Kashin-Beck Disease Cartilage Research Articles

A comparative metabolomic analysis reveals the metabolic variations among cartilage of Kashin-Beck disease and osteoarthritis.

The metabolic variations between the cartilage of osteoarthritis (OA) and Kashin-Beck disease (KBD) remain largely unknown. Our study aimed to address this by conducting a comparative analysis of the metabolic profiles present in the cartilage of KBD and OA. Cartilage samples from patients with KBD (n = 10) and patients with OA (n = 10) were collected during total knee arthroplasty surgery. An untargeted metabolomics approach using liquid chromatography coupled with mass spectrometry (LC-MS) was conducted to investigate the metabolomics profiles of KBD and OA. LC-MS raw data files were converted into mzXML format and then processed by the XCMS, CAMERA, and metaX toolbox implemented with R software. The online Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate the metabolites by matching the exact molecular mass data of samples with those from the database. A total of 807 ion features were identified for KBD and OA, including 577 positive (240 for upregulated and 337 for downregulated) and 230 negative (107 for upregulated and 123 for downregulated) ions. After annotation, LC-MS identified significant expressions of ten upregulated and eight downregulated second-level metabolites, and 183 upregulated and 162 downregulated first-level metabolites between KBD and OA. We identified differentially expressed second-level metabolites that are highly associated with cartilage damage, including dimethyl sulfoxide, uric acid, and betaine. These metabolites exist in sulphur metabolism, purine metabolism, and glycine, serine, and threonine metabolism. This comprehensive comparative analysis of metabolism in OA and KBD cartilage provides new evidence of differences in the pathogenetic mechanisms underlying cartilage damage in these two conditions.

Activation of Notch Signaling Pathway is involved in Extracellular Matrix Degradation in human induced pluripotent stem cells chondrocytes induced by HT-2 toxin

Notch signaling regulates cartilage formation and homeostasis. Kashin-Beck Disease (KBD), an endemic osteochondropathy, is characterized by severe cartilage degradation. The etiology of KBD is related to the exposure of HT-2 toxin, a mycotoxin and primary metabolite of T-2 toxin. This study aims to explore the role of HT-2 toxin in the Notch signaling regulation and extracellular matrix (ECM) metabolism of hiPSCs-Chondrocytes. Immunohistochemistry and qRT-PCR were employed to investigate the expression of Notch pathway molecules in KBD articular cartilage and primary chondrocytes. hiPSCs-Chondrocytes, derived from hiPSCs, were treated with 100 ng/mL HT-2 toxin and the γ-secretase inhibitor (DAPT) for 48h, respectively. The markers related to the Notch signaling pathway and ECM were assessed using qRT-PCR and Western blot. Notch pathway dysregulation was prominent in KBD cartilage. HT-2 toxin exposure caused cytotoxicity in hiPSCs-Chondrocytes, and activated Notch signaling by increasing the mRNA and protein levels of NOTCH1 and HES1. HT-2 toxin also upregulated ECM catabolic enzymes and downregulated ECM components (COL2A1 and ACAN), indicating ECM degradation. DAPT-mediated Notch signaling inhibition suppressed the mRNA and protein level of ADAMTS5 expression while enhancing ECM component expression in hiPSCs-Chondrocytes. This study suggests that HT-2 toxin may induce ECM degradation in hiPSCs-Chondrocytes through activating Notch signaling.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

WISP1 Is Involved in the Pathogenesis of Kashin-Beck Disease via the Autophagy Pathway.

