Objective This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3(LOXL3)affects the autophagy in chondrocytes in osteoarthritis(OA),specifically through the activation of mammalian targe...Objective This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3(LOXL3)affects the autophagy in chondrocytes in osteoarthritis(OA),specifically through the activation of mammalian target of rapamycin complex 1(mTORC1).Methods To establish an OA model,rats underwent anterior cruciate ligament transection(ACLT).Chondrocytes were isolated from cartilage tissues and cultured.Western blotting was performed to assess the expression of LOXL3,Rheb,phosphorylation of p70S6K(p-p70S6K,a downstream marker of mTORC1),and autophagy markers.The autophagy of chondrocytes was observed using an immunofluorescence assay.Results The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage,in comparison to those from the normal cartilage.The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K,as well as an increase in the expression of autophagy-related proteins.Additionally,the effect of LOXL3 could be reversed through the silencing of Rheb.The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy.Conclusion LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.展开更多
Endochondral ossification requires proper control of chondrocyte proliferation,differentiation,survival,and organization.Here we show that knockout ofα-parvin,an integrin-associated focal adhesion protein,from murine...Endochondral ossification requires proper control of chondrocyte proliferation,differentiation,survival,and organization.Here we show that knockout ofα-parvin,an integrin-associated focal adhesion protein,from murine limbs causes defects in endochondral ossification and dwarfism.The mutant long bones were shorter but wider,and the growth plates became disorganized,especially in the proliferative zone.With two-photon time-lapse imaging of bone explant culture,we provide direct evidence showing thatα-parvin regulates chondrocyte rotation,a process essential for chondrocytes to form columnar structure.Furthermore,loss ofα-parvin increased binucleation,elevated cell death,and caused dilation of the resting zones of mature growth plates.Single-cell RNA-seq analyses revealed alterations of transcriptome in all three zones(i.e.,resting,proliferative,and hypertrophic zones)of the growth plates.Our results demonstrate a crucial role ofα-parvin in long bone development and shed light on the cellular mechanism through whichα-parvin regulates the longitudinal growth of long bones.展开更多
Osteoarthritis (OA), the most common form of joint disease, is characterized clinically by joint pain, stiffness,and deformity. OA is now considered a whole joint disease;however, the breakdown of the articular cartil...Osteoarthritis (OA), the most common form of joint disease, is characterized clinically by joint pain, stiffness,and deformity. OA is now considered a whole joint disease;however, the breakdown of the articular cartilage remains themajor hallmark of the disease. Current treatments targeting OA symptoms have a limited impact on impeding orreversing the OA progression. Understanding the molecular and cellular mechanisms underlying OA development isa critical barrier to progress in OA therapy. Recent studies by the current authors’ group and others have revealedthat the nuclear factor of activated T cell 1 (NFAT1), a member of the NFAT family of transcription factors, regulatesthe expression of many anabolic and catabolic genes in articular chondrocytes of adult mice. Mice lacking NFAT1exhibit normal skeletal development but display OA in both appendicular and spinal facet joints as adults. Thisreview mainly focuses on the recent advances in the regulatory role of NFAT1 transcription factor in the activities ofarticular chondrocytes and its implication in the pathogenesis of OA.展开更多
There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchy...There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchymal cells condensing into tissue elements(patterning phase) followed by their differentiation into cartilage(chondrocytes) or bone(osteoblasts) cells within the condensations. During the growth and remodeling phase, bone is formed directly via intramembranous ossification or through a cartilage to bone conversion via endochondral ossification routes. The canonical pathway of the endochondral bone formation process involves apoptosis of hypertrophic chondrocytes followed by vascular invasion that brings in osteoclast precursors to remove cartilage and osteoblast precursors to form bone. However, there is now an emerging role for chondrocyte-to-osteoblast transdifferentiation in the endochondral ossification process. Although the concept of "transdifferentiation" per se is not recent,new data using a variety of techniques to follow the fate of chondrocytes in different bones during embryonic and post-natal growth as well as during fracture repair in adults have identified three different models for chondrocyte-to-osteoblast transdifferentiation(direct transdifferentiation, dedifferentiation to redifferentiation, and chondrocyte to osteogenic precursor). This review focuses on the emerging models of chondrocyte-to-osteoblast transdifferentiation and their implications for the treatment of skeletal diseases as well as the possible signaling pathways that contribute to chondrocyte-to-osteoblast transdifferentiation processes.展开更多
Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are ...Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are unclear. In this study, we found activated Akt signaling in human OA cartilage as well as in a mouse OA model with surgical destabilization of the medial meniscus.Genetic mouse models mimicking sustained Akt signaling in articular chondrocytes via PTEN deficiency driven by either Col2a1-Cre or Col2a1-Cre^(ERT2) developed OA, whereas restriction of Akt signaling reversed the OA phenotypes in PTEN-deficient mice.Mechanistically, prolonged activation of Akt signaling caused an accumulation of reactive oxygen species and triggered chondrocyte senescence as well as a senescence-associated secretory phenotype, whereas chronic administration of the antioxidant N-acetylcysteine suppressed chondrocyte senescence and mitigated OA progression in PTEN-deficient mice. Therefore,inhibition of Akt signaling by PTEN is required for the maintenance of articular cartilage. Disrupted Akt signaling in articular chondrocytes triggers oxidative stress-induced chondrocyte senescence and causes OA.展开更多
Objective To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease(KBD) and in an established T-2 toxin-and selenium(Se) deficiency-induced rat mo...Objective To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease(KBD) and in an established T-2 toxin-and selenium(Se) deficiency-induced rat model. Methods Cartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase d UTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and m RNA levels were determined by real-time reverse transcription polymerase chain reaction. Results Increased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and m RNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet. Conclusion T-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.展开更多
Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis(OA) patients, and explore the relationship between the miRNA-140 expression and OA severity. Methods This study...Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis(OA) patients, and explore the relationship between the miRNA-140 expression and OA severity. Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence(KL) grade 1-2], moderate(KL grade 3) and severe(KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 sn RNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant(F=305.464, P<0.001). miRNA-140 could be detected in synovial fluid of both normal knees and OA knees, its relative expression level was reduced in synovial fluid of OA group compared with normal group, and the between-group difference was statistically significant as well(F=314.245, P<0.001). The relative expression level of miRNA-140 in both chondrocytes and synovial fluid were negatively correlated with the KL grade of OA(r=-0.969, P<0.001; r=-0.970, P<0.001). Conclusion miRNA-140 could be detected in chondrocytes and synovial fluid of OA patients, and its expression was negatively correlated with the severity of OA.展开更多
To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2O2.Methods Murine chondrogenitor cells(ATDC5)were differentiated into hypertrophic chondrocytes by Insulin-Tra...To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2O2.Methods Murine chondrogenitor cells(ATDC5)were differentiated into hypertrophic chondrocytes by Insulin-Transferrin-Selenium(ITS)treatment,and then treated with H2O2.Suitable conditions(concentration,time)were determined by using the MTT assay.After total RNA isolation and cDNA synthesis,the levels of 84 genes were determined using the PCR array,whereas quantitative RT-PCR was carried out to validate the PCR array data.Results We identified 9 up-regulated genes and 12 down-regulated genes,encoding proteins with various functions,such as collagen proteins,transcription factors,proteins involved in skeletal development and bone mineral metabolism,as well as cell adhesion molecules.Quantitative RT-PCR confirmed the altered expression of 5 down-regulated genes(Smad2,Smad4,transforming growth factorβreceptor 1,transforming growth factorβreceptor 3,and matrix metalloproteinase 10).Conclusions H2O2 significantly changed the expression of several genes involved in a variety of biological functions.Because of the link between oxidative damage and Kashin-Beck disease,these genes may also be involved in the deep-zone necrosis of the cartilage observed in Kashin-Beck disease.展开更多
Osteoarthritis(OA)is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity.As a bacterial synthesis of quinine,pyrroloquinoline quinone(PQ...Osteoarthritis(OA)is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity.As a bacterial synthesis of quinine,pyrroloquinoline quinone(PQQ)is a strong redox cofactor with a variety of biological benefits,including antioxidant,anti-inflammation-induced mitochondrial metabolism regulation.This study was designed to investigate the effect of PQQ on TNF-α-induced mitochondrial damage in chondrocytes.Chondrocytes isolated from C57BL/6 mice were exposed to TNF-α50 ng/mL,TNF-α50 ng/mL+PQQ 10µmol/L for 24 h.Then,morphological study,functional study and mechanism study were taken.The results revealed TNF-α-induced chondrocyte mitochondrion damage could be reduced by application of PQQ,evidenced by elevated number of mitochondria,well-kept mtDNA integrity,preserved ATP level,reestablished mitochondrial membrane potential,and prevented mitochondrial function.The present work strongly suggests that the mitochondrion is an important target for OA chondrocyte damage induced by TNF-αand the PQQ protection from this damage ameliorates mitochondrial dysfunction induced by TNF-α.PQQ might be a potential chemical for OA intervention.展开更多
