several studies have demonstrated that various types of osteogenic growth factors are involved in pathological new bone formation (Lories & Dougados, 2012; Ruiz-Heiland et al, 2012; Gonzalez-Chavez et al, 2016). Recently, we demonstrated that inflammation intensity-dependent expression of osteoinductive factors and Wnt family proteins by proinflammatory cells plays a vital role in inflammation-induced pathological new bone formation (Li et al, 2018). Thus, consistent with other studies, we confirmed that enhanced differentiation of osteogenic precursor cells induced by growth factors is an important molecular mechanism of inflammation-induced bone formation in the pathological microenvironment. However, other than the indirect effect through induction of osteogenic growth factors, whether inflammatory cytokines have direct positive regulatory effects on osteogenic differentiation of precursor cells remains unknown. Calcium-sensing receptor (CaSR) is a diametric family of G protein-coupled receptors, which have a critical role in modulating Ca2+ homeostasis via its role in the parathyroid glands and kidneys, which might affect bone mass. Normally, CaSR modulates systemic Ca2+ homeostasis by detecting increasing circulating concentrations of Ca2+, leading to intracellular signalling events that mediate reduced parathyroid hormone (PTH) secretion and a reduction in renal tubular Ca2+ reabsorption, therefore influencing skeletal homeostasis (Gowen et al, 2000; Hannan et al, 2016). Moreover, CaSR is abundantly expressed in cells of osteogenic lineage and in skeleton tissues. Several studies have confirmed that CaSR plays a pivotal role in modulating differentiation and mineralization of osteogenic precursor cells (Chang et al, 2008; Hendy & Canaff, 2016; Hannan et al, 2018b). Activation of CaSR downstream pathways in osteoblasts promotes osteogenic differentiation in vitro, while ablating CaSR expression specifically in osteoblasts suppresses osteogenic activity and the mineralizing function in bony calluses and leads to decreased bone mass in animal models (Chang et al, 2008; Dvorak-Ewell et al, 2011; Gonzalez-Vazquez et al, 2014; Cheng et al, 2020). Interestingly, previous studies showed that hyperstimulation of CaSR is a driving factor of the pathological progression in multiple inflammatory conditions, such as asthma and burn injury (Wu et al, 2015; Yarova et al, 2015; Cheng, 2016; Lee et al, 2017). Some studies reported that several inflammatory cytokines, including IL-6 and IL-1β, drive overexpression of CaSR and worsen the pathological condition in burn injury and sepsis (Hendy & Canaff, 2016; Klein et al, 2016). Given that activation of CaSR and downstream cellular signalling enhances osteogenic differentiation of osteoblasts, while aberrant expression of CaSR is commonly observed under inflammatory conditions, we speculated that CaSR might be involved in the pathological process of pathological new bone formation in the inflammatory microenvironment of AS. In the current study, we explored the role of CaSR in AS and observed overexpression of CaSR in osteoblasts in spinal entheseal tissues collected from AS patients. Furthermore, aberrant expression of CaSR and activation of its downstream signalling pathways was also confirmed in several classic animal models of AS with different hypothetical types of pathogenesis. Inhibition of CaSR activation attenuated pathological new bone formation phenotype in vivo and osteogenic differentiation in cultured hBMSCs in vitro. Furthermore, various inflammatory cytokines were confirmed to enhance CaSR expression in osteoblasts. Hence, our findings revealed a prominent role for CaSR in the interplay between inflammation and the process of ankylosis progression, which might shed more light on the enigma of inflammation-related pathological new bone formation in AS and propose a potential therapeutic target for slowing ankylosis progression. Results CaSR+ osteoblasts accumulate in spinal tissues from AS patients To investigate whether CaSR plays a role in the process of pathological new bone formation in AS, we first assessed expression of CaSR in uncalcified spinal ligament tissues (supraspinous ligament and interspinous ligament) from AS patients who underwent correction