T-2 Toxin Induces Epiphyseal Plate Lesions via Decreased SECISBP2-Mediated Selenoprotein Expression in DA Rats, Exacerbated by Selenium Deficiency.

OBJECTIVE: Both selenium (Se) deficiency and mycotoxin T2 lead to epiphyseal plate lesions, similar to Kashin-Beck disease (KBD). However, regulation of selenoproteins synthesis mediated by SECISBP2, in response to these 2 environmental factors, remained unclear. The present study proposed to explore the mechanism behind the cartilage degradation resulting from Se deficiency and mycotoxin T2 exposure. DESIGN: Deep chondrocyte necrosis and epiphyseal plate lesions were replicated in Dark Agouti (DA) rats by feeding them T2 toxin/Se deficiency artificial synthetic diet for 2 months. RESULTS: Se deficiency led to decreased expression of COL2α1, while T2 treatment reduced the heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) expression, both of which affected the cartilage extracellular matrix metabolism in the rat models. The expression of Col2α1, Acan, Hs6st2, Secisbp2, Gpx1, and Gpx4 were all significantly decreased in cartilage tissues from DA rats, fed a Se-deficient diet or exposed to T2 toxin, contrary to Adamts4, whose expression was increased in both conditions. In addition, T2 treatment led to the decreased expression of SBP2, GPX1, GPX4, and total GPXs activity in C28/I2 cells. CONCLUSION: DA rats exposed to T2 toxin and/or Se-deficient conditions serve as the perfect model of KBD. The 2 environmental risk factors of KBD, which serve as a "double whammy," can intensify the extracellular matrix metabolic imbalance and the antioxidant activity of chondrocytes, leading to articular cartilage degradation and epiphyseal plate abnormalities similar to those observed in KBD.

miR-497 Is Implicated in the Process of Chondrogenesis and Inhibits IHH Gene Expression in Human Chondrocytes.

OBJECTIVE: The aim of this study was to examine differences in microRNA-497 (miR-497) expression during cartilage tissue formation and to test whether miR-497 directly interferes with Indian hedgehog (IHH) gene and inhibits IHH expression in human chondrocytes. DESIGN: At different cartilage development stages and different time points in bone matrix gelatin-induced endochondral ossification (BMG-ECO) rat models, the expression of miR-497 and the Ihh gene was monitored at the mRNA level. Bioinformatic analysis, gene mutation, dual luciferase reporter gene assays and gene expression assays at both the mRNA and protein levels in human chondrocytes were subsequently performed to validate the interaction between miR-497 and the IHH gene. RESULTS: The mRNA expression of miR-497 or the Ihh gene in BMG-ECO rats showed significant differences between the cartilage development stages and between different time points, and the trends in the expression of miR-497 and Ihh were reversed. Bioinformatic and dual luciferase reporter gene assays demonstrated a direct interaction between miR-497 and the IHH gene. Differential mRNA and protein expression profiles of the IHH gene in human chondrocytes after 48 hours of transfection with miR-497 mimics and a negative control indicated that miR-497 inhibited IHH expression. CONCLUSION: Our study provided new clues for further functional and molecular mechanism studies of miR-497 in chondrogenesis and demonstrated a potential target for clinical therapy for cartilage degenerative disease.

The hsa-miR-181a-5p reduces oxidation resistance by controlling SECISBP2 in osteoarthritis.