Free fatty acids(FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis...Free fatty acids(FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis pathogenesis. However, the molecular mechanism by which FFAs exert lipotoxicity and induce osteoarthritis remains largely unknown. We here observed that oleate at the usual clinical range does not exert lipotoxicity while oleate at high pathological ranges exerted lipotoxicity through apoptosis in articular chondrocytes. By investigating the differential effect of oleate at toxic and nontoxic concentrations, we revealed that lipid droplet(LD) accumulation confers articular chondrocytes, the resistance to lipotoxicity. Using high fat diet-induced osteoarthritis models and articular chondrocytes treated with oleate alone or oleate plus palmitate, we demonstrated that articular chondrocytes gain resistance to lipotoxicity through protein kinase casein kinase 2(PKCK2)—six-transmembrane protein of prostate 2(STAMP2)—and fat-specific protein 27(FSP27)-mediated LD accumulation. We further observed that the exertion of FFAs-induced lipotoxicity was correlated with the increased concentration of cellular FFAs freed from LDs, whether FFAs are saturated or not. In conclusion, PKCK2/STAMP2/FSP27-mediated sequestration of FFAs in LD rescues osteoarthritic chondrocytes. PKCK2/STAMP2/FSP27 should be considered for interventions against metabolic OA.展开更多
In our previous studies, we have found that the prepubertal increase in thyroid hormone levels induces osterix(Osx) signaling in hypertrophic chondrocytes to transdifferentiate them into osteoblasts. To test if Osx ex...In our previous studies, we have found that the prepubertal increase in thyroid hormone levels induces osterix(Osx) signaling in hypertrophic chondrocytes to transdifferentiate them into osteoblasts. To test if Osx expressed in chondrocytes directly contributes to transdifferentiation and secondary ossification, we generated Osx^flox/flox;Col2-Cre-ERT2 mice and knocked out Osx with a single injection of tamoxifen at postnatal day(P) 3 prior to evaluation of the epiphyseal bone phenotype by μCT, histology, and immunohistochemistry(IHC) at P21. Vehicle(oil)-treated Osx^flox/flox;Col2-Cre-ERT2 and tamoxifen-treated, Cre-negative Osx^flox/flox mice were used as controls.μCT analysis of tibial epiphyses revealed that trabecular bone mass was reduced by 23% in the Osx conditional knockout(c KO) compared with control mice. Trabecular number and thickness were reduced by 28% and 8%,respectively, while trabecular separation was increased by 24% in the c KO mice. Trichrome staining of longitudinal sections of tibial epiphyses showed that bone area and bone area adjusted for total area were decreased by 22% and 18%, respectively. IHC studies revealed the presence of abundant Osx-expressing prehypertrophic chondrocytes in the epiphyses of control mice at P10, but not in the cKO mice. Furthermore, expression levels of MMP13, COL10, ALP, and BSP were considerably reduced in the epiphyses of cKO mice. We also found that Osx overexpression in ATDC5 chondrocytes increased expression of Col10, Mmp13, Alp, and Bsp. Our data indicate that Osx expressed in chondrocytes plays a significant role in secondary ossification by regulating expression of genes involved in chondrocyte hypertrophy and osteoblast transdifferentiation.展开更多
Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bon...Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bone matrix gelatin and cancellous bone matrix gelatin. Methods To plant human fetal chondrocytes on the BMG, the damage of the cultured chondrocytes was observed by the optical microscope (HE staining). The immunohistochemistry of CD44 was quantitative analysis by the image collection and analysis system. Results With the increasing concentration of T 2 toxin, the damage of chondroytes was more and more evident and CD44 expression was lowered. After adding selenium, the damage was relieved and CD44 expression increased. The density of chondrocytes on the cortical bone matrix gelatin was much higher than that on the cancellous bone matrix gelatin. Conclusion T 2 toxin can lower the CD44 expression on the chondrocytes and adding selenium can relieve the damage caused by T 2toxin and increased CD44 expression. The inducing effect on reestablished cartilage in vitro of cortical bone matrix gelatin was much higher than that of cancellous bone matrix gelatin.展开更多
Cryopreservation is the process of choice for long term preservation of cells and tissues. In this study, the effects of cryoprotective agents, dimethyl sulfoxide(DMSO), glycerol and 1,2 propanediol on the bovine arti...Cryopreservation is the process of choice for long term preservation of cells and tissues. In this study, the effects of cryoprotective agents, dimethyl sulfoxide(DMSO), glycerol and 1,2 propanediol on the bovine articular chondrocyte viability were examined experimentally. The CPA was added at the concentrations of 0 6, 0 9, 1 2 and 1 5 mol/L and at 4 ℃ and 37 ℃ and removed at 37 ℃ in one step. CPA stepwise addition and removal at 0 6 and 1 2 mol/L and at 37 ℃ was also tested as an alternative protocol. Cell volume excursion during DMSO addition and removal was estimated and correlated well with cell survival rates. Solution makeup affects cell survival rate and a stepwise protocol can improve the cell survival rates significantly.展开更多
