• 【TGF-β 通过Smad介导的信号调节鸡胸骨软骨细胞中PTHrP的表达。】 复制标题 收藏 收藏
    DOI:10.1002/jcp.1118 复制DOI
    作者列表:Pateder DB,Ferguson CM,Ionescu AM,Schwarz EM,Rosier RN,Puzas JE,O'Keefe RJ
    BACKGROUND & AIMS: :PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF-beta, BMP-2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF-beta stimulated the expression of PTHrP (5-fold), PTHrP caused a slight inhibition, and BMP-2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP-2 increased, while both PTHrP and TGF-beta decreased the expression of type X collagen. TGF-beta isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF-beta were further evaluated in a PTHrP-promoter CAT construct. The effect of TGF-beta, BMP-2, and PTHrP on the PTHrP-promoter paralleled their effects on mRNA expression, with TGF-beta significantly increasing CAT activity, BMP-2 decreasing CAT activity, and PTHrP having a minimal effect. Co-transfection of the TGF-beta signaling molecule, Smad 3, mimicked the effect of TGF-beta (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF-beta. Furthermore, infection with a Smad 3-expressing retrovirus mimicked the effects of exogenously added TGF-beta, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF-beta, but only partially blocked the effect of TGF-beta on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF-beta through Smad mediated signaling events.
    背景与目标: : PTHrP调节软骨内骨形成过程中软骨细胞分化的速率。连续治疗1、3和5天后,在上胸骨软骨细胞中检查了PTHrP的表达及其对TGF-β,BMP-2和PTHrP的调节。虽然TGF-β 刺激了PTHrP的表达 (5倍),但PTHrP引起了轻微的抑制,并且BMP-2明显抑制了PTHrP mRNA的表达。这些因素对PTHrP表达的影响不仅与细胞的成熟状态有关,因为BMP-2增加,而PTHrP和TGF-β 均降低了x型胶原的表达。TGF-β 亚型1、2和3均刺激PTHrP表达。在PTHrP启动子CAT构建体中进一步评估了TGF-β 诱导PTHrP所涉及的信号事件。TGF-β,BMP-2和PTHrP对PTHrP启动子的作用与它们对mRNA表达的作用平行,TGF-β 显着增加CAT活性,BMP-2降低CAT活性,而PTHrP的作用最小。TGF-β 信号分子Smad 3的共转染模仿了TGF-β (PTHrP启动子的诱导) 的作用,而显性负Smad 3抑制了TGF-β 对PTHrP启动子的诱导。此外,用表达Smad 3的逆转录病毒感染模仿了外源添加TGF-β 的作用,并在感染的软骨细胞培养物中诱导了PTHrP mRNA的表达。相反,显性阴性Smad 3完全抑制了TGF-β 对PTHrP启动子的刺激,但仅部分阻断了TGF-β 对PTHrP mRNA合成的作用。这些发现表明PTHrP在经历软骨内骨化的软骨细胞中表达,并且至少部分地通过Smad介导的信号事件显示出TGF-β 的调节。
  • 【分离兔骨骺软骨细胞在分化的各个阶段。】 复制标题 收藏 收藏
    DOI:10.1007/BF00225811 复制DOI
    作者列表:Ralphs JR,Evans L,Ali SY
    BACKGROUND & AIMS: :Separation of fractions enriched in hypertrophic cells and proliferative cells has been achieved by density gradient centrifugation of cells from collagenase digests of rabbit epiphyseal cartilage. Concentrated suspensions of cells are centrifuged on a continuous Percoll density gradient. Hypertrophic cells remain in the upper part of the gradient and proliferative zone cells move to the lower regions. The resultant fractions show differences in mean cell diameter, alkaline phosphatase activity, morphology and synthetic activity in culture. Fractions rich in hypertrophic cells contain larger cells and more alkaline phosphatase activity than those enriched in proliferative cells. In culture the hypertrophic cells flatten as large irregular polygonal cells, whereas proliferative fractions form smaller spindle-shaped cells. In micromass culture hypertrophic fractions incorporate less 35S-sulphate and 14C-proline, and less tritiated thymidine than do proliferative fractions. These results suggest a general reduction in matrix and DNA synthesis with the attainment of the fully differentiated hypertrophic state, coincident with the expression of alkaline phosphatase activity and mineralisation of the cartilage matrix.
