Extracellular PPM1A promotes mineralization of osteoblasts differentiation in ankylosing spondylitis via the FOXO1A-RUNX2 pathway.

Protein phosphatase magnesium-dependent 1A (PPM1A), serine/threonine protein phosphatase, in sera level was increased in patients with ankylosing spondylitis (AS). Preosteoblasts were differentiated actively to matured osteoblasts by intracellular PPM1A overexpression. However, it was unclear whether extracellular PPM1A contributes to the excessive bone-forming activity in AS. Here, we confirmed that PPM1A and runt-related transcription factor 2 (RUNX2) were increased in facet joints of AS. During osteoblasts differentiation, exogenous PPM1A treatment showed increased matrix mineralization in AS-osteoprogenitor cells accompanied by induction of RUNX2 and factor forkhead box O1A (FOXO1A) protein expressions. Moreover, upon growth condition, exogenous PPM1A treatment showed an increase in RUNX2 and FOXO1A protein expression and a decrease in phosphorylation at ser256 of FOXO1A protein in AS-osteoprogenitor cells, and positively regulated promoter activity of RUNX2 protein-binding motif. Mechanically, exogenous PPM1A treatment induced the dephosphorylation of transcription factor FOXO1A protein and translocation of FOXO1A protein into the nucleus for RUNX2 upregulation. Taken together, our results suggest that high PPM1A concentration promotes matrix mineralization in AS via the FOXO1A-RUNX2 pathway.

Clinical and genetic factors associated with radiographic damage in patients with ankylosing spondylitis.

OBJECTIVES: To identify clinical and genetic factors associated with severe radiographic damage in patients with ankylosing spondylitis (AS). METHODS: We newly generated genome-wide single nucleotide polymorphism data (833K) for 444 patients with AS. The severity of radiographic damage was assessed using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). To identify clinical and genetic factors associated with severe radiographic damage, multiple linear regression analyses were performed. Human AS-osteoprogenitor and control-osteoprogenitor cells were used for functional validation. RESULTS: The significant clinical factors of final mSASSS were baseline mSASSS (ß=0.796, p=3.22×10-75), peripheral joint arthritis (ß=-0.246, p=6.85×10-6), uveitis (ß=0.157, p=1.95×10-3), and smoking (ß=0.130, p=2.72×10-2) after adjusting for sex, age and disease duration. After adjusting significant clinical factors, the Ryanodine receptor 3 (RYR3) gene was associated with severe radiographic damage (p=1.00×10-6). For pathway analysis, the PI3K-Akt signalling pathway was associated with severe radiographic damage in AS (p=2.21×10-4, false discovery rate=0.040). Treatment with rhodamine B, a ligand of RYR3, dose-dependently induced matrix mineralisation of AS osteoprogenitors. However, the rhodamine B-induced accelerated matrix mineralisation was not definitive in control osteoprogenitors. Knockdown of RYR3 inhibited matrix mineralisation in SaOS2 cell lines. CONCLUSIONS: This study identified clinical and genetic factors that contributed to better understanding of the pathogenesis and biology associated with radiographic damage in AS.

CCL20 inhibition for treating inflammation in ankylosing spondylitis.

OBJECTIVES: Th17 cells are known to play a significant role in AS. C-C motif chemokine ligand 20 (CCL20) binds to C-C chemokine receptor 6 (CCR6) on Th17 cells, promoting their migration to inflammation sites. The aim of this research is to examine the effectiveness of CCL20 inhibition in treating inflammation in AS. METHODS: Mononuclear cells from peripheral blood (PBMC) and SF (SFMC) were collected from healthy individuals and AS. Flow cytometry was used to analyse cells producing inflammatory cytokines. CCL20 levels were determined using ELISA. The impact of CCL20 on Th17 cell migration was verified using a Trans-well migration assay. The in vivo efficacy of CCL20 inhibition was evaluated using an SKG mouse model. RESULTS: The presence of Th17 cells and CCL20 expressing cells was higher in SFMCs from AS patients compared with their PBMCs. The CCL20 level in AS SF was significantly higher than in OA patients. The percentage of Th17 cells in PBMCs from AS patients increased when exposed to CCL20, whereas the percentage of Th17 cells in SFMCs from AS patients decreased when treated with CCL20 inhibitor. The migration of Th17 cells was found to be influenced by CCL20, and this effect was counteracted by the CCL20 inhibitor. In the SKG mouse model, the use of CCL20 inhibitor significantly reduced joint inflammation. CONCLUSION: This research validates the critical role of CCL20 in AS and suggests that targeting CCL20 inhibition could serve as a novel therapeutic approach for AS treatment.