Kashin-Beck disease (KBD) is a kind of endemic and chronic osteochondropathy in China. This study aims to explore the functional relevance and potential mechanism of Wnt-inducible signaling pathway protein 1 (WISP1) in the pathogenesis of KBD. KBD and control cartilage specimens were collected for tissue section observation and primary chondrocyte culture. Firstly, the morphological and histopathological observations were made under a light and electron microscope. Then, the expression levels of WISP1 as well as molecular markers related to the autophagy pathway and extracellular matrix (ECM) synthesis were detected in KBD and control chondrocytes by qRT-PCR, Western blot, and immunohistochemistry. Furthermore, the lentiviral transfection technique was applied to make a WISP1 knockdown cell model based on KBD chondrocytes. In vitro intervention experiments were conducted on the C28/I2 human chondrocyte cell line using human recombinant WISP1 (rWISP1). The results showed that the autolysosome appeared in the KBD chondrocytes. The expression of WISP1 was significantly higher in KBD chondrocytes. Additionally, T-2 toxin, a risk factor for KBD onset, could up-regulate the expression of WISP1 in C28/I2. The autophagy markers ATG4C and LC3II were upregulated after the low-concentration treatment of T-2 toxin and downregulated after the high-concentration treatment. After knocking down WISP1 expression in KBD chondrocytes, MAP1LC3B decreased while ATG4C and COL2A1 increased. Moreover, the rWISP1 protein treatment in C28/I2 chondrocytes could upregulate the expression of ATG4C and LC3II at the beginning and downregulate them then. Our study suggested that WISP1 might play a role in the pathogenesis of KBD through autophagy.

A Metabolomics Study of Feces Revealed That a Disturbance of Selenium-Centered Metabolic Bioprocess Was Involved in Kashin-Beck Disease, an Osteoarthropathy Endemic to China.

Background: Kashin-Beck disease (KBD) is a distinct osteoarthropathy in China with an unclear pathogenesis. This study aims to explore whether perturbations in the intestine metabolome could be linked to KBD individuals. Methods: An investigation was conducted in KBD endemic villages and fecal samples were collected. After applying inclusion and exclusion criteria, a total of 75 subjects were enrolled for this study, including 46 KBD (including 19 Grade I KBD and 27 Grade II KBD) and 29 controls. Untargeted metabolomics analysis was performed on the platform of UHPLC-MS. PLS-DA and OPLS-DA were conducted to compare the groups and identify the differential metabolites (DMs). Pathway analysis was conducted on MPaLA platform to explore the functional implication of the DMs. Results: Metabolomics analysis showed that compared with the control group, KBD individuals have a total of 584 differential metabolites with dysregulated levels such as adrenic acid (log2FC = -1.87, VIP = 4.84, p = 7.63 × 10-7), hydrogen phosphate (log2FC = -2.57, VIP = 1.27, p = 1.02 × 10-3), taurochenodeoxycholic acid (VIP = 1.16, log2FC = -3.24, p = 0.03), prostaglandin E3 (VIP = 1.17, log2FC = 2.67, p = 5.61 × 10-4), etc. Pathway analysis revealed several significantly perturbed pathways associated with KBD such as selenium micronutrient network (Q value = 3.11 × 10-3, Wikipathways), metabolism of lipids (Q value = 8.43 × 10-4, Reactome), free fatty acid receptors (Q value = 3.99 × 10-3, Reactome), and recycling of bile acids and salts (Q value = 2.98 × 10-3, Reactome). Subgroup comparisons found a total of 267 differential metabolites were shared by KBD vs. control, KBD II vs. control, and KBD I vs. control, while little difference was found between KBD II and KBD I (only one differential metabolite detected). Conclusions: KBD individuals showed distinct metabolic features characterized by perturbations in lipid metabolism and selenium-related bioprocesses. Our findings suggest that the loss of nutrients metabolism balance in intestine was involved in KBD pathogenesis. Linking the nutrients metabolism (especially selenium and lipid) to KBD cartilage damage should be a future direction of KBD study.

Detection of selenoprotein transcriptome in chondrocytes of patients with Kashin-Beck disease.