    背景与目标: : 通过从兔epi骨软骨的胶原酶消化物中分离细胞的密度梯度离心,可以分离富含肥厚细胞和增殖细胞的级分。在连续的Percoll密度梯度上离心细胞的浓缩悬浮液。肥厚细胞保留在梯度的上部,增殖区细胞移动到下部区域。所得组分在培养物中显示出平均细胞直径,碱性磷酸酶活性,形态和合成活性的差异。富含肥厚细胞的组分比富含增殖细胞的组分含有更大的细胞和更多的碱性磷酸酶活性。在培养物中,肥厚细胞变平为大型不规则多边形细胞,而增殖部分形成较小的纺锤形细胞。在微质量培养中,肥厚级分与增殖级分相比,含有更少的35s硫酸盐和14c-脯氨酸,以及更少的tristiate胸苷。这些结果表明,随着完全分化的肥厚状态的实现,基质和DNA合成普遍减少,与碱性磷酸酶活性的表达和软骨基质的矿化相吻合。
  • 【细胞外核苷酸选择性诱导软骨细胞的迁移和II型胶原的表达。】 复制标题 收藏 收藏
    DOI:10.3390/ijms21155227 复制DOI
    作者列表:Szustak M,Gendaszewska-Darmach E
    BACKGROUND & AIMS: :The migration of chondrocytes from healthy to injured tissues is one of the most important challenges during cartilage repair. Additionally, maintenance of the chondrogenic phenotype remains another limitation, especially during monolayer culture in vitro. Using both the differentiated and undifferentiated chondrogenic ATDC5 cell line, we showed that extracellular nucleotides are able to increase the migration rate of chondrocytes without affecting their chondrogenic phenotype. We checked the potency of natural nucleotides (ATP, ADP, UTP, and UDP) as well as their stable phosphorothioate analogs, containing a sulfur atom in the place of one nonbridging oxygen atom in a phosphate group. We also detected P2y1, P2y2, P2y4, P2y6, P2y12, P2y13, and P2y14 mRNA transcripts for nucleotide receptors, demonstrating that P2y1 and P2y13 are highly upregulated in differentiated ATDC5 cells. We showed that ADPβS, UDPβS, and ADP are the best stimulators of migration of differentiated chondrocytes. Additionally, ADP and ADPβS positively affected the expression of type II collagen, a structural component of the cartilage matrix.
    背景与目标: : 软骨细胞从健康组织向受伤组织的迁移是软骨修复过程中最重要的挑战之一。此外,软骨形成表型的维持仍然是另一个限制,尤其是在体外单层培养期间。使用分化和未分化的软骨源性ATDC5细胞系,我们表明细胞外核苷酸能够增加软骨细胞的迁移率,而不会影响其软骨形成表型。我们检查了天然核苷酸 (ATP,ADP,UTP和UDP) 及其稳定的硫代磷酸酯类似物的效力,其中含有硫原子代替磷酸基团中的一个非桥接氧原子。我们还检测到核苷酸受体的P2y1,P2y2,P2y4,P2y6,P2y12,P2y13和P2y14 mRNA转录本,表明P2y1和P2y13在分化的ATDC5细胞中高度上调。我们发现ADP β s,udp β s和ADP是分化软骨细胞迁移的最佳刺激因子。此外,ADP和ADP β 对软骨基质的结构成分II型胶原的表达产生积极影响。
  • 【NF-κ b调节软骨细胞中Lef1基因的表达。】 复制标题 收藏 收藏
    DOI:10.1016/j.bbrc.2007.03.170 复制DOI
    作者列表:Yun K,Choi YD,Nam JH,Park Z,Im SH
    BACKGROUND & AIMS: :The relation of Wnt/beta-catenin signaling to osteoarthritis progression has been revealed with little information on the underlying molecular mechanism. In this study we found overexpression of Lef1 in cartilage tissue of osteoarthritic patients and elucidated molecular mechanism of NF-kappaB-mediated Lef1 gene regulation in chondrocytes. Treatment of IL-1beta augmented Lef1 upregulation and nuclear translocation of NF-kappaB in chondrocytes. Under IL-1beta signaling, treatment of NF-kappaB nuclear translocation inhibitor SN-50 reduced Lef1 expression. A conserved NF-kappaB-binding site between mouse and human was selected through bioinformatic analysis and mapped at the 14 kb upstream of Lef1 transcription initiation site. NF-kappaB binding to the site was confirmed by chromatin immunoprecipitation assay. Lef1 expression was synergistically upregulated by interactions of NF-kappaB with Lef1/beta-catenin in chondrocytes. Our results suggest a pivotal role of NF-kappaB in Lef1 expression in arthritic chondrocytes or cartilage degeneration.
