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

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.

[1]  F. Flamigni,et al.  Chondrocyte hypertrophy and apoptosis induced by GROα require three‐dimensional interaction with the extracellular matrix and a co‐receptor role of chondroitin sulfate and are associated with the mitochondrial splicing variant of cathepsin B , 2007, Journal of cellular physiology.

[2]  Hana Kim,et al.  A Fourth IκB Protein within the NF-κB Signaling Module , 2007, Cell.

[3]  K. N. Pennington,et al.  IκB Kinase Subunits α and γ Are Required for Activation of NF-κB and Induction of Apoptosis by Mammalian Reovirus , 2006, Journal of Virology.

[4]  A. Hoffmann,et al.  Transcriptional regulation via the NF-κB signaling module , 2006, Oncogene.

[5]  A J Verbout,et al.  Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. , 2006, Tissue engineering.

[6]  S. Jimenez,et al.  NF-κB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis , 2006 .

[7]  Jian Fei Wang,et al.  A Novel Role for GADD45β as a Mediator of MMP-13 Gene Expression during Chondrocyte Terminal Differentiation* , 2005, Journal of Biological Chemistry.

[8]  Michael Karin,et al.  NF-κB: linking inflammation and immunity to cancer development and progression , 2005, Nature Reviews Immunology.

[9]  L. Pfeffer,et al.  Interferon Induces NF-κB-inducing Kinase/Tumor Necrosis Factor Receptor-associated Factor-dependent NF-κB Activation to Promote Cell Survival* , 2005, Journal of Biological Chemistry.

[10]  F. Flamigni,et al.  Polyamine depletion inhibits NF‐κB binding to DNA and interleukin‐8 production in human chondrocytes stimulated by tumor necrosis factor‐α , 2005 .

[11]  R. Borzì,et al.  Cell and matrix morpho‐functional analysis in chondrocyte micromasses , 2005, Microscopy research and technique.

[12]  B Kurz,et al.  Oxygen and reactive oxygen species in cartilage degradation: friends or foes? , 2005, Osteoarthritis and cartilage.

[13]  R. O’Keefe,et al.  Transcriptional regulation of chondrocyte maturation: potential involvement of transcription factors in OA pathogenesis. , 2005, Molecular aspects of medicine.

[14]  R. Zimmer,et al.  Functional Genomics of Osteoarthritis: On the Way to Evaluate Disease Hypotheses , 2004, Clinical orthopaedics and related research.

[15]  R. Spencer,et al.  Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants , 2004, Journal of anatomy.

[16]  H. Anderson,et al.  Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization. , 2004, Bone.

[17]  M. Karin,et al.  The two NF-κB activation pathways and their role in innate and adaptive immunity , 2004 .

[18]  M. Karin,et al.  IκB kinase-α acts in the epidermis to control skeletal and craniofacial morphogenesis , 2004, Nature.

[19]  Z. Werb,et al.  Matrix remodeling during endochondral ossification. , 2004, Trends in cell biology.

[20]  A. Baldwin,et al.  NF-κB mediates inhibition of mesenchymal cell differentiation through a posttranscriptional gene silencing mechanism , 2003 .

[21]  Di Chen,et al.  NF-κB Specifically Activates BMP-2 Gene Expression in Growth Plate Chondrocytes in Vivo and in a Chondrocyte Cell Line in Vitro* , 2003, Journal of Biological Chemistry.

[22]  W. Li,et al.  Induction of matrix metalloproteinase-13 gene expression by TNF-α is mediated by MAP kinases, AP-1, and NF-κB transcription factors in articular chondrocytes , 2003 .

[23]  Brian D. Strahl,et al.  A nucleosomal function for IκB kinase-α in NF-κB-dependent gene expression , 2003, Nature.

[24]  R. Gaynor,et al.  Histone H3 phosphorylation by IKK-α is critical for cytokine-induced gene expression , 2003, Nature.