House dust mite-induced Akt-ERK1/2-C/EBP beta pathway triggers CCL20-mediated inflammation and epithelial-mesenchymal transition for airway remodeling.

House dust mite (HDM) allergens cause inflammatory responses and chronic allergic diseases such as bronchial asthma and atopic dermatitis. Here, we investigate the mechanism by which HDM induces C-C chemokine ligand 20 (CCL20) expression to promote chronic inflammation and airway remodeling in an HDM-induced bronchial asthma mouse model. We showed that HDM increased CCL20 levels via the Akt-ERK1/2-C/EBPß pathway. To investigate the role of CCL20 in chronic airway inflammation and remodeling, we made a mouse model of CCL20-induced bronchial asthma. Treatment of anti-CCL20Ab in this mouse model showed the reduced airway hyper-responsiveness and inflammatory cell infiltration into peribronchial region by neutralizing CCL20. In addition, CCL20 induced the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation through NLRP3 deubiquitination and transcriptional upregulation in BEAS-2B cells. As expected, anti-CCL20Ab markedly suppressed NLRP3 activation induced by CCL20. Moreover, HDM-induced CCL20 leads to epithelial-mesenchymal transition in the lung epithelium which appears to be an important regulator of airway remodeling in allergic asthma. We also found that anti-CCL20Ab attenuates airway inflammation and remodeling in an HDM-induced mouse model of bronchial asthma. Taken together, our results suggest that HDM-induced CCL20 is required for chronic inflammation that contributes airway remodeling in a mouse model of asthma.

IL-6 activates pathologic Th17 cell via STAT 3 phosphorylation in inflammatory joint of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease, which is characterized by inflammation of the axial skeleton and the peripheral arthritis. An increase in the number of Th17 cells in patients with AS has been reported. Although Th17 cells have been involved in the induction of inflammation, recent data suggest that not all Th17 cells are pathogenic, showing regulatory function of Th17 cell. Cells producing both interferon-gamma (IFN-γ) and interleukin (IL)-17 have been reported to be the main pathologic Th17 (pTh17) cells that induce inflammation at sites of joint. Emerging evidence demonstrated that IL-6 has a main role in regulating the balance between inflammatory and regulatory T cells. However, there is no direct study to assess pTh17 cell with IL-6 in AS. Therefore, we evaluated the effect of IL-6 on pTh17 cell activation, and it's mechanism, using ex vivo and mouse model of AS. As a result, we found that pTh17 cell is dependent on the cytokine milieu with IL-6. We confirmed pTh17 cells play a pathogenic role at sites of inflammation. As a mechanism, it was revealed that IL-6 induced STAT 3 phosphorylation contributes to the increased pTh17 responses in AS patients with peripheral arthritis. Though validation of our results is required, IL-6 inhibitor can be a promising treatment for inflammation in AS patients with peripheral arthritis.

Biological insights into systemic lupus erythematosus through an immune cell-specific transcriptome-wide association study.