    背景与目标: : Wnt/β-catenin信号与骨关节炎进展的关系已被揭示,但有关潜在分子机制的信息很少。在这项研究中,我们发现了骨关节炎患者软骨组织中Lef1的过表达,并阐明了NF-κ b介导的软骨细胞中Lef1基因调控的分子机制。IL-1beta治疗可增强软骨细胞中NF-κ b的Lef1上调和核移位。在IL-1beta信号下,NF-κ b核易位抑制剂的处理SN-50降低了Lef1的表达。通过生物信息学分析选择了小鼠和人之间保守的NF-κ b结合位点,并定位在Lef1转录起始位点上游的14 kb处。通过染色质免疫沉淀法证实NF-κ b与该位点的结合。软骨细胞中NF-κ b与Lef1/β-catenin的相互作用协同上调Lef1表达。我们的结果表明,NF-κ b在关节炎软骨细胞或软骨变性中Lef1表达中起关键作用。
  • 【通过在3D可生物降解的支架上与猪软骨细胞共培养的人真皮成纤维细胞在体外形成腔结构。】 复制标题 收藏 收藏
    DOI:10.1007/s10529-007-9457-8 复制DOI
    作者列表:Liu X,Zhou G,Liu W,Zhang W,Cui L,Cao Y
    BACKGROUND & AIMS: :Dermal fibroblasts (DF) possess chondrogenic differentiation potential but whether DF, or a subpopulation of DF, can form a typical cartilage structure in culture is unknown. In this study, human DF were co-cultured with porcine articular chondrocytes on a biodegradable scaffold of polylactic acid/polyglycolic acid. Histological analysis demonstrated that some DFs can be induced to form cartilage lacuna structure showing the existence of a chondrogenic subpopulation of human DF. Moreover, the 3D-co-culture system can serve as an optimal model for directing stem cell differentiation in vitro.
    背景与目标: : 真皮成纤维细胞 (DF) 具有软骨分化潜力,但是DF或DF的亚群是否可以在培养物中形成典型的软骨结构尚不清楚。在这项研究中,人DF与猪关节软骨细胞在可生物降解的聚乳酸/聚乙醇酸支架上共培养。组织学分析表明,可以诱导某些DFs形成软骨腔结构,表明存在人DF的软骨形成亚群。此外,3d共培养系统可以作为指导干细胞体外分化的最佳模型。
  • 【诱导型一氧化氮合酶的NF-κ b增强子元件中CpG位点甲基化的丧失是人类关节软骨细胞基因诱导的原因。】 复制标题 收藏 收藏
    DOI:10.1002/art.37806 复制DOI
    作者列表:de Andrés MC,Imagawa K,Hashimoto K,Gonzalez A,Roach HI,Goldring MB,Oreffo RO
    BACKGROUND & AIMS: OBJECTIVE:To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. METHODS:Expression of iNOS was quantified by quantitative reverse transcriptase-polymerase chain reaction. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Cotransfections with expression vectors encoding NF-κB subunits were carried out to analyze iNOS promoter and enhancer activities in response to changes in methylation status. RESULTS:The 1,000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both control and OA samples. The CpG site at -289 and the sites in the starting coding region were largely unmethylated in both groups. The NF-κB enhancer region at -5.8 kb was significantly demethylated in OA samples compared with control samples. This enhancer element was transactivated by cotransfection with the NF-κB subunit p65, alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in a reporter assay. CONCLUSION:These findings demonstrate the association between demethylation of specific NF-κB-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, importantly, show association with the OA process.