Chondrocyte dysfunction has been demonstrated to be a major inducer of osteoarthritis(OA).The pathological mechanism of chondrocyte dysfunction is definitely multifactoral,but oxidative stressis regarded as one of the...Chondrocyte dysfunction has been demonstrated to be a major inducer of osteoarthritis(OA).The pathological mechanism of chondrocyte dysfunction is definitely multifactoral,but oxidative stressis regarded as one of the leading causes of apoptosis,autophagy,senescence,and mitochondrial dysfunctionin chondrocytes.Strategies for arresting oxidative stress-induced chondrocyte dysfunction have been considered as potential therapeutic targets for OA.Recently,fork head box O(Fox O)transcription factors have been determined to play a protective role in chondrocytes through the regulation of autophagy and defense against oxidative stress;they also regulate growth,maturation,and matrix synthesis.To explore Fox O′s potential role in the treatment of OA,we first discussed the recent advances in the field of oxidative stress-induced chondrocyte dysfunction and then emphasized the protective role of fox otranscription factors as a potential molecular target for the treatment of OA.Understanding the function of fox otranscription factors will be important in designing next-generation therapies to prevent or reverse the development of OA.展开更多
Articular cartilage damage is a universal health problem.Despite recent progress,chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration.Loss-of-function chang...Articular cartilage damage is a universal health problem.Despite recent progress,chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration.Loss-of-function changes are frequently observed in chondrocyte expansion and other pathological conditions,but the characteristics and intermediate molecular mechanisms remain unclear.In this study,we demonstrate a time-lapse atlas of chondrocyte dedifferentiation to provide molecular details and informative biomarkers associated with clinical chondrocyte evaluation.We performed various assays,such as single-cell RNA sequencing(scRNA-seq),live-cell metabolic assays,and assays for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq),to develop a biphasic dedifferentiation model consisting of early and late dedifferentiation stages.Early-stage chondrocytes exhibited a glycolytic phenotype with increased expression of genes involved in metabolism and antioxidation,whereas late-stage chondrocytes exhibited ultrastructural changes involving mitochondrial damage and stress-associated chromatin remodeling.Using the chemical inhibitor BTB06584,we revealed that early and late dedifferentiated chondrocytes possessed distinct recovery potentials from functional phenotype loss.Notably,this two-stage transition was also validated in human chondrocytes.An image-based approach was established for clinical use to efficiently predict chondrocyte plasticity using stage-specific biomarkers.Overall,this study lays a foundation to improve the quality of chondrocytes in clinical use and provides deep insights into chondrocyte dedifferentiation.展开更多
Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissu...Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.The expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κ B(NF-κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species(ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively. Results: The chondrocytes viability was significantly reduced after 12 h incubation with AGEs(P<0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF-α and NF-κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis. Conclusions: Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF-α and NF-κB expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEsinduced adverse effects.展开更多
Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chondrocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins we...Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chondrocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins were extracted from the cells and subjected to 2-dimensional differential image gel electrophoresis (2D-DIGE). Proteins of interest were identified by mass spectrometry. Results: Eighty-six protein spots showed significantly different intensities with each reagent or reagent combination. AAA+ protein, HSP47/Serpin, cAMP-dependent protein kinase type II-beta regulatory subunit, alpha-actin-4 and tubulin decreased with the addition of celecoxib, while apolipoprotein A-V, glutamate carboxipeptide 2, mitochondrial stress-70 protein, sorting nexin-9 and GRP78 increased with the addition of celecoxib. GRP78 is a stress protein and may be chondroprotective. Celecoxib modulated IL-1β stimulated chondrocytes, and CD200R and moesin were identified as such resulting proteins. Conclusion: Protein profiles of OA chondrocytes changed after administration of celecoxib. Further investigation is needed to elucidate the function of each protein in OA chondrocytes.展开更多
The damage effects of oxy free radical and fulvic acid on cultivated chicken embryo chondrocytes were studied. The results show that the growrth of chondrocytes is inhibited and the morphology of the cells altered. Th...The damage effects of oxy free radical and fulvic acid on cultivated chicken embryo chondrocytes were studied. The results show that the growrth of chondrocytes is inhibited and the morphology of the cells altered. The collagen synthesizing capability of the damaged cell changes somewhat. A noteworthy change of the type of collagen synthesized by the abnormal cells was observed by CMC-chromatography and amino acid analysis. The results indicated that the abnonml cells tend to synthesize type I instead of type II collagen, which is synthesized and secreted by the intact chondrocyte.展开更多
基金the National Natural Science Foundation of China(No.81702187)Natural Science Foundation of Jiangxi Province(No.20202BAB206019)+4 种基金Science Fund for Distinguished Young Scholars of Jiangxi Province(No.20224ACB216018)Scientific Talents Grants of Jiangxi Province(No.S2018LQCQ0800)Scientific Grants of Health Commission of Jiangxi Province(No.20194048)Scientific Innovation Talents Grants of Ganzhou(No.2019-60-08)Leading Talents Grants and Ph.D.Programs Foundation of Ganzhou People’s Hospital(No.Bsqd2019003)and Academic leaders Program of Ganzhou Institutes of Health.