Background: Kashin-Beck disease (KBD) is a deformed osteochondral disease with a chronic progression that is restrictively distributed in eastern Siberia, North Korea, and some areas of China, and selenium deficiency has been identified as an important factor in the pathogenesis of this disease in recent years. Objective: The aim of this study is to investigate the selenoprotein transcriptome in chondrocytes and define the contribution of selenoprotein to KBD pathogenesis. Methods: Three cartilage samples were collected from the lateral tibial plateau of adult KBD patients and normal controls paired by age and sex for real-time quantitative polymerase chain reaction (RT-qPCR) to detect the mRNA expression of 25 selenoprotein genes in chondrocytes. Six other samples were collected from adult KBD patients and normal controls. In addition, immunohistochemistry was used on four adolescent KBD samples and seven normal controls (IHC) to determine the expression of proteins screened by RT-qPCR results that had different gene levels. Results: Increased mRNA expression of GPX1 and GPX3 was observed in chondrocytes, and stronger positive staining was displayed in the cartilage from both adult and adolescent patients. The mRNA levels of DIO1, DIO2, and DIO3 were increased in KBD chondrocytes; however, the percentage of positive staining decreased in the KBD cartilage of adults. Conclusion: The selenoprotein transcriptome, mainly the glutathione peroxidase (GPX) and deiodinase (DIO) families were altered in KBD and might play a vital role in the pathogenesis of KBD.

Abnormal expression of TSG-6 disturbs extracellular matrix homeostasis in chondrocytes from endemic osteoarthritis.

Background and aims: Kashin-Beck disease (KBD) is a unique endemic osteochondropathy with unclear pathogenesis in China. T-2 toxin exposure has been identified as a significant risk factor of KBD. However, the mechanism of articular cartilage damage induced by T-2 toxin is a conundrum. We explored the role of the extracellular matrix-related gene TSG-6 in the articular chondrocyte damage process under the exposure of HT-2 toxin. Methods: TSG-6 was identified as a candidate gene by mining our previous gene expression profiling of KBD and verified by qRT-PCR and immunohistochemistry. Then, TSG-6 was silenced by RNA interference technology and overexpressed induction by TNF-α. Gradient concentrations of HT-2 toxin were added to intervene with C28/I2 chondrocytes. MTT was used to observe the proliferation and cell viability of chondrocytes, and qRT-PCR was utilized to detect the expression changes of MMP1, MMP3, MMP13, COL2A1, and proteoglycan before and after treatments for verification. Results: TSG-6 was upregulated in KBD chondrocytes at the mRNA level and upregulated in the superficial, middle, and deep zones of KBD cartilage. After TSG-6 silencing, the expression of MMP1, MMP3, MMP13, and proteoglycan was significantly decreased while COL2A1 expression was significantly increased, which was reversed after the overexpression of TSG-6 induced by TNF-α (p < 0.05). The survival rate of chondrocytes was correspondingly reduced with an increase in the HT-2 toxin concentration. Compared with the blank control group, the expression of MMPs was increased in the intervention group of HT-2 toxin, while the expression of proteoglycan and COL2A1 decreased (p < 0.05). Conclusion: The upregulation of the TSG-6 gene may play a role in promoting the damage and degradation of the extracellular matrix in KBD chondrocytes under the exposure of HT-2 toxin.

Identification of N-Glycoproteins of Knee Cartilage from Adult Osteoarthritis and Kashin-Beck Disease Based on Quantitative Glycoproteomics, Compared with Normal Control Cartilage.

Glycoproteins are involved in the development of many diseases, while the type and content of N-glycoproteins in the cartilage of osteoarthritis (OA) and Kashin–Beck disease (KBD) are still unclear. This research aims to identify N-glycoproteins in knee cartilage patients with OA and KBD compared with normal control (N) adults. The cartilage samples were collected from gender- and age-matched OA (n = 9), KBD (n = 9) patients, and N (n = 9) adults. Glycoproteomics and label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) obtained N-glycoproteins of KBD and OA. A total of 594 N-glycoproteins and 1146 N-glycosylation peptides were identified. The identified data were further compared and analyzed with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein–Protein Interactions (PPI). Pairwise comparison of the glycoproteins detected in the three groups showed that integrin beta-1 (ITGB1), collagen alpha-1 (II) chain (COL2A1), collagen alpha-1 (VII) chain (COL7A1), carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 4 (CHST-4), thrombospondin 2 (THBS2), bone morphogenetic protein 8A (BMP8A), tenascin-C (TNC), lysosome-associated membrane protein (LAMP2), and beta-glucuronidase (GUSB) were significantly differentially expressed. GO results suggested N-glycoproteins mainly belonged to protein metabolic process, single-multicellular and multicellular organism process, cell adhesion, biological adhesion, and multicellular organism development. KEGG and PPI results revealed that key N-glycoproteins were closely related to pathways for OA and KBD, such as phagosome, ECM-receptor interaction, lysosome, focal adhesion, protein digestion, and absorption. These results reflected glycoprotein expression for OA and KBD in the process of ECM degradation, material transport, cell–cell or cell–ECM interaction, and information transduction. These key significantly differentially expressed N-glycoproteins and pathways lead to the degeneration and degradation of the cartilage of OA and KBD mainly by disrupting the synthesis and catabolism of basic components of ECM and chondrocytes and interfering with the transfer of material or information. The key N-glycoproteins or pathways in this research are potential targets for pathological mechanisms and therapies of OA and KBD.