    背景与目标:
  • 【地塞米松刺激软骨细胞中C型利钠肽的表达。】 复制标题 收藏 收藏
    DOI:10.1186/1471-2474-7-87 复制DOI
    作者列表:Agoston H,Baybayan L,Beier F
    BACKGROUND & AIMS: BACKGROUND:Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation--such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias)--generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. METHODS:Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX) for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. RESULTS:We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc). In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II) and Npr3 (natriuretic peptide decoy receptor) genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor), as well as the Npr2 gene (encoding the CNP receptor). CONCLUSION:Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine factors.
    背景与目标:
  • 【重组马interleukin-1beta对马关节软骨细胞胰岛素样生长因子-I系统的转录和蛋白水解调节。】 复制标题 收藏 收藏
    DOI:10.1002/jcp.20762 复制DOI
    作者列表:Porter RM,Akers RM,Howard RD,Forsten-Williams K
    BACKGROUND & AIMS: :Interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I), which have opposing effects on matrix metabolism within articular cartilage, are thought to play prominent roles in the pathogenesis of osteoarthritis. To better understand the link between these anabolic (IGF-I) and catabolic (IL-1) stimuli, we examined exogenous IL-1 regulation of the IGF-I signaling system of articular chondrocytes (ACs). Equine ACs from non-arthritic stifle joints were expanded in monolayer culture, encapsulated for 10 days in alginate beads, and stimulated as high-density monolayers with recombinant equine IL-1beta (0, 1, 10 ng/ml) for 48 h. IL-1beta enhanced expression of IGF-IR levels, as determined by both [125I]-IGF-I binding studies and Western blotting, while reducing the concentration of endogenous IGF-I detected in conditioned media by radioimmunoassay. Western ligand blotting revealed that chondrocytes primarily secreted IGF binding proteins (IGFBPs) with molecular weights of 28-30 and 32-34 kDa, which were identified as IGFBPs 5 and 2, respectively, and that IL-1beta treatment diminished IGFBP-2, the prominent homolog in conditioned media. Northern blot analysis suggested IL-1beta regulation of IGF-I and, to some extent, IGF-IR was mediated by transcription; however, the cytokine did not affect IGFBP-2 expression. To test for evidence of proteolysis by matrix metalloproteinases (MMPs), additional cultures were co-incubated with inhibitors for MMPs 2/9, 3, and 8. IGFBP-2 suppression was partially reversed by gelatinase (MMP-2/9) inhibition. In summary, these findings further delineate the role of IL-1 as a key regulator of the IGF-I system within articular cartilage, demonstrating that regulation occurs through both direct (transcriptional) and indirect (proteolytic) mechanisms.
    背景与目标: : Interleukin-1 (IL-1) 和胰岛素样生长因子-I (igf-i) 对关节软骨内基质代谢具有相反作用,被认为在骨关节炎的发病机理中起着重要作用。为了更好地了解这些合成代谢 (igf-i) 和分解代谢 (IL-1) 刺激之间的联系,我们检查了关节软骨细胞 (ACs) 的igf-i信号系统的外源性IL-1调节。来自非关节炎窒息关节的马ACs在单层培养中扩增,在藻酸盐珠中封装10天,并用重组马IL-1beta (0,1,10 ng/ml) 刺激为高密度单层48小时。通过 [125I]-igf-i结合研究和Western印迹测定,IL-1beta增强了igf-ir水平的表达,同时降低了通过放射免疫测定法在条件培养基中检测到的内源性igf-i的浓度。Western配体印迹显示,软骨细胞主要分泌分子量为28-30和32-34 kDa的IGF结合蛋白 (IGFBPs),分别被鉴定为IGFBPs 5和2,并且IL-1beta治疗减少了IGFBP-2,这是条件培养基中的主要同源物。Northern印迹分析表明igf-i的IL-1beta调节,并且在某种程度上,igf-ir是通过转录介导的; 然而,细胞因子不影响IGFBP-2的表达。为了测试基质金属蛋白酶 (mmp) 蛋白水解的证据,将另外的培养物与mmp 2/9、3和8的抑制剂共同孵育。IGFBP-2抑制被明胶酶 (MMP-2/9) 抑制部分逆转。总之,这些发现进一步描述了IL-1作为关节软骨内igf-i系统的关键调节因子的作用,表明调节通过直接 (转录) 和间接 (蛋白水解) 机制发生。