surgeries (Fig 1A). Immunohistochemical staining showed that increased CaSR+ cells were accumulated at the entheseal site of the spinal ligament attaching the spinous process from patients with AS than in age- and sex-matched controls. Cells that expressed Runx2, an osteogenic marker of naive osteoblasts, also accumulated at the same site as CaSR+ cells in the AS group (Fig 1B and C). RT–qPCR and Western blot analysis showed that expression of CaSR was increased in spinal ligament tissues of AS (Fig 1D and E). Immunofluorescence staining revealed that cells highly expressing CaSR were primarily Runx2+ naive osteoblasts (Fig 1F and G). SOFG (Safranine O-Fast Green) staining showed that spinous process and calcified ligament were indistinguishable. Meanwhile, increased cells accumulated at the uncalcified zone in the AS group (Fig 1H). In addition, immunofluorescence staining of the same region showed that the majority of CaSR+ cells expressed OCN, a mature osteoblast marker (Fig 1H and I). These results suggested that CaSR+ osteoblasts accumulated at potential pathological new bone-forming site of enthesis of the spinal ligament in AS patients. Figure 1. CaSR+ osteoblasts accumulated in the spinal entheseal tissues from AS patients An illustration of spinal ligament tissues collection. SOFG staining of spinal entheseal tissue and immunohistochemical analysis of CaSR+ and Runx2+ cells in human spinal tissue. The bottom panels show higher magnification of the boxed area in the top panels. Quantitative analysis of the number of CaSR+ and Runx2+ cells per area (mm2). n = 5, Student's t-test. RT–qPCR analysis of expression of CaSR in human spinal tissue. n = 3, Student's t-test. Western blot analysis of expression of CaSR in human spinal tissue. Immunofluorescence analysis of CaSR+ (Green) and Runx2+ (Red) cells in human spinal tissue. Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number per area (mm2) and CaSR+ cell percentage. n = 5, Student's t-test. Immunofluorescence analysis of CaSR+ (Green) and OCN+ (Red) cells in human spinal tissue. Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number per area (mm2). n = 5, Student's t-test. Data information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD. **P < 0.01 compared between groups. Scale bar: 100 μm. Download figure Download PowerPoint CaSR+ osteoblasts accumulate at pathological new bone-forming sites in animal models of AS A proteoglycan-induced spondylitis (PGIS) mouse model that exhibits both axial and peripheral inflammation was established to observe pathologic changes in spinal ankylosis (Hanyecz et al, 2004; Szabo et al, 2005; Adarichev & Glant, 2006; Berlo et al, 2006). The result of μCT analysis showed that spinal ankylosis gradually developed 24 weeks after PG induction (Fig 2A). The incidence of spinal ankylosis per cage increased from 0% to 33.3 ± 11.55% 8 weeks later and continued rising to 73.33 ± 11.55% at 24 weeks (Fig 2B). The bone volume (BV) of pathological new bone was increased 16 and 24 weeks after PG induction (Fig 2C). Meanwhile, RT–qPCR analysis showed that expression of CaSR was increased in spinal ligament tissues of the PGIS model (Fig 2D). H&E staining revealed pathological new bone formation at spinal entheseal sites attaching to the vertebral growth plate at 24 weeks. SOFG staining showed pathological process of new bone formation at 16 and 24 weeks through endochondral ossification (Fig 2E). Immunofluorescence staining revealed that cells expressing CaSR were primarily Runx2+ osteoblasts, which are analogous to human ligament tissues from AS patients. The number of CaSR+ Runx2+ osteoblasts was increased at spinal ligament tissues 16 and 24 weeks after PG induction compared to baseline (Fig 2E and F). Thirty weeks after PG immunization, most CaSR+ cells accumulated at spinal entheseal sites expressed OCN compared to baseline and 8 weeks (Fig 2G and H). Figure 2. CaSR+ osteoblasts mediate new bone formation in PGIS model μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis. n = 5 per group. μCT images of new bone (red area) in hind paws (2D and 3D reconstruction) in DBA/1 model compared to baseline. Incidence of spinal ankylosis in PGIS model. n = 5 per cage of total 3 cages per group, Fisher's exact test. Quantitative analysis of structural parameters of spinal ankylosis by μCT analysis. n = 5 per group, one-way ANOVA, Bonferroni post hoc. RT–qPCR analysis of expression of CaSR in spinal ligament tissue. n = 3, Student's t-test. H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model compare to baseline. n = 5 per group. Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in the spine specimen of PGIS model. n = 5 per group, one-way ANOVA, Bonferroni post hoc. H&E, SOFG and immunofluorescence analyses of the spine specimen of PGIS model at 30 weeks compared to baseline. GP: Growth plate. n = 5 per group. Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in the spine specimen of PGIS model. n = 5 per group, one-way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; GP: Growth plate; NB: New bone. Data shown as mean ± SD. **P < 0.01 compared between groups. Scale bar: 100 μm. Download figure Download PowerPoint An ageing DBA/1 arthritis animal model spontaneously developed pathological new bone in the hind paws without spinal involvement compared to the PGIS animal model. This model shares multiple similarities with AS in humans, including enthesitis and entheseal new bone formation (Matthys et al, 2003). μCT analysis showed that pathological new bone formation (red area) in the hind paws was increased at 16 and 20 weeks of age (Fig 3A). The incidence of pathological new bone formation in the hind paws per cage increased from 0% at 8 weeks of age to 29.63 ± 6.41% and 74.08 ± 6.41% at 16 and 20 weeks of age, respectively (Fig 3B). BV of pathological new bone was gradually increased at 16 and 20 weeks of age (Fig 3C). RT–qPCR analysis showed that expression of CaSR was increased in hind paw tissues (Fig 3D). H&E and SOFG staining demonstrated pathological new bone formation at entheseal sites on dorsal surfaces at 16 and 20 weeks of age (Fig 3E). Immunofluorescence staining revealed that cells highly expressing CaSR were mostly Runx2+ osteoblasts analogous to the phenomenon observed in human ligament tissues from AS patients. The number of CaSR+ Runx2+ osteoblasts increased at entheseal sites on dorsal surfaces at 16 and 20 weeks of age (Fig 3E and F). At 24 weeks of age, most CaSR+ cells were accumulated at entheseal sites on dorsal surfaces and expressed OCN compared to baseline (Fig 3G and H). Figure 3. CaSR+ osteoblasts mediate new bone formation in DBA/1 model μCT images of pathological new bone formation in DBA/1 model. n = 9 per group. Incidence of ankle pathological new bone formation in DBA/1 model. n = 9 per cage of total 3 cages per group, Fisher's exact test. Quantitative analysis of structural parameters of pathological new bone by μCT analysis. n = 9 per group, one-way ANOVA, Bonferroni post hoc. RT–qPCR analysis of expression of CaSR in hind paw tissue. n = 3, Student's t-test. H&E, SOFG and immunofluorescence analyses of pathological new bone formation in hind paws. n = 9 per group. Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in pathological new bone formation sites. n = 9 per group, one-way ANOVA, Bonferroni post hoc. H&E, SOFG and immunofluorescence analyses of pathological new bone in hind paws. n = 9 per group. Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in pathological new bone formation sites. n = 9 per group, one-way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone. Data shown as mean ± SD. **P < 0.01 compared between groups. Scale bar: 100 μm. Download figure Download PowerPoint A semi-Achilles tendon transection (SMTS) model was established to study pathological new bone formation due to the disruption of stress transmission at the posterior calcaneal tuberosity (PCT), as mechanical stress plays a prominent role in experimental AS (McClure, 1983; Jacques & McGonagle, 2014; Wang et al, 2018b). The results of μCT analysis showed that the entheseal bony projection gradually enlarged at 4 and 8 weeks compared to the sham-operated group (Fig EV1A). H&E and SOFG staining revealed that areas of both uncalcified fibrocartilage (UF) (with rounded chondrocytes morphology) and calcified fibrocartilage (CF) (with calcified extracellular matrix and separated from UF by a tidemark) of the Achilles tendon were increased 4 and 8 weeks after surgery (Fig EV1B–D) (Raspanti et al, 1996; Hibino et