OBJECTIVE: Genome-wide association studies (GWAS) have identified >100 risk loci for systemic lupus erythematosus (SLE), but the disease genes at most loci remain unclear, hampering translation of these genetic discoveries. We aimed to prioritise genes underlying the 110 SLE loci that were identified in the latest East Asian GWAS meta-analysis. METHODS: We built gene expression predictive models in blood B cells, CD4+ and CD8+ T cells, monocytes, natural killer cells and peripheral blood cells of 105 Japanese individuals. We performed a transcriptome-wide association study (TWAS) using data from the latest genome-wide association meta-analysis of 208 370 East Asians and searched for candidate genes using TWAS and three data-driven computational approaches. RESULTS: TWAS identified 171 genes for SLE (p<1.0×10-5); 114 (66.7%) showed significance only in a single cell type; 127 (74.3%) were in SLE GWAS loci. TWAS identified a strong association between CD83 and SLE (p<7.7×10-8). Meta-analysis of genetic associations in the existing 208 370 East Asian and additional 1498 cases and 3330 controls found a novel single-variant association at rs72836542 (OR=1.11, p=4.5×10-9) around CD83. For the 110 SLE loci, we identified 276 gene candidates, including 104 genes at recently-identified SLE novel loci. We demonstrated in vitro that putative causal variant rs61759532 exhibited an allele-specific regulatory effect on ACAP1, and that presence of the SLE risk allele decreased ACAP1 expression. CONCLUSIONS: Cell-level TWAS in six types of immune cells complemented SLE gene discovery and guided the identification of novel genetic associations. The gene findings shed biological insights into SLE genetic associations.

Transient receptor potential melastatin 2 governs stress-induced depressive-like behaviors.

Major depressive disorder (MDD) is a devastating disease that arises in a background of environmental risk factors, such as chronic stress, that produce reactive oxygen species (ROS) in the brain. The chronic stress-induced ROS production involves Ca2+ signals; however, the mechanism is poorly understood. Transient receptor potential melastatin type 2 (TRPM2) is a Ca2+-permeable cation channel that is highly expressed in the brain. Here we show that in animal models of chronic unpredictable stress (CUS), deletion of TRPM2 (Trpm2-/- ) produces antidepressant-like behaviors in mice. This phenotype correlates with reduced ROS, ROS-induced calpain activation, and enhanced phosphorylation of two Cdk5 targets including synapsin 1 and histone deacetylase 5 that are linked to synaptic function and gene expression, respectively. Moreover, TRPM2 mRNA expression is increased in hippocampal tissue samples from patients with MDD. Our findings suggest that TRPM2 is a key agent in stress-induced depression and a possible target for treating depression.

Thick ascending limb claudins are altered to increase calciuria and magnesiuria in metabolic acidosis.

Urinary calcium and magnesium wasting is a characteristic feature of metabolic acidosis, and this study focused on the role of the thick ascending limb of Henle's loop in metabolic acidosis-induced hypercalciuria and hypermagnesiuria because thick ascending limb is an important site of paracellular calcium and magnesium reabsorption. Male Sprague-Dawley rats were used to determine the effects of acid loading (by adding NH4Cl, 7.2 mmol/220 g body wt/day to food slurry for 7 days) on renal expression of claudins and then to evaluate whether the results were reversed by antagonizing calcium-sensing receptor (using NPS-2143). At the end of each animal experiment, the kidneys were harvested for immunoblotting, immunofluorescence microscopy, and quantitative PCR (qPCR) analysis of claudins and the calcium-sensing receptor. As expected, NH4Cl loading lowered urinary pH and increased excretion of urinary calcium and magnesium. In NH4Cl-loaded rats, renal protein and mRNA expression of claudin-16, and claudin-19, were decreased compared with controls. However, claudin-14 protein and mRNA increased in NH4Cl-loaded rats. Consistently, the calcium-sensing receptor protein and mRNA were up-regulated in NH4Cl-loaded rats. All these changes were reversed by NPS-2143 coadministration and were confirmed using immunofluorescence microscopy. Hypercalciuria and hypermagnesiuria in NH4Cl-loaded rats were significantly ameliorated by NPS-2143 coadministration as well. We conclude that in metabolic acidosis, claudin-16 and claudin-19 in the thick ascending limb are down-regulated to produce hypercalciuria and hypermagnesiuria via the calcium-sensing receptor.NEW & NOTEWORTHY This study found that the thick ascending limb of Henle's loop is involved in the mechanisms of hypercalciuria and hypermagnesiuria in metabolic acidosis. Specifically, expression of claudin-16/19 and claudin-14 was altered via up-regulation of calcium-sensing receptor in NH4Cl-induced metabolic acidosis. Our novel findings contribute to understanding the regulatory role of paracellular tight junction proteins in the thick ascending limb.