BACKGROUND: The phenotypes of osteoarthritis (OA) consist of cartilage extracellular matrix (ECM) metabolism disorder and the breakdown of cartilage homeostasis, which are induced by pro-inflammatory factors and oxidative stress. Selenoproteins regulated by selenocysteine insertion sequence binding protein 2 (SBP2) are highly effective antioxidants, but their regulatory mechanisms, particularly the involvement of miRNAs, are not fully understood. METHODS: To explore whether miR-181a-5p and SBP2 are involved in OA pathogenesis, we established an IL-1ß model using the chondrocyte SW1353 cell line. Next, we up- or down-regulated SBP2 and miRNA-181a-5p expression in the cells. Finally, we measured the expression of miRNA-181a-5p, SBP2 and three selenoproteins in OA cartilage and peripheral blood. RESULTS: The results showed that IL-1ß increased hsa-miR-181a-5p and decreased SBP2 in a time- and dose-dependent manner. GPX1 and GPX4, which encode crucial glutathione peroxidase antioxidant enzymes, were up-regulated along with SBP2 and miR-181a-5p. Furthermore, SBP2 showed a significant negative correlation with miR-181a-5p during induced ATDC5 cell differentiation. There was lower GPX1 and GPX4 mRNA expression and SBP2 protein expression in damaged cartilage than in smooth cartilage from the same OA sample, and hsa-miR-181a-5p expression on the contrary. Similar results were observed in peripheral blood. In conclusion, we have reported a novel pathway in which pro-inflammatory factors, miRNA, SBP2 and selenoproteins are associated with oxidation resistance in cartilage. CONCLUSION: Overall, this study provides the first comprehensive evidence that pro-inflammatory factors cause changes in the cartilage antioxidant network and describes the discovery of novel mediators of cartilage oxidative stress and OA pathophysiology. Our data suggest that miR-181a-5p may be used to develop novel early-stage diagnostic and therapeutic strategies for OA.

Selenium-sensitive miRNA-181a-5p targeting SBP2 regulates selenoproteins expression in cartilage.

Selenium (Se) deficiency brings about defects in the biosynthesis of several selenoproteins and has been associated with aberrant chondrogenesis. Selenocysteine (Sec) Insertion Sequence (SECIS) and SECIS binding protein 2 (SBP2) interaction is a very critical node for the metabolic balance between Se and selenoproteins. The Gpx1, Gpx4 and SelS have different binding affinities with SBP2 in cells. According to our results, both miR-181a-5p and SBP2 appeared to be selenium-sensitive and regulated the expression of selenoproteins in C28/I2 cells under Se sufficient environment. However, they showed significantly opposite expression trend in Se deficiency rats cartilage and SeD C28/I2 cells. The SBP2 is a direct target gene of miR-181a-5p in C28/I2 cells as determined by reporter gene and off-target experiments. And the miR-181a-5p could regulate SBP2 and the selenoproteins in C28/I2 cells. Depending upon the Se supply levels, C28/I2 cells were divided into three groups, that is normal Se, SeD and SeS, which underwent through a 7-day Se deprivation process, then SBP2 was knocked-down and overexpressed in all the groups. Moreover, the selected selenoproteins were down-regulated in second-generation low Se diet rat cartilage. The selenoproteins expression was decreased by Se deficiency which depended on the Selenium-sensitive miR-181a-5p to participate and regulate SBP2 at post-transcriptional level. It involves a series of antioxidant and ECM (extracellular matrix) genes, to overcome the ROS-related stress for the protection of essential physiological functions and to maintain the balance between anabolism and catabolism of the cartilage.

Down-regulated in OA cartilage, SFMBT2 contributes to NF-κB-mediated ECM degradation.

The interplay between anabolic and catabolic factors regulates cartilage matrix homoeostasis. In OA, this balance is disrupted which results in cartilage degradation involving a plethora of inflammatory factors. Here, we identify a novel gene "Scm-like with four MBT domains protein 2" (SFMBT2) negatively regulated in OA cartilage. Articular cartilage from human OA patients undergoing knee arthroplasty surgery exhibited significantly decreased levels of SFMBT2 compared to the normal controls. Down-regulation of SFMBT2 by specific siRNA disturbed the metabolic homoeostasis and led to decreased expression of anabolic genes (SOX9, COL2A1) while increasing the expression of catabolic genes (MMP13 and ADAMTS4), in human chondrocytes. Finally, we revealed that SFMBT2 intervention by siRNA contributed to the catabolic phenotype of human chondrocytes mediated by NF-kB pathway.

Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.