文摘Objective This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3(LOXL3)affects the autophagy in chondrocytes in osteoarthritis(OA),specifically through the activation of mammalian target of rapamycin complex 1(mTORC1).Methods To establish an OA model,rats underwent anterior cruciate ligament transection(ACLT).Chondrocytes were isolated from cartilage tissues and cultured.Western blotting was performed to assess the expression of LOXL3,Rheb,phosphorylation of p70S6K(p-p70S6K,a downstream marker of mTORC1),and autophagy markers.The autophagy of chondrocytes was observed using an immunofluorescence assay.Results The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage,in comparison to those from the normal cartilage.The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K,as well as an increase in the expression of autophagy-related proteins.Additionally,the effect of LOXL3 could be reversed through the silencing of Rheb.The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy.Conclusion LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.
基金supported by the National Natural Science Foundation of China Grant 82273308,Inno HK@Health,Theme-based Research Scheme (Tl3-602/21-N)Guangdong-Dongguan Joint Research Scheme Guangdong-Hong Kong-Macao Program (2021B1515130004)+4 种基金the Natural Science Foundation of Guangdong Province Grant 2017B030301018the Special Support Program for Training High-Level Talents in Guangdong Grant 2019TQ05Y518the Shenzhen Innovation Committee of Science and Technology Grant JCYJ20220530112817040,ZDSYS20220606101604009the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital&Shenzhen Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Shenzhen E010122002supported by the Lombardi and Shinozuka Experimental Pathology Research Endowment Fund,University of Pittsburgh。
文摘Endochondral ossification requires proper control of chondrocyte proliferation,differentiation,survival,and organization.Here we show that knockout ofα-parvin,an integrin-associated focal adhesion protein,from murine limbs causes defects in endochondral ossification and dwarfism.The mutant long bones were shorter but wider,and the growth plates became disorganized,especially in the proliferative zone.With two-photon time-lapse imaging of bone explant culture,we provide direct evidence showing thatα-parvin regulates chondrocyte rotation,a process essential for chondrocytes to form columnar structure.Furthermore,loss ofα-parvin increased binucleation,elevated cell death,and caused dilation of the resting zones of mature growth plates.Single-cell RNA-seq analyses revealed alterations of transcriptome in all three zones(i.e.,resting,proliferative,and hypertrophic zones)of the growth plates.Our results demonstrate a crucial role ofα-parvin in long bone development and shed light on the cellular mechanism through whichα-parvin regulates the longitudinal growth of long bones.
基金supported by the United States National Institutes of Health(NIH)under Award Number R01 AR059088(to J.W.)the Mary A.and Paul R.Harrington Distinguished Professorship Endowment.
文摘Osteoarthritis (OA), the most common form of joint disease, is characterized clinically by joint pain, stiffness,and deformity. OA is now considered a whole joint disease;however, the breakdown of the articular cartilage remains themajor hallmark of the disease. Current treatments targeting OA symptoms have a limited impact on impeding orreversing the OA progression. Understanding the molecular and cellular mechanisms underlying OA development isa critical barrier to progress in OA therapy. Recent studies by the current authors’ group and others have revealedthat the nuclear factor of activated T cell 1 (NFAT1), a member of the NFAT family of transcription factors, regulatesthe expression of many anabolic and catabolic genes in articular chondrocytes of adult mice. Mice lacking NFAT1exhibit normal skeletal development but display OA in both appendicular and spinal facet joints as adults. Thisreview mainly focuses on the recent advances in the regulatory role of NFAT1 transcription factor in the activities ofarticular chondrocytes and its implication in the pathogenesis of OA.
基金supported by funding from the National Institutes of Arthritis and Musculoskeletal Diseases RO1 grant (AR048139)Veterans Administration BLR&D merit review grant (101-BX-001396) to S.M
文摘There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchymal cells condensing into tissue elements(patterning phase) followed by their differentiation into cartilage(chondrocytes) or bone(osteoblasts) cells within the condensations. During the growth and remodeling phase, bone is formed directly via intramembranous ossification or through a cartilage to bone conversion via endochondral ossification routes. The canonical pathway of the endochondral bone formation process involves apoptosis of hypertrophic chondrocytes followed by vascular invasion that brings in osteoclast precursors to remove cartilage and osteoblast precursors to form bone. However, there is now an emerging role for chondrocyte-to-osteoblast transdifferentiation in the endochondral ossification process. Although the concept of "transdifferentiation" per se is not recent,new data using a variety of techniques to follow the fate of chondrocytes in different bones during embryonic and post-natal growth as well as during fracture repair in adults have identified three different models for chondrocyte-to-osteoblast transdifferentiation(direct transdifferentiation, dedifferentiation to redifferentiation, and chondrocyte to osteogenic precursor). This review focuses on the emerging models of chondrocyte-to-osteoblast transdifferentiation and their implications for the treatment of skeletal diseases as well as the possible signaling pathways that contribute to chondrocyte-to-osteoblast transdifferentiation processes.