An integrative analysis of DNA methylation and transcriptome showed the dysfunction of MAPK pathway was involved in the damage of human chondrocyte induced by T-2 toxin

BackgroundT-2 toxin is thought to induce the growth plate and articular cartilage damage of Kashin-Beck disease (KBD), an endemic osteochondropathy in China. This study aims to explore the potential underlying mechanism of such toxic effects by integrating DNA methylation and gene expression profiles.MethodsIn this study, C28/I2 chondrocytes were treated with T-2 toxin (5 ng/mL) for 24 h and 72 h. Global DNA methylation level of chondrocyte was tested by Enzyme-Linked Immuno Sorbent Assay. Genome-wide DNA methylation and expression profiles were detected using Illumina Infinium HumanMethylation850 BeadChip and RNA-seq technique, respectively. Differentially methylated genes (DMGs) and differentially expressed genes (DEGs) were identified mainly for two stages including 24 h group versus Control group and 72 h group versus 24 h group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed by Metascape. DMGs and DEGs were further validated by Sequenom MassARRAY system and quantitative real-time polymerase chain reaction.ResultsThe global DNA methylation levels of chondrocytes exposed to T-2 toxin were significantly increased (P < 0.05). For 24 h group versus Control group (24 VS C), 189 DEGs and 590 DMGs were identified, and 4 of them were overlapping. For 72 h group versus 24 h group (72 VS 24), 1671 DEGs and 637 DMGs were identified, and 45 of them were overlapping. The enrichment analysis results of DMGs and DEGs both showed that MAPK was the one of the mainly involved signaling pathways in the regulation of chondrocytes after T-2 toxin exposure (DEGs: P24VSc = 1.62 × 10− 7; P72VS24 = 1.20 × 10− 7; DMGs: P24VSc = 0.0056; P72VS24 = 3.80 × 10− 5).ConclusionsThe findings depicted a landscape of genomic methylation and transcriptome changes of chondrocytes after T-2 toxin exposure and suggested that dysfunction of MAPK pathway may play important roles in the chondrocytes damage induced by T-2 toxin, which could provide new clues for understanding the potential biological mechanism of KBD cartilage damage induced by T-2 toxin.

Mechanism of DNA methylation-mediated downregulation of O6-methylguanine-DNA methyltransferase in cartilage injury of Kashin-Beck disease.

Kashin-Beck disease (KBD) is an endemic osteoarthropathy, in which excessive apoptosis of chondrocytes occurs. O6-methylguanine-DNA methyltransferase (MGMT), a DNA damage repair gene, plays an important role in apoptosis, but the mechanism is unclear in KBD cartilage injury. This study was to investigate the expression and promoter methylation of MGMT in KBD patients and its role in DNA damage and apoptosis of chondrocytes. MGMT mRNA and protein level were detected by quantitative real-time PCR and immunohistochemistry. Demethylation of MGMT was carried out using 5-Aza-2'-deoxycytidine, and the methylation level of MGMT promoter was measured by quantitative methylation specific PCR. Next, small hairpin RNA was used to knockdown the expression of MGMT. Cell viability, apoptosis and DNA damage were determined by MTT assay, flow cytometry, Hoechst 33342 staining and alkaline comet assay following T-2 toxin and selenium treatment. MGMT protein expression and mRNA levels were decreased (P = 0.02, P = 0.007) and promoter methylation was increased (P = 0.008) in KBD patients. Meanwhile, MGMT level was upregulated by 5-Aza-2'-deoxycytidine in chondrocytes (P = 0.0002). DNA damage and apoptosis rates were increased in MGMT-silenced chondrocytes (all P < 0.0001). Furthermore, DNA damage and apoptosis were increased in chondrocytes treated with T-2 toxin (all P < 0.0001), but were decreased after selenium treatment (P < 0.0001, P = 0.01). Decreased mRNA level and increased methylation of MGMT were found in the T-2 toxin group (P = 0.005, P = 0.002), while selenium reversed it (P = 0.02, P = 0.004). MGMT might play a crucial part in the pathogenesis of KBD cartilage injury, which could provide a therapeutic target for KBD.