Abnormality of epiphyseal plate induced by selenium deficiency diet in two generation DA rats.

This study aimed to observe the effects of Se deficiency on epiphyseal plates of two generation DA rats fed with artificial total synthetic low Se diet. All F0 and F1 DA rats were fed with synthetic low Se diet (SeD group) and low Se diet supplied with Se (SeS group). The levels of selenium and enzyme activities of GPx were detected in plasma of the rats. General growth of bone and articular cartilage was measured macroscopically and microscopically. The epiphyseal plate of femur heads or tibia were obtained to histological and immunohistochemical examinations. The cartilage from left knee joints and femur heads was used to detect the gene expression of collagens, ADAMTSs and several selenoproteins by RT-qPCR. Two generation SeD rats showed Se insufficiency status. The thicknesses of the femur and tibial epiphyseal plates in both F0 and F1 SeD rats were significantly less than that of SeS rats. In F1 generation, SeD rats showed much fewer proliferative chondrocyte layers than SeS ones. Importantly, two generation SeD rats both showed significantly more serious pathological changes of epiphyseal plates. In two generation rats, gene expressions of COL II, GPx1 and GPx4 were significantly down-regulated in SeD rats than SeS ones; meanwhile ADAMTS-4 showed an up-regulated expression in cartilage. Dietary Se deficiency can apparently cause epiphyseal plate lesion and decrease cartilage type II collagen production and GPx1 activity in two generation DA rats fed with the artificial total synthesis low Se diet.

MicroRNAs associated with osteoarthritis differently expressed in bone matrix gelatin (BMG) rat model.

Osteoarthritis (OA) is characterized by degeneration of articular cartilage, limited intraarticular inflammation with synovitis, and changes in peri-articular and subchondral bone. In recent years, more and more evidence demonstrated that microRNAs (miRNAs) play important roles in the molecular mechanisms in OA by suppressing gene expression at the post-transcriptional level. In current study, histological staining of toluidine blue and cartilage-specific gene express revealed that the bone matrix gelatin (BMG) rat model could demonstrate the different development of cartilage. In current study, we tested whether some miRNAs associated with OA differently expressed in BMG rat model. We verified that miR-140 and miR-455 were associated with cartilage development, and further revealed that miR-140-5p and miR-455-3p might play more important function than miR-140-3p and miR-455-5p in the BMG rat model. Moreover, we found that miR-9 and miR-98 were involved in the endochondral ossification, suggesting they may be also the key regulators in the process of endochondral ossification. In fact, many miRNAs worked as a miRNA-mediated regulatory network in the process of cartilage development and OA. Further functional discovery will clarify the roles of individual miRNAs and their targets, and serve as a strong foundation for translating these findings to the clinic therapy for OA.

Selenium effect on selenoprotein transcriptome in chondrocytes.

Selenium is an essential micronutrient and exerts its biological functions predominantly through selenoproteins. Selenium deficiency is associated with cartilage function. This study demonstrated that all 24 selenoprotein transcripts in mouse genome were detectable in ATDC5 chondrocytes except deiodinase 1 (DIO1), DIO2, and selenoprotein V (Sel V), while all 25 selenoprotein transcripts in human genome were detectable in C28/I2 chondrocytes except glutathione peroxidase 6 (GPx6) and DIO1. In addition, gene expression of five selenoproteins (GPx1, Sel H, Sel N, Sel P, and Sel W) was up-regulated and two selenoproteins (SPS2 and Sel O) was down-regulated by sodium selenite (Se) in both ATDC5 and C28/I2 cells. Gene expression of six selenoproteins (TrxR1, Sel I, Sel M, Sel R, Sel S, Sel T) and one selenoprotein (GPx3) was up-regulated by Se in ATDC5 and C28/I2 cells, respectively. Gene expression of one selenoprotein (TrxR2) was down-regulated by Se only in ATDC5 cells. Further transcription inhibition assay showed that both transcriptional and posttranscriptional mechanisms involved in Se-regulated gene expression of GPx1, TrxR1, TrxR2, SPS2, Sel O, and Sel S. However, Se-regulated gene expression of Sel H, Sel I, Sel M, Sel N, Sel P, Sel R, Sel T, and Sel W mainly at posttranscriptional level. Moreover, new protein synthesis inhibition assay indicated that Se-mediated new protein synthesis also played roles in Se-regulated gene expression of GPx1, TrxR1, TrxR2, Sel H, Sel O, Sel P, Sel R, and Sel W. In summary, this study described the selenoprotein transcriptome, Se-regulated selenoproteins and possible mechanisms involved in chondrocytes.