Meta-analysis of 208370 East Asians identifies 113 susceptibility loci for systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE), an autoimmune disorder, has been associated with nearly 100 susceptibility loci. Nevertheless, these loci only partially explain SLE heritability and their putative causal variants are rarely prioritised, which make challenging to elucidate disease biology. To detect new SLE loci and causal variants, we performed the largest genome-wide meta-analysis for SLE in East Asian populations. METHODS: We newly genotyped 10 029 SLE cases and 180 167 controls and subsequently meta-analysed them jointly with 3348 SLE cases and 14 826 controls from published studies in East Asians. We further applied a Bayesian statistical approach to localise the putative causal variants for SLE associations. RESULTS: We identified 113 genetic regions including 46 novel loci at genome-wide significance (p<5×10-8). Conditional analysis detected 233 association signals within these loci, which suggest widespread allelic heterogeneity. We detected genome-wide associations at six new missense variants. Bayesian statistical fine-mapping analysis prioritised the putative causal variants to a small set of variants (95% credible set size ≤10) for 28 association signals. We identified 110 putative causal variants with posterior probabilities ≥0.1 for 57 SLE loci, among which we prioritised 10 most likely putative causal variants (posterior probability ≥0.8). Linkage disequilibrium score regression detected genetic correlations for SLE with albumin/globulin ratio (rg=-0.242) and non-albumin protein (rg=0.238). CONCLUSION: This study reiterates the power of large-scale genome-wide meta-analysis for novel genetic discovery. These findings shed light on genetic and biological understandings of SLE.

STAT3 phosphorylation inhibition for treating inflammation and new bone formation in ankylosing spondylitis.

OBJECTIVE: AS is a rheumatic disease characterized by chronic inflammation and bony ankylosis. This study was to evaluate whether a signal transducer and activator of transcription 3 phosphorylation inhibitor (stat3-p Inh) could treat both chronic inflammation and bone formation in AS. METHODS: Primary AS osteoprogenitor cells and spinal entheseal cells were examined for osteogenic differentiation. SF mononuclear cells (SFMCs) and lamina propria mononuclear cells (LPMCs) were obtained from AS patients. Inflammatory cytokine-producing cells were analysed using flow cytometry and ELISA. Female SKG mice were treated with stat3-p Inh, IL-17A blocker or vehicle. Inflammation and new bone formation were evaluated using immunohistochemistry, PET and micro-CT. RESULTS: In the SKG mouse model, stat3-p Inh significantly suppressed arthritis, enthesitis, spondylitis and ileitis. In experiments culturing SFMCs and LPMCs, the frequencies of IFN-γ-, IL-17A- and TNF-α-producing cells were significantly decreased after stat3-p Inh treatment. When comparing current treatments for AS, stat3-p Inh showed a comparable suppression effect on osteogenesis to Janus kinase inhibitor or IL-17A blocker in AS-osteoprogenitor cells. Stat3-p Inh suppressed differentiation and mineralization of AS-osteoprogenitor cells and entheseal cells toward osteoblasts. Micro-CT analysis of hind paws revealed less new bone formation in stat3-p Inh-treated mice than vehicle-treated mice (P = 0.005). Hind paw and spinal new bone formation were similar between stat3-p Inh- and anti-IL-17A-treated SKG mice (P = 0.874 and P = 0.117, respectively). CONCLUSION: Stat-3p inhibition is a promising treatment for both inflammation and new bone formation in AS.

TGFß1 Suppressed Matrix Mineralization of Osteoblasts Differentiation by Regulating SMURF1-C/EBPß-DKK1 Axis.