Downregulation of Insulin-Like Growth Factor-1 Receptor Mediates Chondrocyte Death and Matrix Degradation in Kashin-Beck Disease.

To explore the relationship between insulin-like growth factor (IGF)-1R expression and the pathological progression of Kashin-Beck disease (KBD). KBD cartilage samples were collected from 5 patients. Additionally, T-2 toxin was administered to rats fed a selenium (Se)-deficient diet, and their knee joints were collected. Human C28/I2 chondrocytes and mouse hypertrophic ATDC5 chondrocytes were cultured in vitro and treated with T-2 toxin and Se supplementation. Subsequently, the cultured human and mouse chondrocytes were treated with the IGF-1R inhibitor, picropodophyllin. Chondrocyte death and caspase-3 activity were analyzed using flow cytometry and a specific kit, respectively. Protein and mRNA expression levels of IGF-1R and matrix molecules were measured using immunohistochemistry, western blotting, and quantitative real-time reverse transcription-polymerase chain reaction analyses. The cartilages from patients with KBD and T-2 toxin-treated rats on a Se-deficient diet showed significantly decreased expression of IGF-1R compared to cartilages from controls. T-2 toxin decreased IGF-1R mRNA and protein levels in both C28/I2 and hypertrophic ATDC5 chondrocytes in a dose-dependent manner; however, Se supplementation reduced the decrease of IGF-1R induced by T-2 toxin. Furthermore, inhibition of IGF-1R resulted in chondrocyte death of C28/I2 and hypertrophic ATDC5 chondrocytes, as well as decreased type II collagen expression and increased MMP-13 expression at the mRNA and protein levels. Downregulation of IGF-1R was associated with KBD cartilage destruction. Therefore, inhibition of IGF-1R may mediate chondrocyte death and extracellular matrix degeneration related to the pathological progression of KBD.

Reduction of Smad2 caused by oxidative stress leads to necrotic death of hypertrophic chondrocytes associated with an endemic osteoarthritis.

The occurrence and development of an endemic OA, Kashin-Beck disease (KBD), is closely related to oxidative stress induced by free radicals. The aim of the study was to find the key signalling molecules or pathogenic factors as a potential treatment strategy for KBD. Real-time PCR and western blotting were performed to detect the mRNA and protein expression levels in cells and tissues. Immunohistochemical staining was assayed in rat models and human samples obtained from children. The type of cell death was identified by annexin V and propidium iodide staining with flow cytometry. Oxidative stress decreased levels of Smad2 and Smad3 in hypertrophic chondrocytes both in vitro and in vivo. In the cartilage of KBD patients, the expression of Smad2 and Smad3 proteins in the middle and deep zone was significantly decreased with an observed full deletion in the deep zone of some samples. Reduction of Smad2 protein induced necrotic death of hypertrophic chondrocytes, while reduction of Smad3 protein induced apoptosis. The reduction of Smad2 protein was not accompanied by Smad3 protein reduction in hypertrophic chondrocyte necrosis. Furthermore, the reduction of Smad2 also impaired the construction of tissue-engineered cartilage in vitro. These studies reveal that oxidative stress causes necrosis of hypertrophic chondrocytes by downregulating Smad2 protein, which increases the pathogenesis of KBD cartilage. The importance of Smad2 in the development of KBD provides a new potential target for the treatment of KBD.