基金supported by grants from the State Key Program of National Natural Science of China (31630093)the National Natural Science Foundation of China (31571512, 31871476, and 81241062)+1 种基金the Beijing Nova Program (Z161100004916146)the National Basic Research Program of China (2012CB966904)
文摘Osteoarthritis(OA) is an age-related disorder that is strongly associated with chondrocyte senescence. The causal link between disruptive PTEN/Akt signaling and chondrocyte senescence and the underlying mechanism are unclear. In this study, we found activated Akt signaling in human OA cartilage as well as in a mouse OA model with surgical destabilization of the medial meniscus.Genetic mouse models mimicking sustained Akt signaling in articular chondrocytes via PTEN deficiency driven by either Col2a1-Cre or Col2a1-Cre^(ERT2) developed OA, whereas restriction of Akt signaling reversed the OA phenotypes in PTEN-deficient mice.Mechanistically, prolonged activation of Akt signaling caused an accumulation of reactive oxygen species and triggered chondrocyte senescence as well as a senescence-associated secretory phenotype, whereas chronic administration of the antioxidant N-acetylcysteine suppressed chondrocyte senescence and mitigated OA progression in PTEN-deficient mice. Therefore,inhibition of Akt signaling by PTEN is required for the maintenance of articular cartilage. Disrupted Akt signaling in articular chondrocytes triggers oxidative stress-induced chondrocyte senescence and causes OA.
基金supported by the National Natural Science Foundation of China(No.81573102 and No.81273006)the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry(11-01)
文摘Objective To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease(KBD) and in an established T-2 toxin-and selenium(Se) deficiency-induced rat model. Methods Cartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase d UTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and m RNA levels were determined by real-time reverse transcription polymerase chain reaction. Results Increased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and m RNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet. Conclusion T-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.
基金Supported by the National Natural Science Foundation of China(No.81672219No.81601936)the Science and Technology Support Program of Sichuan province(No.2014SZ0023-2)
文摘Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis(OA) patients, and explore the relationship between the miRNA-140 expression and OA severity. Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence(KL) grade 1-2], moderate(KL grade 3) and severe(KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 sn RNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant(F=305.464, P<0.001). miRNA-140 could be detected in synovial fluid of both normal knees and OA knees, its relative expression level was reduced in synovial fluid of OA group compared with normal group, and the between-group difference was statistically significant as well(F=314.245, P<0.001). The relative expression level of miRNA-140 in both chondrocytes and synovial fluid were negatively correlated with the KL grade of OA(r=-0.969, P<0.001; r=-0.970, P<0.001). Conclusion miRNA-140 could be detected in chondrocytes and synovial fluid of OA patients, and its expression was negatively correlated with the severity of OA.
基金Supported by the National Natural Science Foundation of China(81573102 and 81273006)the Natural Science Fund Projects of Shaanxi Province(2017JM812)
文摘To identify the osteogenesis genes whose expression is altered in hypertrophic chondrocytes treated with H2O2.Methods Murine chondrogenitor cells(ATDC5)were differentiated into hypertrophic chondrocytes by Insulin-Transferrin-Selenium(ITS)treatment,and then treated with H2O2.Suitable conditions(concentration,time)were determined by using the MTT assay.After total RNA isolation and cDNA synthesis,the levels of 84 genes were determined using the PCR array,whereas quantitative RT-PCR was carried out to validate the PCR array data.Results We identified 9 up-regulated genes and 12 down-regulated genes,encoding proteins with various functions,such as collagen proteins,transcription factors,proteins involved in skeletal development and bone mineral metabolism,as well as cell adhesion molecules.Quantitative RT-PCR confirmed the altered expression of 5 down-regulated genes(Smad2,Smad4,transforming growth factorβreceptor 1,transforming growth factorβreceptor 3,and matrix metalloproteinase 10).Conclusions H2O2 significantly changed the expression of several genes involved in a variety of biological functions.Because of the link between oxidative damage and Kashin-Beck disease,these genes may also be involved in the deep-zone necrosis of the cartilage observed in Kashin-Beck disease.
基金the National Natural Science Foundation of China(No.81171760).
文摘Osteoarthritis(OA)is a degenerative disease characterized by matrix degradation and cell death leading to a gradual loss of articular cartilage integrity.As a bacterial synthesis of quinine,pyrroloquinoline quinone(PQQ)is a strong redox cofactor with a variety of biological benefits,including antioxidant,anti-inflammation-induced mitochondrial metabolism regulation.This study was designed to investigate the effect of PQQ on TNF-α-induced mitochondrial damage in chondrocytes.Chondrocytes isolated from C57BL/6 mice were exposed to TNF-α50 ng/mL,TNF-α50 ng/mL+PQQ 10µmol/L for 24 h.Then,morphological study,functional study and mechanism study were taken.The results revealed TNF-α-induced chondrocyte mitochondrion damage could be reduced by application of PQQ,evidenced by elevated number of mitochondria,well-kept mtDNA integrity,preserved ATP level,reestablished mitochondrial membrane potential,and prevented mitochondrial function.The present work strongly suggests that the mitochondrion is an important target for OA chondrocyte damage induced by TNF-αand the PQQ protection from this damage ameliorates mitochondrial dysfunction induced by TNF-α.PQQ might be a potential chemical for OA intervention.