  • 【辛伐他汀依赖的肌动蛋白细胞骨架重排通过兔关节软骨细胞的细胞外信号调节激酶1/2和p38激酶途径调节分化。】 复制标题 收藏 收藏
    DOI:10.1016/j.ejphar.2018.07.016 复制DOI
    作者列表:Han Y,Kim SJ
    BACKGROUND & AIMS: :Alterations in cell morphology involve changes in the actin cytoskeleton and play crucial roles in determining chondrocyte phenotypes. Although the effects of simvastatin (SV) have been demonstrated in various cell types, the mechanisms and effects of SV on chondrocyte differentiation and actin cytoskeletal rearrangement are still unclear. Here, we investigated the roles of actin filament rearrangement on SV-induced differentiation of rabbit articular chondrocytes. Treatment with SV caused actin remodeling in comparison with that in untreated chondrocytes, as determined by immunofluorescence staining. Moreover, treatment with cytochalasin D (CD) and jasplakinolide (JAS), which modulate actin filament formation, resulted in reorganization of the actin cytoskeleton compared with that induced by SV in chondrocytes. In addition, CD synergistically enhanced the SV-induced increase in type II collagen expression, whereas JAS dramatically inhibited SV-induced differentiation. We also found that differentiation via SV-dependent actin cytoskeleton changes was regulated by the extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase pathways. These results demonstrated that actin cytoskeletal rearrangement by SV regulated type II collagen expression and suggested that ERK-1/2 and p38 kinase pathways may play important roles in SV-induced type II collagen expression by altering actin cytoskeletal reorganization in rabbit articular chondrocytes.
    背景与目标: : 细胞形态的改变涉及肌动蛋白细胞骨架的改变,并在确定软骨细胞表型中起关键作用。尽管辛伐他汀 (SV) 的作用已在各种细胞类型中得到证实,但SV对软骨细胞分化和肌动蛋白细胞骨架重排的机制和作用仍不清楚。在这里,我们研究了肌动蛋白丝重排在SV诱导的兔关节软骨细胞分化中的作用。通过免疫荧光染色确定,与未处理的软骨细胞相比,SV治疗引起肌动蛋白重塑。此外,与SV诱导的软骨细胞相比,调节肌动蛋白丝形成的细胞松弛素D (CD) 和jasplakinolide (JAS) 治疗可导致肌动蛋白细胞骨架的重组。此外,CD协同增强SV诱导的II型胶原表达的增加,而JAS显着抑制SV诱导的分化。我们还发现,通过SV依赖性肌动蛋白细胞骨架变化的分化受细胞外信号调节激酶 (ERK)-1/2和p38激酶途径的调节。这些结果表明,SV引起的肌动蛋白细胞骨架重排调节了II型胶原的表达,并表明ERK-1/2和p38激酶途径可能通过改变兔关节软骨细胞的肌动蛋白细胞骨架重组在SV诱导的II型胶原表达中起重要作用。
  • 【microRNA-203a的下调通过Smad3信号抑制人软骨细胞中il-1β 诱导的炎症和软骨降解。】 复制标题 收藏 收藏
    DOI:10.1042/BSR20192723 复制DOI
    作者列表:An Y,Wan G,Tao J,Cui M,Zhou Q,Hou W
    BACKGROUND & AIMS: :Osteoarthritis (OA) is a chronic and prevalent degenerative musculoskeletal disorder, which is characterized by articular cartilage degradation and joint inflammation. MicroRNA-203a (miR-203a) has been shown to be involved in multiple pathological processes during OA, but little is known about its function in chondrocyte extracellular matrix (ECM) degradation. In the present study, we aimed to elucidate the effects of miR-203a on articular cartilage degradation and joint inflammation. We observed that the miR-203a level was significantly up-regulated in OA tissues and in an in vitro model of OA, respectively. Inhibition of miR-203a significantly alleviated the interleukin (IL)-1β-induced inflammatory response and ECM degradation in chondrocytes. Moreover, mothers against decapentaplegic homolog 3 (Smad3), a key factor in maintaining chondrocyte homeostasis, was identified as a putative target of miR-203a in chondrocytes. More importantly, inhibition of Smad3 impaired the inhibitory effects of the miR-203a on IL-1β-induced inflammatory response and ECM degradation. Collectively, these results demonstrated that miR-203a may contribute to articular cartilage degradation of OA by targeting Smad3, suggesting a novel therapeutic target for the treatment of OA.