Transforming growth factor ß1 (TGFß1) is a major mediator in the modulation of osteoblast differentiation. However, the underlying molecular mechanism is still not fully understood. Here, we show that TGFß1 has a dual stage-dependent role in osteoblast differentiation; TGFß1 induced matrix maturation but inhibited matrix mineralization. We discovered the underlying mechanism of the TGFß1 inhibitory role in mineralization using human osteoprogenitors. In particular, the matrix mineralization-related genes of osteoblasts such as osteocalcin (OCN), Dickkopf 1 (DKK1), and CCAAT/enhancer-binding protein beta (C/EBPß) were dramatically suppressed by TGFß1 treatment. The suppressive effects of TGFß1 were reversed with anti-TGFß1 treatment. Mechanically, TGFß1 decreased protein levels of C/EBPß without changing mRNA levels and reduced both mRNA and protein levels of DKK1. The degradation of the C/EBPß protein by TGFß1 was dependent on the ubiquitin-proteasome pathway. TGFß1 degraded the C/EBPß protein by inducing the expression of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (SMURF1) at the transcript level, thereby reducing the C/EBPß-DKK1 regulatory mechanism. Collectively, our findings suggest that TGFß1 suppressed the matrix mineralization of osteoblast differentiation by regulating the SMURF1-C/EBPß-DKK1 axis.

Identification and characterization of human bone-derived cells.

This study was designed to identify and characterize primary bone-derived cells (BdCs) and investigate the potential role of osteoblast differentiation. Primary BdCs were isolated from surgical bone for comparative analysis with mesenchymal stem cells (MSCs) and fetal osteoblasts (FOBs) and for potential differentiation to mature osteoblasts. Using three different cells, we successfully cultivated human osteoblast differentiation and activity which were evaluated using microarray and biochemical methods. BdCs are more correlated to MSCs in bioinformatics result and similar with FOBs in gene expression. In particular, Osterix, osteoprogenitor marker, was high expressed in BdCs, while the expression in MSCs and FOBs were very low. Furthermore, BdCs exhibited a marked alkaline phosphatase (ALP) expression, early stage of osteogenic marker, and retained osteogenic properties and physiological changes into maturation as in FOBs. BdCs also showed an increase in bone morphogenic protein 2 (BMP2), osteopontin (OPN), and osteocalcin (OCN) mRNA expressions during differentiation. This study suggests that BdCs may be osteoprogenitor cells or undifferentiated preosteoblasts with strong capacity to differentiate toward mature osteoblasts.

Accelerated osteogenic differentiation of human bone-derived cells in ankylosing spondylitis.

Ankylosing spondylitis (AS) is characterized by excessive bone formation with syndesmophytes, leading to bony ankylosis. The contribution of osteoblasts to the pathogenesis of ankylosis is poorly understood. The aim of this study was to determine molecular differences between disease controls (Ct) and AS bone-derived cells (BdCs) during osteogenic differentiation with or without inflammation using AS patient serum. We confirmed osteoblastic differentiation of Ct and AS BdCs under osteogenic medium by observing morphological changes and measuring osteoblastic differentiation markers. Osteoblast differentiation was detected by alkaline phosphatase (ALP) staining and activity, and alizarin red and hydroxyapatite staining. Osteoblast-specific markers were analyzed by quantitative reverse-transcriptase-polymerase chain reaction, immunoblotting, and immunostaining. To examine the effects of inflammation, we added AS and healthy control serum to Ct and AS BdCs, and then analyzed osteoblast-specific markers. AS BdCs showed elevated basal intercellular and extracellular ALP activity compared to Ct. When osteoblast differentiation was induced, AS BdCs exhibited higher expression of osteoblast-specific marker genes and faster mineralization than Ct, indicating that these cells differentiated more rapidly into osteoblasts. ALP activity and mineralization accelerated when serum from AS patients was added to Ct and AS BdCs. Our results revealed that AS BdCs showed significantly increased osteoblastic activity and differentiation capacity by regulating osteoblast-specific transcription factors and proteins compared to Ct BdCs. Active inflammation of AS serum accelerated osteoblastic activity. Our study could provide useful basic data for understanding the molecular mechanism of ankylosis in AS.

PCGF2 negatively regulates arsenic trioxide-induced PML-RARA protein degradation via UBE2I inhibition in NB4 cells.

Arsenic trioxide (ATO) is a therapeutic agent for acute promyelocytic leukemia (APL) which induces PML-RARA protein degradation via enhanced UBE2I-mediated sumoylation. PCGF2, a Polycomb group protein, has been suggested as an anti-SUMO E3 protein by inhibiting the sumoylation of UBE2I substrates, HSF2 and RANGAP1, via direct interaction. Thus, we hypothesized that PCGF2 might play a role in ATO-induced PML-RARA degradation by interacting with UBE2I. PCGF2 protein was down-regulated upon ATO treatment in human APL cell line, NB4. Knockdown of PCGF2 in NB4 cells, in the absence of ATO treatment, was sufficient to induce sumoylation-, ubiquitylation- and PML nuclear body-mediated degradation of PML-RARA protein. Moreover, overexpression of PCGF2 protected ATO-mediated degradation of ectopic and endogenous PML-RARA in 293T and NB4 cells, respectively. In 293T cells, UBE2I-mediated PML-RARA degradation was reduced upon PCGF2 co-expression. In addition, UBE2I-mediated sumoylation of PML-RARA was reduced upon PCGF2 co-expression and PCGF2-UBE2I interaction was confirmed by co-immunoprecipitation. Likewise, endogenous PCGF2-UBE2I interaction was detected by co-immunoprecipitation and immunofluorescence assays in NB4 cells. Intriguingly, upon ATO-treatment, such interaction was disrupted and UBE2I was co-immunoprecipitated or co-localized with its SUMO substrate, PML-RARA. Taken together, our results suggested a novel role of PCGF2 in ATO-mediated degradation of PML-RARA that PCGF2 might act as a negative regulator of UBE2I via direct interaction.

A novel role for bone-derived cells in ankylosing spondylitis: Focus on IL-23.

The main aim of this study are to explore the role of bone-derived cells (BdCs) in ankylosing spondylitis (AS) and determine the underlying molecular mechanisms of IL-23 production. Primary BdCs were isolated from diced bone of facet joints obtained during surgery from seven AS patients and seven disease control (Ct) patients. Osteoblastic activity of BdCs was assessed by measuring their alkaline phosphatase activity and by alizarin red staining. Osteoblast and endoplasmic reticulum (ER) stress-related genes were assessed by quantitative PCR, immunoblotting, immunofluorescence, and immunohistochemistry. In addition, expression of IL-23 in response to BIX (selective BIP inducer X)-induced ER stress was evaluated by qPCR and ELISA. Protein interaction and binding to IL-23 promoter were confirmed by Immunoprecipitation and Chromatin immunoprecipitation, respectively. Transcript levels of genes involved in osteoblast function, as well as of the ER stress marker were higher in the AS group than the Ct group, and elevated RUNX2, BiP and IL-23 expression were observed in the BdCs, serum, and bone biopsies from the AS group. BIX-induced ER stress stimulated osteoblastic activity and IL-23 secretion by upregulating RUNX2 expression. Furthermore, in AS BdCs, RUNX2 interacted with C/EBPß to bind to IL-23 promoter and RUNX2 knockdown suppressed IL-23 secretion. These finding may provide a molecular mechanism involved in sustained ER stress in AS BdCs stimulates the activation of RUNX2 and C/EBPß genes, leading to IL-23 production.

Swimming exercise alleviates pathological bone features in curdlan-injected SKG mice by inducing irisin expression.