基金supported by a grant from the National Research Foundation of Korea(NRF)funded by the Korean Government(MISP)(No.2015R1A2A1A10051603)
文摘Free fatty acids(FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis pathogenesis. However, the molecular mechanism by which FFAs exert lipotoxicity and induce osteoarthritis remains largely unknown. We here observed that oleate at the usual clinical range does not exert lipotoxicity while oleate at high pathological ranges exerted lipotoxicity through apoptosis in articular chondrocytes. By investigating the differential effect of oleate at toxic and nontoxic concentrations, we revealed that lipid droplet(LD) accumulation confers articular chondrocytes, the resistance to lipotoxicity. Using high fat diet-induced osteoarthritis models and articular chondrocytes treated with oleate alone or oleate plus palmitate, we demonstrated that articular chondrocytes gain resistance to lipotoxicity through protein kinase casein kinase 2(PKCK2)—six-transmembrane protein of prostate 2(STAMP2)—and fat-specific protein 27(FSP27)-mediated LD accumulation. We further observed that the exertion of FFAs-induced lipotoxicity was correlated with the increased concentration of cellular FFAs freed from LDs, whether FFAs are saturated or not. In conclusion, PKCK2/STAMP2/FSP27-mediated sequestration of FFAs in LD rescues osteoarthritic chondrocytes. PKCK2/STAMP2/FSP27 should be considered for interventions against metabolic OA.
基金supported by the National Institutes of Health grant AR 048139 to S.M.
文摘In our previous studies, we have found that the prepubertal increase in thyroid hormone levels induces osterix(Osx) signaling in hypertrophic chondrocytes to transdifferentiate them into osteoblasts. To test if Osx expressed in chondrocytes directly contributes to transdifferentiation and secondary ossification, we generated Osx^flox/flox;Col2-Cre-ERT2 mice and knocked out Osx with a single injection of tamoxifen at postnatal day(P) 3 prior to evaluation of the epiphyseal bone phenotype by μCT, histology, and immunohistochemistry(IHC) at P21. Vehicle(oil)-treated Osx^flox/flox;Col2-Cre-ERT2 and tamoxifen-treated, Cre-negative Osx^flox/flox mice were used as controls.μCT analysis of tibial epiphyses revealed that trabecular bone mass was reduced by 23% in the Osx conditional knockout(c KO) compared with control mice. Trabecular number and thickness were reduced by 28% and 8%,respectively, while trabecular separation was increased by 24% in the c KO mice. Trichrome staining of longitudinal sections of tibial epiphyses showed that bone area and bone area adjusted for total area were decreased by 22% and 18%, respectively. IHC studies revealed the presence of abundant Osx-expressing prehypertrophic chondrocytes in the epiphyses of control mice at P10, but not in the cKO mice. Furthermore, expression levels of MMP13, COL10, ALP, and BSP were considerably reduced in the epiphyses of cKO mice. We also found that Osx overexpression in ATDC5 chondrocytes increased expression of Col10, Mmp13, Alp, and Bsp. Our data indicate that Osx expressed in chondrocytes plays a significant role in secondary ossification by regulating expression of genes involved in chondrocyte hypertrophy and osteoblast transdifferentiation.
文摘Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bone matrix gelatin and cancellous bone matrix gelatin. Methods To plant human fetal chondrocytes on the BMG, the damage of the cultured chondrocytes was observed by the optical microscope (HE staining). The immunohistochemistry of CD44 was quantitative analysis by the image collection and analysis system. Results With the increasing concentration of T 2 toxin, the damage of chondroytes was more and more evident and CD44 expression was lowered. After adding selenium, the damage was relieved and CD44 expression increased. The density of chondrocytes on the cortical bone matrix gelatin was much higher than that on the cancellous bone matrix gelatin. Conclusion T 2 toxin can lower the CD44 expression on the chondrocytes and adding selenium can relieve the damage caused by T 2toxin and increased CD44 expression. The inducing effect on reestablished cartilage in vitro of cortical bone matrix gelatin was much higher than that of cancellous bone matrix gelatin.
文摘Cryopreservation is the process of choice for long term preservation of cells and tissues. In this study, the effects of cryoprotective agents, dimethyl sulfoxide(DMSO), glycerol and 1,2 propanediol on the bovine articular chondrocyte viability were examined experimentally. The CPA was added at the concentrations of 0 6, 0 9, 1 2 and 1 5 mol/L and at 4 ℃ and 37 ℃ and removed at 37 ℃ in one step. CPA stepwise addition and removal at 0 6 and 1 2 mol/L and at 37 ℃ was also tested as an alternative protocol. Cell volume excursion during DMSO addition and removal was estimated and correlated well with cell survival rates. Solution makeup affects cell survival rate and a stepwise protocol can improve the cell survival rates significantly.
