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Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes.

ABSTRACT: To determine the ability of basic calcium phosphate (BCP) crystals to induce (a) mitogenesis, matrix metalloproteinase (MMP)-1, and MMP-13 in human osteoarthritic synovial fibroblasts (HOAS) and (b) MMP-13 in cultured porcine articular chondrocytes.Mitogenesis of HOAS was measured by [3H]thymidine incorporation assay and counts of cells in monolayer culture. MMP messenger RNA (mRNA) accumulation was determined either by northern blot analysis or reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA from chondrocytes or HOAS treated with BCP crystals. MMP-13 secretion was identified by immunoprecipitation and MMP-1 secretion by western blot of conditioned media.BCP crystals caused a 4.5-fold increase in [3H]thymidine incorporation by HOAS within 20 hours compared with untreated control cultures (p< or =0.05). BCP crystals induced MMP-13 mRNA accumulation and MMP-13 protein secretion by articular chondrocytes. In contrast, in HOAS, MMP-13 mRNA induced by BCP crystals was detectable only by RT-PCR, and MMP-13 protein was undetectable. BCP crystals induced MMP-1 mRNA accumulation and MMP-1 protein secretion by HOAS. MMP-1 expression was further augmented when HOAS were co-incubated with either BCP and tumour necrosis factor alpha (TNFalpha; threefold) or BCP and interleukin 1alpha (IL1alpha; twofold).These data confirm the ability of BCP crystals to activate HOAS, leading to the induction of mitogenesis and MMP-1 production. MMP-13 production in response to BCP crystals is substantially more detectable in porcine articular chondrocytes than in HOAS. These data support the active role of BCP crystals in osteoarthritis and suggest that BCP crystals act synergistically with IL1alpha and TNFalpha to promote MMP production and subsequent joint degeneration.

SUBMITTER: McCarthy GM

PROVIDER: S-EPMC1753595 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes.

McCarthy G M GM Westfall P R PR Masuda I I Christopherson P A PA Cheung H S HS Mitchell P G PG

Annals of the rheumatic diseases 20010401 4

<h4>Objective</h4>To determine the ability of basic calcium phosphate (BCP) crystals to induce (a) mitogenesis, matrix metalloproteinase (MMP)-1, and MMP-13 in human osteoarthritic synovial fibroblasts (HOAS) and (b) MMP-13 in cultured porcine articular chondrocytes.<h4>Methods</h4>Mitogenesis of HOAS was measured by [3H]thymidine incorporation assay and counts of cells in monolayer culture. MMP messenger RNA (mRNA) accumulation was determined either by northern blot analysis or reverse transcri ...[more]

PMID: 11247873

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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:INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

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.

Dataset Information

Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes.

Publications

Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes.

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