This study assessed the therapeutic potential of swimming exercise in the curdlan-injected SKG mouse model and investigated the modulatory effects of irisin on inflammation. Curdlan-injected SKG were randomly assigned to either a home-cage group or a swimming group for 6 weeks. Changes in clinical arthritis scores and ankle thickness were measured weekly. Post-swimming program, mice were anesthetized for collection of vastus lateralis muscle and blood, which was followed by histological analysis, micro-CT imaging of the ankle joints, and the measurement of pro-inflammatory cytokines and irisin levels. Additionally, curdlan-injected SKG mice were intravenously injected with recombinant irisin protein and observed. Finally, serum levels of irisin in healthy control and ankylosing spondylitis (AS) patient groups were measured by ELISA. The swimming group of curdlan-injected SKG mice exhibited significant improvements in arthritis and enthesitis compared to the home-cage group. In particular, micro-CT and histological analyses revealed a notable reduction in pathological bone features in the swimming group compared to the home-cage group. Muscle endurance was also enhanced in the swimming group compared to the home-cage group, as determined by the wire-hanging test. Intriguingly, irisin levels not only were statistically increased in the swimming group but, also, TNF-α, IL-1ß, and IL-6 levels were decreased. Additionally, injection of irisin protein slightly attenuated both arthritis and enthesitis in curdlan-injected SKG mice. Meanwhile, irisin serum levels were declined in AS patients. Overall, we found that swimming exercise attenuated pathological bone features in an AS animal model, potentially mediated by increased irisin serum levels with associated anti-inflammatory effects.

HIF-1α and MIF enhance neutrophil-driven type 3 immunity and chondrogenesis in a murine spondyloarthritis model.

The hallmarks of spondyloarthritis (SpA) are type 3 immunity-driven inflammation and new bone formation (NBF). Macrophage migration inhibitory factor (MIF) was found to be a key driver of the pathogenesis of SpA by amplifying type 3 immunity, yet MIF-interacting molecules and networks remain elusive. Herein, we identified hypoxia-inducible factor-1 alpha (HIF1A) as an interacting partner molecule of MIF that drives SpA pathologies, including inflammation and NBF. HIF1A expression was increased in the joint tissues and synovial fluid of SpA patients and curdlan-injected SKG (curdlan-SKG) mice compared to the respective controls. Under hypoxic conditions in which HIF1A was stabilized, human and mouse neutrophils exhibited substantially increased expression of MIF and IL-23, an upstream type 3 immunity-related cytokine. Similar to MIF, systemic overexpression of IL-23 induced SpA pathology in SKG mice, while the injection of a HIF1A-selective inhibitor (PX-478) into curdlan-SKG mice prevented or attenuated SpA pathology, as indicated by a marked reduction in the expression of MIF and IL-23. Furthermore, genetic deletion of MIF or HIF1A inhibition with PX-478 in IL-23-overexpressing SKG mice did not induce evident arthritis or NBF, despite the presence of psoriasis-like dermatitis and blepharitis. We also found that MIF- and IL-23-expressing neutrophils infiltrated areas of the NBF in curdlan-SKG mice. These neutrophils potentially increased chondrogenesis and cell proliferation via the upregulation of STAT3 in periosteal cells and ligamental cells during endochondral ossification. Together, these results provide supporting evidence for an MIF/HIF1A regulatory network, and inhibition of HIF1A may be a novel therapeutic approach for SpA by suppressing type 3 immunity-mediated inflammation and NBF.

The complement factor H-related protein-5 (CFHR5) exacerbates pathological bone formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease, characterized by excessive new bone formation. We previously reported that the complement factor H-related protein-5 (CFHR5), a member of the human factor H protein family, is significantly elevated in patients with AS compared to other rheumatic diseases. However, the pathophysiological mechanism underlying new bone formation by CFHR5 is not fully understood. In this study, we revealed that CFHR5 and proinflammatory cytokines (TNF, IL-6, IL-17A, and IL-23) were elevated in the AS group compared to the HC group. Correlation analysis revealed that CFHR5 levels were not significantly associated with proinflammatory cytokines, while CFHR5 levels in AS were only positively correlated with the high CRP group. Notably, treatment with soluble CFHR5 has no effect on clinical arthritis scores and thickness at hind paw in curdlan-injected SKG, but significantly increased the ectopic bone formation at the calcaneus and tibia bones of the ankle as revealed by micro-CT image and quantification. Basal CFHR5 expression was upregulated in AS-osteoprogenitors compared to control cells. Also, treatment with CFHR5 remarkedly induced bone mineralization status of AS-osteoprogenitors during osteogenic differentiation accompanied by MMP13 expression. We provide the first evidence demonstrating that CFHR5 can exacerbate the pathological bone formation of AS. Therapeutic modulation of CFHR5 could be promising for future treatment of AS. KEY MESSAGES: Serum level of CFHR5 is elevated and positively correlated with high CRP group of AS patients. Recombinant CFHR5 protein contributes to pathological bone formation in in vivo model of AS. CFHR5 is highly expressed in AS-osteoprogenitors compared to disease control. Recombinant CFHR5 protein increased bone mineralization accompanied by MMP13 in vitro model of AS.