    背景与目标: : 骨关节炎 (OA) 是一种慢性且普遍的退行性肌肉骨骼疾病,其特征是关节软骨退化和关节炎症。MicroRNA-203a (miR-203a) 已被证明参与OA期间的多个病理过程,但对其在软骨细胞细胞外基质 (ECM) 降解中的功能知之甚少。在本研究中,我们旨在阐明miR-203a对关节软骨退化和关节炎症的影响。我们观察到,在OA组织和OA的体外模型中,miR-203a水平分别显着上调。抑制miR-203a可显著减轻白细胞介素 (IL)-1β 诱导的软骨细胞炎症反应和ECM降解。此外,抗去胸截瘫同源物3 (Smad3) 是维持软骨细胞稳态的关键因素,被确定为软骨细胞miR-203a的推定靶标。更重要的是,Smad3的抑制损害了miR-203a对il-1β 诱导的炎症反应和ECM降解的抑制作用。总的来说,这些结果表明,miR-203a可能通过靶向Smad3来促进OA的关节软骨降解,从而为OA的治疗提供了新的治疗靶标。
  • 【蛋白激酶C和细胞外信号调节蛋白激酶维持关节软骨细胞分化表型。】 复制标题 收藏 收藏
    DOI:10.1074/jbc.M110608200 复制DOI
    作者列表:Yoon YM,Kim SJ,Oh CD,Ju JW,Song WK,Yoo YJ,Huh TL,Chun JS
    BACKGROUND & AIMS: The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC alpha was essentially the same as that of type II collagen during de- and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC alpha, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC alpha in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC alpha did not affect type II collagen levels, suggesting that PKC alpha expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by down-regulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of N-cadherin, alpha-catenin, and beta-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.

    背景与目标: 软骨细胞的分化表型在体外培养过程中通过称为 “去分化” 的过程迅速丧失。在这项研究中,我们研究了蛋白激酶C (PKC) 和细胞外信号调节蛋白激酶 (ERK) 在维持分化的软骨细胞表型中的作用。从兔关节软骨分离的软骨细胞在连续单层培养后进行去分化,停止II型胶原表达和蛋白聚糖合成,这通过在藻酸盐凝胶中培养去分化细胞而被逆转。PKC α 在去分化和再分化过程中的表达模式与II型胶原的表达模式基本相同,因为在去分化过程中表达降低,而在再分化过程中表达增加。与PKC α 相反,去分化过程中ERK活性增加了15倍。这种增强的活性在再分化过程中终止。0代软骨细胞中PKC α 的下调导致去分化。然而,PKC α 的过表达并未影响II型胶原水平,这表明PKC α 的表达不足以维持分化的表型。然而,PD98059对ERK的抑制增强了第0代细胞中II型胶原的表达和蛋白聚糖的合成,延缓了单层培养过程中的去分化,并逆转了PKC下调引起的去分化。与PKC依赖性的ERK调节软骨形成不同,PKC和ERK独立调节软骨细胞去分化,这通过观察证实,PKC下调和ERK抑制分别不会改变ERK磷酸化和PKC表达。此外,在表型改变过程中与II型胶原相反调节的N-钙粘蛋白,α-连环蛋白和 β-连环蛋白的表达在软骨形成过程中受到PKC和ERK的调节,但没有去分化,支持这两种蛋白激酶调节软骨细胞分化和维持分化表型的独特机制。
  • 【非甾体抗炎药对胎鼠骨骺关节软骨细胞增殖和死亡的影响。】 复制标题 收藏 收藏
    DOI:10.1016/j.tox.2006.08.028 复制DOI
    作者列表:Chang JK,Wu SC,Wang GJ,Cho MH,Ho ML
    BACKGROUND & AIMS: :Previous reports indicated that non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone repair. Our previous study further found that ketorolac delayed the endochondral bone formation, and the critical effective timing was at the early stage of repair. Furthermore, we found that NSAIDs suppressed proliferation and induced cell death of cultured osteoblasts. In this study, we hypothesized that chondrocytic proliferation and death, which plays an important role at the early stage of endochondral bone formation, might be affected by NSAIDs. Non-selective NSAIDs, indomethacin, ketorolac, diclofenac and piroxicam; cyclooxygenase-2 (COX-2) selective NSAIDs, celecoxib and DFU (an analog of rofecoxib); prostaglandins (PGs), PGE1, PGE2 and PGF2alpha; and each NSAID plus each PG were tested. The effects of NSAIDs on proliferation, cell cycle kinetics, cytotoxicity and cell death of epiphyseal-articular chondrocytes of fetal rats were examined. The results showed that all the tested NSAIDs, except DFU, inhibited thymidine incorporation of chondrocytes at a concentration