Dataset Information

Differential requirements for IKKalpha and IKKbeta in the differentiation of primary human osteoarthritic chondrocytes.

ABSTRACT: Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKalpha and IKKbeta are essential kinases that activate NF-kappaB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy.Expression of IKKalpha or IKKbeta was ablated in primary human chondrocytes by retro-transduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed.The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKKalpha or IKKbeta. Silencing of IKKbeta markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKKalpha dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKKalpha-deficient cells retained the phenotype of cells in a pre-hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses.IKKalpha and IKKbeta exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKKalpha were more profound and pleotrophic in nature, our observations suggest that exacerbated IKKalpha activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.

SUBMITTER: Olivotto E

PROVIDER: S-EPMC2927097 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Differential requirements for IKKalpha and IKKbeta in the differentiation of primary human osteoarthritic chondrocytes.

Olivotto Eleonora E Borzi Rosa Maria RM Vitellozzi Roberta R Pagani Stefania S Facchini Annalisa A Battistelli Michela M Penzo Marianna M Li Xiang X Flamigni Flavio F Li Jun J Falcieri Elisabetta E Facchini Andrea A Marcu Kenneth B KB

Arthritis and rheumatism 20080101 1

<h4>Objective</h4>Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKalpha and IKKbeta are essential kinases that activate NF-kappaB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy.<h4>Methods< ...[more]

PMID: 18163512

Similar Datasets

Project description:BackgroundCellular senescence is a critical factor contributing to osteoarthritis (OA). Overexpression of chromobox homolog 4 (CBX4) in a mouse system was demonstrated to alleviate post-traumatic osteoarthritis (PTOA) by reducing cellular senescence. Additionally, replicative cellular senescence of WI-38 fibroblasts can be attenuated by CBX4. However, the mechanisms underlying this senomorphic function of CBX4 are not fully understood. In this study, we aimed to investigate the role of CBX4 in cellular senescence in human primary osteoarthritic chondrocytes and to identify the functional domains of CBX4 necessary for its function in modulating senescence.MethodsChondrocytes, isolated from 6 individuals undergoing total knee replacement for OA, were transduced with wild-type CBX4, mutant CBX4, and control lentiviral constructs. Senescence-related phenotypic outcomes included the following: multiple flow cytometry-measured markers (p16INK4A, senescence-associated β-galactosidase [SA-β-gal] activity and dipeptidyl peptidase-4 [DPP4], and proliferation marker EdU), multiplex ELISA-measured markers in chondrocyte culture media (senescence-associated secretory phenotypes [SASPs], including IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-3, and MMP-9), and PCR array-evaluated senescence-related genes.ResultsCompared with control, CBX4 overexpression in OA chondrocytes decreased DPP4 expression and SASP secretion and increased chondrocyte proliferation confirming CBX4 senomorphic effects on primary human chondrocytes. Point mutations of the chromodomain domain (CDM, involved in chromatin modification) alone were sufficient to partially block the senomorphic activity of CBX4 (p16INK4A and DPP4 increased, and EdU decreased) but had minimal effect on SASP secretion. Although having no effect on p16INK4A, DPP4, and EdU, deletion of two small-ubiquitin-like-modifier-interaction motifs (CBX4 ΔSIMs) led to increased SASP secretion (IL-1β, TNF-α, IL-8). The combination CBX4 CDMΔSIMs altered all these measures adversely and to a greater degree than the single domain mutants. Deletion of the C-terminal (CBX4 ΔC-box) involved with transcriptional silencing of polycomb group proteins increased IL-1β slightly but significantly but altered none of the other senescence outcome measures.ConclusionsCBX4 has a senomorphic effect on human osteoarthritic chondrocytes. CDM is critical for CBX4-mediated regulation of senescence. The SIMs are supportive but not indispensable for CBX4 senomorphic function while the C-box is dispensable.

Project description:Objectives: Zeel T (Ze14) is a multicomponent medicinal product. Initial preclinical data suggested a preventive effect on cartilage degradation. Clinical observational studies demonstrated that Ze14 reduced symptoms of osteoarthritis (OA), including stiffness and pain. This study aimed to explore these effects further to better understand the mode of action of Ze14 on human OA chondrocytes in vitro. Methods: Primary chondrocytes were obtained from the knees of 19 OA patients and cultured either as monolayers or in alginate beads. The cultures were treated with 20% or 10% (v/v) Ze14 or placebo. For RNA-seq, reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19). Differential expression analysis between Ze14 and placebo was performed in R using the DESeq2 package. Protein quantification by ELISA was performed on selected genes from the culture medium and/or the cellular fractions of primary human OA chondrocyte cultures. Results: In monolayer cultures, Ze14 20% (v/v) significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05: EGR1, FOS, NR4A1, DUSP1, ZFP36, ZFP36L1, NFKBIZ, and CCN1 were upregulated and ATF7IP, TXNIP, DEPP1, CLEC3A, and MMP13 were downregulated after 24 h Ze14 treatment. Ze14 significantly increased (mean 2.3-fold after 24 h, p = 0.0444 and 72 h, p = 0.0239) the CCN1 protein production in human OA chondrocytes. After 72 h, Ze14 significantly increased type II collagen pro-peptide production by mean 27% (p = 0.0147). For both time points CCN1 production by OA chondrocytes was correlated with aggrecan (r = 0.66, p = 0.0004) and type II collagen pro-peptide (r = 0.64, p = 0.0008) production. In alginate beads cultures, pro-MMP-13 was decreased by Ze14 from day 7-14 (from -16 to -25%, p < 0.05) and from day 17-21 (-22%, p = 0.0331) in comparison to controls. Conclusion: Ze14 significantly modified the expression of DUSP1, DEPP1, ZFP36/ZFP36L1, and CLEC3A, which may reduce MMP13 expression and activation. Protein analysis confirmed that Ze14 significantly reduced the production of pro-MMP-13. As MMP-13 is involved in type II collagen degradation, Ze14 may limit cartilage degradation. Ze14 also promoted extracellular matrix formation arguably through CCN1 production, a growth factor well correlated with type II collagen and aggrecan production.

Dataset Information

Differential requirements for IKKalpha and IKKbeta in the differentiation of primary human osteoarthritic chondrocytes.

Publications

Differential requirements for IKKalpha and IKKbeta in the differentiation of primary human osteoarthritic chondrocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets