Dataset Information

Biological Effects of Phosphocitrate on Osteoarthritic Articular Chondrocytes.

ABSTRACT: BACKGROUND:Phosphocitrate (PC) inhibits osteoarthritis (OA) in Hartley guinea pigs. However, the underlying molecular mechanisms remain poorly understood. OBJECTIVE:This study sought to examine the biological effect of PC on OA chondrocytes and test the hypothesis that PC may exert its OA disease modifying effect, in part, by inhibiting the expression of genes implicated in OA disease process and stimulating the production of extracellular matrices. METHOD:OA chondrocytes were cultured in the absence or presence of PC. Total RNA was extracted and subjected to microarray analyses. The effect of PC on proliferation and chondrocyte-mediated calcification were examined in monolayer culture. The effect of PC on the production of extracellular matrices was examined in micromass culture. RESULTS:PC downregulated the expression of numerous genes classified in proliferation and apoptosis while upregulating the expression of many genes classified in transforming growth factor-? (TGF-?) receptor signaling pathway and ossification. PC also downregulated the expressions of many genes classified in inflammatory response and Wnt receptor signaling pathways. Consistent with its effect on the expression of genes classified in proliferation, ossification, and skeletal development, PC inhibited the proliferation of OA chondrocytes and chondrocyte-mediated calcification while stimulating the production of extracellular matrices. CONCLUSION:PC may exert its OA disease modifying effect, in part, through a crystal-independent mechanism or by inhibiting the expressions of many genes implicated in OA disease process, and at the same time, stimulating the expression of genes implicated in chondroprotection and production of extracellular matrices.

SUBMITTER: Sun Y

PROVIDER: S-EPMC5470061 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Biological Effects of Phosphocitrate on Osteoarthritic Articular Chondrocytes.

Sun Yubo Y Franklin Atiya M AM Mauerhan David R DR Hanley Edward N EN

The open rheumatology journal 20170531

<h4>Background</h4>Phosphocitrate (PC) inhibits osteoarthritis (OA) in Hartley guinea pigs. However, the underlying molecular mechanisms remain poorly understood.<h4>Objective</h4>This study sought to examine the biological effect of PC on OA chondrocytes and test the hypothesis that PC may exert its OA disease modifying effect, in part, by inhibiting the expression of genes implicated in OA disease process and stimulating the production of extracellular matrices.<h4>Method</h4>OA chondrocytes w ...[more]

PMID: 28659999

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Project description:A switch from autophagy to apoptosis is implicated in chondrocytes during the osteoarthritis (OA) progression with currently unknown mechanism(s). In this study we utilized a flow fluid shear stress (FFSS) model in cultured chondrocytes and a unilateral anterior crossbite (UAC) animal model. We found that both FFSS and UAC actively induced endoplasmic reticulum stress (ERS) in the temporomandibular joints (TMJ) chondrocytes, as demonstrated by dramatic increases in expression of HSPA5, p-EIF2AK3, p-ERN1 and ATF6. Interestingly, both FFSS and UAC activated not only pro-death p-EIF2AK3-mediated ERS-apoptosis programs but also pro-survival p-ERN1-mediated autophagic flux in chondrocytes. Data from FFSS demonstrated that MTORC1, a downstream of p-ERN1, suppressed autophagy but promoted p-EIF2AK3 mediated ERS-apoptosis. Data from UAC model demonstrated that at early stage both the p-ERN1 and p-EIF2AK3 were activated and MTORC1 was inhibited in TMJ chondrocytes. At late stage, MTORC1-p-EIF2AK3-mediated ERS apoptosis were predominant, while p-ERN1 and autophagic flux were inhibited. Inhibition of MTORC1 by TMJ local injection of rapamycin in rats or inducible ablation of MTORC1 expression selectively in chondrocytes in mice promoted chondrocyte autophagy and suppressed apoptosis, and reduced TMJ cartilage loss induced by UAC. In contrast, MTORC1 activation by TMJ local administration of MHY1485 or genetic deletion of Tsc1, an upstream MTORC1 suppressor, resulted in opposite effects. Collectively, our results establish that aberrant mechanical loading causes cartilage degeneration by activating, at least in part, the MTORC1 signaling which modulates the autophagy and apoptosis programs in TMJ chondrocytes. Thus, inhibition of MTORC1 provides a novel therapeutic strategy for prevention and treatment of OA.Abbreviations : ACTB: actin beta; ATF6: activating transcription factor 6; BECN1: beclin 1; BFL: bafilomycin A1; CASP12: caspase 12; CASP3: caspase 3; DAPI: 4',6-diamidino-2-phenylindole; DDIT3: DNA-damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FFSS: flow fluid shear stress; HSPA5/GRP78/BiP: heat shock protein 5; LAMP2: lysosome-associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; OA: osteoarthritis; PRKAA1/2/AMPK1/2: protein kinase, AMP-activated, alpha 1/2 catalytic subunit; RPS6: ribosomal protein S6; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: thapsigargin; TMJ: temporomandibular joints; TSC1/2: tuberous sclerosis complex 1/2; UAC: unilateral anterior crossbite; UPR: unfolded protein response; XBP1: x-box binding protein 1.