range (10(-8) to 10(-4)M) covering the theoretic therapeutic concentrations. Cell cycle was arrested by NSAIDs at the G(0)/G(1) phase. Upon a 24h treatment, LDH leakage and cell death (both apoptosis and necrosis) were significantly induced by the four non-selective NSAIDs in chondrocyte cultures. However, COX-2 inhibitors revealed non-significant effects on cytotoxicity of chondrocytes except higher concentration of celecoxib (10(-4)M). Replenishments of PGE1, PGE2 or PGF2alpha could not reverse the effects of NSAIDs on chondrocytic proliferation and cytotoxicity. In this study, we found that therapeutic concentrations of non-selective NSAIDs caused proliferation suppression and cell death of chondrocytes, suggesting these adverse effects may be one of the reasons that NSAIDs delay the endochondral ossification during bone repair found in previous studies. Furthermore, these effects of NSAIDs may act via PG-independent mechanisms. COX-2 selective NSAIDs showed less deleterious effects on chondrocytic proliferation and death.
    背景与目标: : 以前的报道表明非甾体抗炎药 (NSAIDs) 抑制骨修复。我们先前的研究进一步发现,酮洛尔可延迟软骨内骨的形成,并且关键的有效时机是在修复的早期。此外,我们发现NSAIDs抑制了培养的成骨细胞的增殖并诱导了细胞死亡。在这项研究中,我们假设NSAIDs可能会影响软骨细胞的增殖和死亡,这在软骨内骨形成的早期阶段起着重要作用。非选择性NSAIDs,吲哚美辛,酮咯酸,双氯芬酸和吡罗昔康; cyclooxygenase-2 (COX-2) 选择性NSAIDs,塞来昔布和DFU (罗非考昔的类似物); 前列腺素 (PGs),PGE1,PGE2和pgf2α; 以及每个NSAID加上每个PG。研究了NSAIDs对胎鼠epi关节软骨细胞增殖,细胞周期动力学,细胞毒性和细胞死亡的影响。结果表明,除DFU外,所有测试的NSAIDs均在覆盖理论治疗浓度的浓度范围 (10(-8) 至10(-4)M) 下抑制胸苷掺入软骨细胞。NSAIDs在G(0)/G(1) 期阻止了细胞周期。处理24小时后,软骨细胞培养物中的四种非选择性NSAIDs显着诱导了LDH泄漏和细胞死亡 (凋亡和坏死)。然而,除了较高浓度的塞来昔布 (10(-4)M) 外,COX-2抑制剂对软骨细胞的细胞毒性无显着影响。补充PGE1,PGE2或PGF2alpha不能逆转NSAIDs对软骨细胞增殖和细胞毒性的影响。在这项研究中,我们发现治疗浓度的非选择性NSAIDs导致软骨细胞增殖抑制和细胞死亡,这可能是先前研究发现的NSAIDs延迟骨修复过程中软骨内骨化的原因之一。此外,NSAIDs的这些作用可能通过PG独立机制起作用。COX-2选择性NSAIDs对软骨细胞增殖和死亡的有害作用较小。
  • 【非经典雌激素受体GPR30在人软骨和软骨细胞中的表达和功能。】 复制标题 收藏 收藏
    DOI:10.1002/jcp.29691 复制DOI
    作者列表:Ribeiro M,Sousa C,Rufino AT,Judas F,Mendes AF
    BACKGROUND & AIMS: :Estrogen hormones are important for cartilage homeostasis, but nothing is known regarding the expression and role of the membrane G protein-coupled estrogen receptor (GPER), G protein-coupled receptor 30 (GPR30), in adult articular chondrocytes. Using immunohistochemistry of cartilage sections, quantitative real-time polymerase chain reaction and Western blot of chondrocyte extracts, we found that these cells express GPR30. Nonetheless, the pattern of bands detected by two distinct antibodies does not overlap, suggesting that the proteins detected represent partially degraded forms of the receptor. Treatment with GPR30 agonists did not induce Akt or ERK1/2 phosphorylation, two known GPR30-activated signaling pathways, suggesting that GPR30 is not functional in human chondrocytes. Therefore, the protective anti-osteoarthritic role of estrogen hormones in cartilage homeostasis is likely independent of GPR30. This study was performed using human cartilage collected from the distal femoral condyles of multiorgan donors at the Bone and Tissue Bank of the University and Hospital Center of Coimbra.