基金supported by National Natural Science Foundation of China(81560662)China Postdoctoral Science Foundation(2017M610543)
文摘Chondrocyte dysfunction has been demonstrated to be a major inducer of osteoarthritis(OA).The pathological mechanism of chondrocyte dysfunction is definitely multifactoral,but oxidative stressis regarded as one of the leading causes of apoptosis,autophagy,senescence,and mitochondrial dysfunctionin chondrocytes.Strategies for arresting oxidative stress-induced chondrocyte dysfunction have been considered as potential therapeutic targets for OA.Recently,fork head box O(Fox O)transcription factors have been determined to play a protective role in chondrocytes through the regulation of autophagy and defense against oxidative stress;they also regulate growth,maturation,and matrix synthesis.To explore Fox O′s potential role in the treatment of OA,we first discussed the recent advances in the field of oxidative stress-induced chondrocyte dysfunction and then emphasized the protective role of fox otranscription factors as a potential molecular target for the treatment of OA.Understanding the function of fox otranscription factors will be important in designing next-generation therapies to prevent or reverse the development of OA.
基金supported by the National Key R&D Program of China(2017YFA0104900)National Natural Science Foundation of China(T2121004,31830029,82002319)。
文摘Articular cartilage damage is a universal health problem.Despite recent progress,chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration.Loss-of-function changes are frequently observed in chondrocyte expansion and other pathological conditions,but the characteristics and intermediate molecular mechanisms remain unclear.In this study,we demonstrate a time-lapse atlas of chondrocyte dedifferentiation to provide molecular details and informative biomarkers associated with clinical chondrocyte evaluation.We performed various assays,such as single-cell RNA sequencing(scRNA-seq),live-cell metabolic assays,and assays for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq),to develop a biphasic dedifferentiation model consisting of early and late dedifferentiation stages.Early-stage chondrocytes exhibited a glycolytic phenotype with increased expression of genes involved in metabolism and antioxidation,whereas late-stage chondrocytes exhibited ultrastructural changes involving mitochondrial damage and stress-associated chromatin remodeling.Using the chemical inhibitor BTB06584,we revealed that early and late dedifferentiated chondrocytes possessed distinct recovery potentials from functional phenotype loss.Notably,this two-stage transition was also validated in human chondrocytes.An image-based approach was established for clinical use to efficiently predict chondrocyte plasticity using stage-specific biomarkers.Overall,this study lays a foundation to improve the quality of chondrocytes in clinical use and provides deep insights into chondrocyte dedifferentiation.
文摘Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.The expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κ B(NF-κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species(ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively. Results: The chondrocytes viability was significantly reduced after 12 h incubation with AGEs(P<0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF-α and NF-κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis. Conclusions: Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF-α and NF-κB expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEsinduced adverse effects.
基金funded by the Natural Science Basic Research Plan of Shaanxi Province,China(2014JM4170)the Department of disease control of Shaanxi Health and Family Planning Commission,China(2010/2012)
文摘Objective: To study a comprehensive proteomic analysis of celecoxib in oseteoarthritis (OA) chondrocytes. Methods: OA chondrocytes were stimulated with celecoxib, IL-1β and IL-1β together with celecoxib. Proteins were extracted from the cells and subjected to 2-dimensional differential image gel electrophoresis (2D-DIGE). Proteins of interest were identified by mass spectrometry. Results: Eighty-six protein spots showed significantly different intensities with each reagent or reagent combination. AAA+ protein, HSP47/Serpin, cAMP-dependent protein kinase type II-beta regulatory subunit, alpha-actin-4 and tubulin decreased with the addition of celecoxib, while apolipoprotein A-V, glutamate carboxipeptide 2, mitochondrial stress-70 protein, sorting nexin-9 and GRP78 increased with the addition of celecoxib. GRP78 is a stress protein and may be chondroprotective. Celecoxib modulated IL-1β stimulated chondrocytes, and CD200R and moesin were identified as such resulting proteins. Conclusion: Protein profiles of OA chondrocytes changed after administration of celecoxib. Further investigation is needed to elucidate the function of each protein in OA chondrocytes.
文摘The damage effects of oxy free radical and fulvic acid on cultivated chicken embryo chondrocytes were studied. The results show that the growrth of chondrocytes is inhibited and the morphology of the cells altered. The collagen synthesizing capability of the damaged cell changes somewhat. A noteworthy change of the type of collagen synthesized by the abnormal cells was observed by CMC-chromatography and amino acid analysis. The results indicated that the abnonml cells tend to synthesize type I instead of type II collagen, which is synthesized and secreted by the intact chondrocyte.