The integrative analysis of DNA methylation and mRNA expression profiles confirmed the role of selenocompound metabolism pathway in Kashin-Beck disease

ABSTRACT Kashin-Beck disease (KBD) is an endemic chronic osteochondropathy. The etiology of KBD remains unknown. In this study, we conducted an integrative analysis of genome-wide DNA methylation and mRNA expression profiles between KBD and normal controls to identify novel candidate genes and pathways for KBD. Articular cartilage samples from 17 grade III KBD patients and 17 healthy controls were used in this study. DNA methylation profiling of knee cartilage and mRNA expression profile data were obtained from our previous studies. InCroMAP was performed to integrative analysis of genome-wide DNA methylation profiles and mRNA expression profiles. Gene ontology (GO) enrichment analysis was conducted by online DAVID 6.7. The quantitative real-time polymerase chain reaction (qPCR), Western blot, immunohistochemistry (IHC), and lentiviral vector transfection were used to validate one of the identified pathways. We identified 298 common genes (such as COL4A1, HOXA13, TNFAIP6 and TGFBI), 36 GO terms (including collagen function, skeletal system development, growth factor), and 32 KEGG pathways associated with KBD (including Selenocompound metabolism pathway, PI3K-Akt signaling pathway, and TGF-beta signaling pathway). Our results suggest the dysfunction of many genes and pathways implicated in the pathogenesis of KBD, most importantly, both the integrative analysis and in vitro study in KBD cartilage highlighted the importance of selenocompound metabolism pathway in the pathogenesis of KBD for the first time.

DR4-Associated Death Receptor Signal Promotes Cartilage Damage in Patients With Kashin-Beck Disease.

Purpose. To explore the relationship between the death receptor (DR) and the pathological progression of Kashin-Beck disease (KBD). Design. KBD cartilage samples were collected from 15 patients diagnosed according to the "National Diagnostic Criteria of KBD" in China. In vitro monolayer chondrocytes were cultured in complete medium. Caspase-3 and caspase-8 activities in chondrocytes were analyzed using a kit. Nuclear morphology was observed by Hoechst 33258 staining, apoptosis was verified by flow cytometry analysis, and DR molecules were detected using Western blotting and quantitative real-time reverse transcription polymerase chain reaction analysis. Results. Early apoptotic rates of KBD and osteoarthritis (OA) chondrocytes were higher than those of normal control (NC) cells. Excessive apoptotic nuclei were observed in OA and KBD cells after Hoechst 33258 staining. Activities of both caspase-3 and caspase-8 were higher in KBD and OA cells than in NC cells. The average DR4 mRNA level in KBD cells was 3.301-fold higher than that in NC cells, Fas-associating protein with death domain (FADD) transcript level in KBD cells was 2.528-fold higher than that in NC cells. Western blot analyses showed that FAS, DR4, DR5, caspase-3, and FADD were upregulated in the KBD and OA groups compared with the NC group. High expression of caspase-8 in KBD compared with NC was verified, whereas cellular FLICE-inhibitory protein (c-FLIP) in KBD was significantly downregulated. Conclusions. KBD and OA chondrocytes showed obvious FADD-caspase-dependent apoptosis, which is related to the DR pathway. Apoptosis in KBD articular cartilage is mainly related to FAS/DR4-FADD-caspase signaling, and OA is associated with FAS/DR4/DR5-FADD-caspase signaling.

Expression and localization of the small proteoglycans decorin and biglycan in articular cartilage of Kashin-Beck disease and rats induced by T-2 toxin and selenium deficiency.

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy of uncertain etiology. Our study sought to identify a correlation between small proteoglycans decorin and biglycan expression and Kashin-Beck Disease. Immunohistochemistry was used to assess the decorin and biglycan levels in cartilage specimens from both child KBD patients, and rats fed with T-2 toxin under a selenium-deficient condition. Real-time PCR and Western blot were used to assess mRNA and protein levels of decorin and biglycan in rat cartilages, as well as in C28/I2 chondrocytes stimulated by T-2 toxin and selenium in vitro. The result showed that decorin was reduced in all zones of KBD articular cartilage, while the expression of biglycan was prominently increased in KBD cartilage samples. Increased expression of biglycan and reduced expression of decorin were observed at mRNA and protein levels in the cartilage of rats fed with T-2 toxin and selenium- deficiency plus T-2 toxin diet, when compared with the normal diet group. Moreover, In vitro stimulation of C28/I2 cells with T-2 toxin resulted in an upregulation of biglycan and downregulation of decorin, T-2 toxin induction of biglycan and decorin levels were partly rescued by selenium supplement. This study highlights the focal nature of the degenerative changes that occur in KBD cartilage and may suggest that the altered expression pattern of decorin and biglycan have an important role in the onset and pathogenesis of KBD.