Project description:Our previous study suggested that growth arrest and DNA damage-inducible protein 45beta (GADD45beta) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45beta plays a role in adult articular cartilage.Gene expression profiles of cartilage from patients with late-stage osteoarthritis (OA) were compared with those from patients with early OA and normal controls in 2 separate microarray analyses. Histologic features of cartilage were graded using the Mankin scale, and GADD45beta was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)-GADD45beta or GADD45beta-FLAG. GADD45beta and COL2A1 messenger RNA (mRNA) levels were analyzed by real-time reverse transcriptase-polymerase chain reaction, and promoter activities were analyzed by transient transfection. Cell death was detected by Hoechst 33342 staining of condensed chromatin.GADD45beta was expressed at higher levels in cartilage from normal donors and patients with early OA than in cartilage from patients with late-stage OA. All chondrocyte nuclei in normal cartilage immunostained for GADD45beta. In early OA cartilage, GADD45beta was distributed variably in chondrocyte clusters, in middle and deep zone cells, and in osteophytes. In contrast, COL2A1, other collagen genes, and factors associated with skeletal development were up-regulated in late OA, compared with early OA or normal cartilage. In overexpression and knockdown experiments, GADD45beta down-regulated COL2A1 mRNA and promoter activity. NF-kappaB overexpression increased GADD45beta promoter activity, and siRNA-GADD45beta decreased cell survival per se and enhanced tumor necrosis factor alpha-induced cell death in human articular chondrocytes.These observations suggest that GADD45beta might play an important role in regulating chondrocyte homeostasis by modulating collagen gene expression and promoting cell survival in normal adult cartilage and in early OA.

Project description:Shenmai injection (SMI) has been widely used as a therapy to treat a number of diseases. However, its anti-osteoarthritic properties have not yet been fully investigated. In the present study, the protective effect of SMI on knee articular cartilage of anterior cruciate ligament transected rabbits and interleukin-1β (IL-1β)-stimulated human chondrocytes was investigated. For the in vivo study, knee osteoarthritis (KOA) was induced in female New Zealand white rabbits by anterior cruciate ligament transection (ACLT) in the knee of right hind limb. Rabbits either underwent sham surgery or ACLT surgery. Out of the rabbits receiving ACLT surgery, half of the rabbits received one 0.3 ml Shenmai intra-articular injection in the knee per week for four weeks, following ACLT surgery. The other rabbits received the same volume of normal saline solution. The cartilage was subsequently collected for histological evaluation. For the in vitro study, cultured human chondrocytes were treated with 10 ng/ml IL-1β in the presence or absence of 5 and 2% (v/v) SMI for 24 h. Nitric oxide (NO) and prostaglandin E2 (PGE2) levels in cell culture supernatant were assessed using a Griess reaction and ELISA respectively. The mRNA expression of cyclooxgenase-2 (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-1, MMP-13 and tissue inhibitors of metalloproteinase-1 (TIMP-1) in chondrocytes were detected by reverse transcription-quantitative polymerase chain reaction. The results of the current study revealed that treatment with SMI ameliorated cartilage degradation in the ACLT rabbit model, and decreased levels of NO and PGE2. Furthermore, treatment with SMI decreased levels of COX-2, iNOS, MMP-1 and MMP-13 mRNA expression and increased TIMP-1 mRNA expression in IL-1β-stimulated human chondrocytes. These results indicate that SMI suppresses inflammation and ameliorated cartilage degradation, making it a potential and promising therapeutic option to treat KOA.

Dataset Information

Biological Effects of Phosphocitrate on Osteoarthritic Articular Chondrocytes.

Publications

Biological Effects of Phosphocitrate on Osteoarthritic Articular Chondrocytes.

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