T-2 toxin enhances catabolic activity of hypertrophic chondrocytes through ROS-NF-κB-HIF-2α pathway.

T-2 toxin (T-2), one of the most important and toxic trichothecene mycotoxins, can cause many medical problems, such as diarrhea, nervous disorders, immunodepression and death, and is also believed as an etiological factor of Kashin-Beck disease, an endemic osteochondropathy prevailing in North China. However, the molecular mechanisms underlying T-2 effects on tissue damage remain elusive. We differentiated ATDC5 chondrogenic cells into hypertrophic chondrocytes, and found that T-2 reduced the expression of anabolic genes, and increased the expression of catabolic genes. To uncover the mechanism that T-2 influenced metabolic homeostasis of hypertrophic chondrocytes, we observed that T-2 increased the production of reactive oxygen species (ROS) and the degradation of IκB-α, and up-regulated the expression of hypoxia-induced factor-2α (HIF-2α). Bay11-7085 (an inhibitor of NF-κB pathway) inhibited the up-regulation of HIF-2α, and N-acetyl-l-cysteine (a ROS scavenger) inhibited both the decrease of IκB-α and the up-regulation of HIF-2α. Our results demonstrate that ROS-NF-κB-HIF-2α pathway participates in the effects of T-2 on hypertrophic chondrocytes, and HIF-2α plays an important role as a key mediator in this process.

Genetic evidence for the association between the early growth response 3 (EGR3) gene and schizophrenia.

Recently, two genome scan meta-analysis studies have found strong evidence for the association of loci on chromosome 8p with schizophrenia. The early growth response 3 (EGR3) gene located in chromosome 8p21.3 was also found to be involved in the etiology of schizophrenia. However, subsequent studies failed to replicate this finding. To investigate the genetic role of EGR3 in Chinese patients, we genotyped four SNPs (average interval ∼2.3 kb) in the chromosome region of EGR3 in 470 Chinese schizophrenia patients and 480 healthy control subjects. The SNP rs35201266 (located in intron 1 of EGR3) showed significant differences between cases and controls in both genotype frequency distribution (P = 0.016) and allele frequency distribution (P = 0.009). Analysis of the haplotype rs35201266-rs3750192 provided significant evidence for association with schizophrenia (P = 0.0012); a significant difference was found for the common haplotype AG (P = 0.0005). Furthermore, significant associations were also found in several other two-, and three-SNP tests of haplotype analyses. The meta-analysis revealed a statistically significant association between rs35201266 and schizophrenia (P = 0.0001). In summary, our study supports the association of EGR3 with schizophrenia in our Han Chinese sample, and further functional exploration of the EGR3 gene will contribute to the molecular basis for the complex network underlying schizophrenia pathogenesis.

Evidence for transmission disequilibrium at the DAOA gene locus in a schizophrenia family sample.

Recently, the DAOA gene locus on chromosome 13q32-q34 has been implicated in the etiology of schizophrenia. We genotyped three single-nucleotide polymorphisms (SNPs: rs778294, rs779293 and rs3918342) in this region in 126 Chinese family trios. In this study, we have identified statistically significant transmission disequilibrium in two markers rs778293 (P=0.01) and rs3918342 (P=0.02), and a highly significant under-transmission between haplotype CAT (P=0.0005) and schizophrenia. The results provide further evidence to support that DAOA gene locus is involved in conferring susceptibility to schizophrenia.