Decreased Expression of CHST-12, CHST-13, and UST in the Proximal Interphalangeal Joint Cartilage of School-Age Children with Kashin-Beck Disease: an Endemic Osteoarthritis in China Caused by Selenium Deficiency.

The objective of this study is to investigate changes in the expression of enzymes involved in chondroitin sulfate (CS) sulfation in distal articular surface of proximal interphalangeal joint isolated from school-age children patients with Kashin-Beck disease (KBD), using normal children as controls. Articular cartilage samples were collected from four normal and four KBD children (7-12years old), and these children were assigned to control and KBD groups. Hematoxylin and eosin (H&E), toluidine blue (TB), and immunohistochemical (IHC) stainings were utilized to evaluate changes in joint pathology and expression of enzymes involved in CS sulfation, including carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), and uronyl 2-O-sulfotransferase (UST). The correspondence results were examined by semi-quantitative analysis. Compared with the control group, the KBD group showed the following: a significant decrease of total chondrocytes in superficial, middle, and deep layers and deposition of sulfated glycosaminoglycans in extracellular matrix of KBD cartilage were observed; positive staining chondrocytes of CHST-12, CHST-13, and UST were significantly less in superficial zone of KBD cartilage; and CHST-13 positive staining chondrocytes was reduced in deep zone of KBD cartilage. In contrast, the positive staining rates of CHST-12, CHST-13, and UST in KBD were significantly higher than those in the control group. The decreased expression of these enzymes and the physiologic compensatory reaction may be the signs of early-stage KBD. The alterations of CS structure modifying sulfotransferases in finger articular cartilage might play an important role in the onset and pathogenesis of school-age KBD children.

Death of chondrocytes in Kashin-Beck disease: Apoptosis, necrosis or necroptosis?

The purpose of this paper was to investigate chondrocyte distribution and death in the cartilage in Kashin-Beck disease (KBD). Apoptotic chondrocytes were detected by TUNEL assay. Ultrastructural changes were examined by transmission electron microscope (TEM). Biochemical markers associated with apoptosis (eg, caspase-3) and necroptosis (eg, RIP3) were investigated by immunohistochemistry. In KBD cartilage chondrocyte death was characterized by paler staining of the cells. Multiple chondral cell clusters surrounded the areas lacking cells in the deep zone. The per cent of TUNEL-positive and RIP3-positive chondrocytes were higher in the middle zones of KBD samples; however, there was some positive staining for TUNEL but negative staining for caspase-3. Immunohistochemistry failed to detect significant differences in caspase-3 levels in KBD children compared to controls, suggesting that beside apoptosis necroptosis dominates as a cell death mechanism in the middle zone of cartilage from KBD children. To clarify further the presence of chondrocyte necroptosis in KBD, we performed TUNEL, caspase-3 and RIP3 staining in a rat KBD model which is based upon T-2 toxin treatment under selenium-deficient conditions. Apoptosis and necroptosis co-existed in the middle zone in this rat KBD model. Ultrastructural analysis of chondrocyte from deep cartilage revealed abnormal cells with numerous morphological changes, such as plasma membrane breakdown, generalized swelling of the cytoplasm and loss of identifiable organelles. Chondrocyte death by necrosis in the deep zone of cartilages in KBD may be a result of exposure to T-2 toxin from bone marrow or bloodstream under selenium-deficient nutrition status in KBD endemic areas. Chondrocyte death plays a key role in either the initiation or the progression of KBD pathogenesis.