[25]  A. Lindahl,et al.  Phenotypic Plasticity of Human Articular Chondrocytes , 2003, The Journal of bone and joint surgery. American volume.

[26]  G. W. Peet,et al.  IKKα, IKKβ, and NEMO/IKKγ Are Each Required for the NF-κB-mediated Inflammatory Response Program* , 2002, The Journal of Biological Chemistry.

[27]  A. Zien,et al.  Functional genomics of osteoarthritis. , 2002, Pharmacogenomics.

[28]  R. Bernards,et al.  Stable suppression of tumorigenicity by virus-mediated RNA interference. , 2002, Cancer cell.

[29]  K. Ostergaard,et al.  Histochemical studies of the extracellular matrix of human articular cartilage--a review. , 2002, Osteoarthritis and cartilage.

[30]  E. Schmidt,et al.  IKKα Provides an Essential Link between RANK Signaling and Cyclin D1 Expression during Mammary Gland Development , 2001, Cell.

[31]  C. Brinckerhoff,et al.  Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors , 2001, Arthritis Research & Therapy.

[32]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[33]  Thomas Aigner,et al.  Articular cartilage and changes in Arthritis: Cell biology of osteoarthritis , 2001, Arthritis Research & Therapy.

[34]  R. Santerre,et al.  Semi-Quantitative Fluorescence Analysis of Calcein Binding as a Measurement of In Vitro Mineralization , 2000, Calcified Tissue International.

[35]  B. Swoboda,et al.  Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage. , 2000, Osteoarthritis and cartilage.

[36]  C. Brinckerhoff,et al.  Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3. , 2000, Arthritis and rheumatism.

[37]  V. Lefebvre,et al.  Potent Inhibition of the Master Chondrogenic FactorSox9 Gene by Interleukin-1 and Tumor Necrosis Factor-α* , 2000, The Journal of Biological Chemistry.

[38]  A. Hollander,et al.  Expression of Type X Collagen and Matrix Calcification in Three‐Dimensional Cultures of Immortalized Temperature‐Sensitive Chondrocytes Derived from Adult Human Articular Cartilage , 1998, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[39]  C. Snapper,et al.  Nuclear Factor (NF)-κB2 (p100/p52) Is Required for Normal Splenic Microarchitecture and B Cell–mediated Immune Responses , 1998, The Journal of experimental medicine.

[40]  P. Roughley,et al.  Aggrecan degradation in human intervertebral disc and articular cartilage. , 1997, The Biochemical journal.

[41]  C. Rorabeck,et al.  Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. , 1997, The Journal of clinical investigation.

[42]  Q. Chen,et al.  Progression and recapitulation of the chondrocyte differentiation program: cartilage matrix protein is a marker for cartilage maturation. , 1995, Developmental Biology.

[43]  T. Aigner,et al.  Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis , 1995, The Journal of cell biology.

[44]  W. Landis,et al.  Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system , 1991, The Journal of cell biology.

[45]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.

[46]  N. Perkins,et al.  Integrating cell-signalling pathways with NF-κB and IKK function , 2007, Nature Reviews Molecular Cell Biology.

[47]  M. Goldring,et al.  The control of chondrogenesis , 2006, Journal of cellular biochemistry.

[48]  Ying Li,et al.  Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. , 2005, Osteoarthritis and cartilage.

[49]  Kozo Nakamura,et al.  Distinct roles of Sox5, Sox6, and Sox9 in different stages of chondrogenic differentiation , 2005, Journal of Bone and Mineral Metabolism.

[50]  R. Borzì,et al.  A role for chemokines in the induction of chondrocyte phenotype modulation. , 2004, Arthritis and rheumatism.

[51]  M. Attur,et al.  Osteoarthritis or osteoarthrosis: the definition of inflammation becomes a semantic issue in the genomic era of molecular medicine. , 2002, Osteoarthritis and cartilage.