SUZ12 inhibition attenuates cell proliferation of glioblastoma via post-translational regulation of CDKN1B.

BACKGROUND: Human gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. Differential expression of Polycomb repressive complex 2 (PRC2) has been reported in various subtypes of glioma. However, the role of PRC2 in uncontrolled growth in glioma and its underlying molecular mechanisms remain to be elucidated. OBJECTIVE: We aimed to investigate the functional role of PRC2 in human glioblastoma cell growth by silencing SUZ12, the non-catalytic core component of PRC2. METHODS: Knockdown of SUZ12 was achieved by infecting T98G cells with lentivirus carrying sequences specifically targeting SUZ12 (shSUZ12). Gene expression was examined by quantitative PCR and western analysis. The impact of shSUZ12 on cell growth was assessed using a cell proliferation assay. Cell cycle distribution was analyzed by flow cytometry, and protein stability was evaluated in cycloheximide-treated cells. Subcellular localization was examined through immunofluorescence staining and biochemical cytoplasmic-nuclear fractionation. Gene expression analysis was also performed on human specimens from normal brain and glioblastoma patients. RESULTS: SUZ12 knockdown (SUZ12 KD) led to widespread decrease in the PRC2-specific histone mark, accompanied by a slowdown of cell proliferation through G1 arrest. In SUZ12 KD cells, the degradation of CDKN1B protein was reduced, resulting from alterations in the MYC-SKP2-CDKN1B axis. Furthermore, nuclear localization of CDKN1B was enhanced in SUZ12 KD cells. Analysis of human glioblastoma samples yielded increased expression of EZH2 and MYC along with reduced CDKN1B compared to normal human brain tissue. CONCLUSION: Our findings suggest a novel role for SUZ12 in cell proliferation through post-translational regulation of CDKN1B in glioblastoma.

Comparison of a Cryopneumatic Compression Device and Ice Packs for Cryotherapy Following Anterior Cruciate Ligament Reconstruction.

Background: The purpose of the current study was to evaluate and compare the effectiveness of a cryopneumatic compression device with that of standard ice packs following arthroscopic anterior cruciate ligament (ACL) reconstruction, with a primary focus on early postoperative pain. Methods: Participants were divided into two groups: cryopneumatic compression device group (CC group) and standard ice pack group (IP group). Patients in the CC Group (28 patients) received a cryopneumatic compression device (CTC-7, Daesung Maref) treatment, while patients in the IP group (28 patients) received standard ice pack cryotherapy postoperatively. All cryotherapy was applied three times (every 8 hours) per day for 20 minutes until discharge (postoperative day 7). Pain scores were assessed preoperatively and at 4, 7, and 14 days after surgery, and the primary outcome for analysis was pain at postoperative day 4 assessed using a visual analog scale (VAS). Other variables were opioid and rescue medication use, knee and thigh circumferences, postoperative drainage, and joint effusion quantified by a three-dimensional magnetic resonance imaging (MRI) reconstruction model. Results: The mean pain VAS score and difference in VAS relative to the preoperative measurements for postoperative day 4 were significantly lower in the CC group than in the IP group (p = 0.001 and p = 0.007, respectively). The sum of postoperative drainage and effusion quantified by MRI showed a significant reduction of postoperative effusion in the CC group compared to the IP group (p = 0.015). The average total rescue medication consumption was comparable between the two groups. Circumferential measurements at days 7 and 14 postoperatively relative to those at day 4 (index day) demonstrated no significant differences between the groups. Conclusions: Compared to standard ice packs, application of cryopneumatic compression was associated with a significant reduction in VAS pain scores and joint effusion during the early postoperative period following ACL reconstruction.