al, 2007). RT–qPCR analysis showed that expression of CaSR was increased in entheseal tissues in SMTS model (Fig EV1E). Immunofluorescence staining demonstrated that cells highly expressing CaSR were mostly Runx2+ osteoblasts, and the number of CaSR+ Runx2+ cells increased in both UF and CF areas 4 and 8 weeks after surgery (Fig EV1F and G). At 4 and 8 weeks, a large amount CaSR+ cells had accumulated at the UF and CF of the Achilles tendon and expressed OCN, indicating CaSR+ mature osteoblasts were involved in the development of bony projections (Fig EV1Hand I). Click here to expand this figure. Figure EV1. CaSR+ osteoblasts mediate pathological new bone formation of SMTS model μCT images of the PCT in SMTS model. Arrowhead shows bony projection. (2D). H&E and SOFG staining of Achilles tendon enthesis compartment in SMTS model. AT: Achilles tendon. Quantitative analysis of area of UF. n = 5, one-way ANOVA, Bonferroni post hoc. Quantitative analysis of area of CF. n = 5, one-way ANOVA, Bonferroni post hoc. RT–qPCR analysis of CaSR in SMTS model. n = 3, Student's t-test. Immunofluorescence analyses of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in Achilles tendon enthesis compartment. n = 5, one-way ANOVA, Bonferroni post hoc. Immunofluorescence analyses of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in Achilles tendon enthesis compartment. n = 5, one-way ANOVA, Bonferroni post hoc. Data information: Data shown as mean ± SD. *P < 0.05, **P < 0.01 compared between groups. Scale bar: 100μm. Download figure Download PowerPoint These results suggested that CaSR+ osteoblasts might be involved in pathological new bone formation in AS animal models with different types of hypothetical pathogenesis (Vieira-Sousa et al, 2015). Systemic inhibition of CaSR suppresses the ankylosing phenotype in animal models of AS To validate the critical role of CaSR in pathological new bone formation, a selective CaSR antagonist, NPS-2143, was administered systemically during the process of pathological new bone formation in these animal models. Following treatment with NPS-2143 in PGIS, μCT analysis revealed that spinal ankylosis was ameliorated compared to the control group (DMSO administration) at 24 weeks (Fig 4A). H&E and SOFG staining demonstrated that spinal pathological new bone formation was attenuated after NPS-2143 treatment (Fig 4B). Meanwhile, histological scores in the NPS-2143 group were decreased at 16 and 24 weeks (Fig EV2A). The incidence of spinal ankylosis and pathological new bone formation was decreased compared to the control group (DMSO administration) at 24 weeks (Fig 4C and D). Similarly, following treatment with NPS-2143 in the DBA/1 model, μCT analysis showed that pathological new bone formation (red area) in the hind paws was suppressed at 20 weeks of age (Fig 4E). Furthermore, the incidence of ankle pathological new bone formation was decreased compared to the control group (DMSO administration) at 20 weeks (Fig 4F and G). H&E and SOFG staining revealed that pathological new bone formation on both plantar and dorsal surfaces was decreased at 20 weeks of age in response to NPS-2143 treatment (Fig 4H). However, the clinical severity score of the NPS-2143 group did not decrease compared to the DMSO group (Fig EV2B). Similarly, in the SMTS model treated with NPS-2143, μCT analysis demonstrated reduction of bony projections 8 weeks after surgery (Fig 4I). H&E and SOFG staining showed both UF and CF areas were decreased in SMTS mice in response to NPS-2143 treatment (Fig 4J–L). Figure 4. Systemic inhibition of CaSR suppresses the ankylosing phenotype of AS animal models μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis. H&E and SOFG staining of spine in PGIS model. Incidence of spinal ankylosis in PGIS model. n = 5 per cage of total 6 cages per group, Fisher's exact test. Quantitative analysis of structural parameters of new bone by μCT analysis. n = 5, one-way ANOVA, Bonferroni post hoc. μCT images of new bone formation (red area) in hind paws (2D and 3D reconstruction). Incidence of new bone formation in DBA/1 model. n = 9 per cage of total 3 cages per group, Fisher's exact test. Quantitative analysis of structural parameters of new bone by μC
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