Chondrocytes damage induced by T-2 toxin via Wnt/β-catenin signaling pathway is involved in the pathogenesis of an endemic osteochondropathy, Kashin-Beck disease

Kashin-Beck disease (KBD), an endemic osteochondropathy, is characterized by cartilage degeneration which is caused by abnormal catabolism in the extracellular matrix (ECM). In this study, we investigated the expression of the Wnt/β-catenin signaling pathway in KBD pathogenesis. Among the proteins involved in the Wnt/β-catenin signaling pathway, WNT-3A, FZD1, SOX9, and β-catenin were up-regulated, while FRZB was down-regulated in KBD cartilage. C28/I2 cells were evaluated for cell viability using the MTT assay after exposure to T-2 toxin, a suspicious environmental pathogenic factors of KBD. C28/I2 cells were treated with different intervening concentrations (0.001μg/mL,0.005μg/mL and 0.01μg/mL) of T-2 toxin for 24h. The expression of FZD1 and CTNNB1 (i.e.,β-catenin) was significantly reduced and SOX9 expression was significantly increased in chondrocytes after treatment with different intervening concentrations of T-2 toxin. Our results indicate that alterations in the Wnt/β-catenin signaling pathway in articular cartilage play an important role in the onset and pathogenesis of KBD.

Genome-wide DNA methylation profiling of articular cartilage reveals significant epigenetic alterations in Kashin-Beck disease and osteoarthritis.

To determine genome-wide DNA methylation profiles of knee cartilage from patients with Kashin-Beck disease (KBD) and osteoarthritis (OA). Knee cartilage was collected from 14 grade III KBD patients, 5 primary OA patients and 13 healthy subjects. The genome-wide methylation profiles of 5 KBD cartilage, 5 OA cartilage and 5 normal cartilage were determined by Illumina HumanMethylation450 array. Illumina Methylation Analyzer package was employed for identifying differentially methylated CpG sites. Functional annotation and enrichment analysis of differentially methylated genes (DMG) were conducted using GeneRIF database, Ingenuity Pathway Analysis (IPA) and The Database for Annotation, Visualization and Integrated Discovery (DAVID). Mass spectrometry (MS) and immunohistochemistry (IHC) were conducted to validate the functional relevance of identified KBD associated gene. We identified a total of 1212 differentially methylated CpG sites in KBD vs Normal, annotated to 264 hypermethylated and 368 hypomethylated genes. Comparing the DNA methylation profiles of KBD vs Normal and OA vs Normal detected overlap of 367 differentially methylated CpG sites (annotated to 182 genes) as well as 845 KBD-specific differentially methylated CpG sites (annotated to 471 unique genes). MS and IHC confirmed the hypermethylation status and decreased protein expression of HAPLN1 gene in KBD cartilage. Our data implicate epigenetic dysregulation of a host of genes in KBD and OA. Furthermore, we observed common causal epigenetic changes shared by KBD and OA.

Altered expression of chondroitin sulfate structure modifying sulfotransferases in the articular cartilage from adult osteoarthritis and Kashin-Beck disease.

To investigate the expression of enzymes involved in chondroitin sulfate (CS) sulfation in the articular cartilage isolated from adult patients with osteoarthritis (OA) and Kashin-Beck disease (KBD), using normal adults as controls. Articular cartilage samples were collected from normal, OA and KBD adults aged 38-60 years old, and divided into three groups with six individual subjects in each group. The morphology and pathology grading of knee joint cartilage was examined by Safranin O staining. The localization and expression of enzymes involved in CS sulfation (CHST-3, CHST-11, CHST-12, CHST-13, carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15 - CHST-15, and uronyl 2-O-sulfotransferase - UST) were examined by immunohistochemical (IHC) staining and semi-quantitative analysis. Positive staining rates for anabolic enzymes CHST-3, CHST-12, CHST-15, and UST were lower in the KBD and OA groups than those in the control group. Meanwhile, reduced levels of CHST-11, and CHST-13 in KBD group were observed, in contrast to those in OA and control groups. The expressions of all six CS sulfation enzymes were less detected in the superficial and deep zones of KBD cartilage compared with control and OA cartilage. The reduced expression of the CS structure modifying sulfotransferases in the chondrocytes of both KBD and OA adult patients may provide explanations for their cartilage damages, and therapeutic targets for their treatment.