    背景与目标: : 雌激素激素对软骨稳态很重要,但关于成年关节软骨细胞中膜g蛋白偶联雌激素受体 (GPER),g蛋白偶联受体30 (GPR30) 的表达和作用尚不清楚。使用软骨切片的免疫组织化学,实时定量聚合酶链反应和软骨细胞提取物的蛋白质印迹,我们发现这些细胞表达gpr30。尽管如此,两种不同抗体检测到的条带模式不会重叠,这表明检测到的蛋白质代表了受体的部分降解形式。用GPR30激动剂治疗不会诱导Akt或ERK1/2磷酸化,这是两种已知的GPR30-activated信号通路,表明GPR30在人软骨细胞中不起作用。因此,雌激素在软骨稳态中的保护性抗骨关节炎作用可能与gpr30无关。这项研究是使用从科英布拉大学和医院中心的骨骼和组织库的多器官供体的股骨远端con收集的人类软骨进行的。
  • 【硫酸氨基葡萄糖对牛原代软骨细胞内UDP-己糖胺和UDP-葡萄糖醛酸水平的影响。】 复制标题 收藏 收藏
    DOI:10.1016/j.joca.2007.01.010 复制DOI
    作者列表:Qu CJ,Jauhiainen M,Auriola S,Helminen HJ,Lammi MJ
    BACKGROUND & AIMS: OBJECTIVE:To analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes. METHODS:Chondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using (35)S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis. RESULTS:Levels of UDP-HexN and UDP-GlcA were unchanged at 10microM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100microM or 1mM of GS for 24h. CONCLUSION:Physiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.
    背景与目标:
  • 【MEG3通过miR-361-5p/FOXO1轴促进骨关节炎软骨细胞的增殖和抑制凋亡。】 复制标题 收藏 收藏
    DOI:10.1186/s12920-019-0649-6 复制DOI
    作者列表:Wang A,Hu N,Zhang Y,Chen Y,Su C,Lv Y,Shen Y
    BACKGROUND & AIMS: BACKGROUND:This study aimed to investigate the role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) and related molecular mechanisms, in osteoarthritis (OA). METHODS:Cartilage tissues of OA patients and healthy volunteers were isolated and cultured. After transfection with the appropriate constructs, chondrocytes were classified into Blank, pcDNA3.1-NC, pcDNA3.1-MEG3, si-NC, si-MEG3, pcDNA3.1-NC + mimics NC, pcDNA3.1-MEG3 + mimics NC, pcDNA3.1-NC + miR-361-5p mimics and pcDNA3.1-MEG3 + miR-361-5p mimics groups. qRT-PCR was used to detect the expression of MEG3, miR-361-5p and FOXO1. Western blot, luciferase reporter assay, RIP, CCK-8, and flow cytometry analysis were performed to reveal the morphology, proliferation, and apoptotic status of cartilage cells. Histological analysis and immunostaining were conducted in the OA rat model. RESULTS:Expression of MEG3 and FOXO1 was significantly decreased in OA compared with the normal group, while the expression of miR-361-5p was increased. MEG3 might serve as a ceRNA of miR-361-5p in OA chondrocytes. Moreover, using western blot analyses and the CCK-8 assay, MEG3 was shown to target miR-361-5p/FOXO1, elevate cell proliferation, and impair cell apoptosis. Functional analysis in vivo showed that MEG3 suppressed degradation of the cartilage matrix. CONCLUSION:MEG3 can contribute to cell proliferation and inhibit cell apoptosis and degradation of extracellular matrix (ECM) via the miR-361-5p/FOXO1 axis in OA chondrocytes.
    背景与目标:

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