Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
The Expression of the Anti-Apoptotic Transcription Factor NFkappaB-P65 Is Markedly Diminished in Chondrocytes of Murine Osteoarthritic Cartilage and in a Subset of Human Osteoarthritic Cartilage Samples.
A. J. van de Loo, Fons, J. Arntz, Onno, B. Bennink, Miranda, Blaney-Davidson, Esmeralda, M. van der Kraan, Peter, B. van den Berg, Wim
Chondrocytes play a central role in cartilage pathology as seen in rheumatoid arthritis (RA) and osteoarthritis (OA) patients by a deranged synthesis of extracellular matrix (ECM) components and the enhanced release of ECM destructive metalloproteinases (MMPs). Nuclear factor-kappaB (NF-kappaB) is an important transcription factor in the regulation of MMPs, but is also regarded as a survival factor in cells. We studied the regulation of NF-kappaB-P65 in chondrocytes in rheumatoid arthritis, osteoarthritis, mouse models of arthritis and osteoarthritis and the functional consequences of decreased level of NFkappaB-P65.
NF-kappaB-P65 was measured in primary chondrocytes of arthritic cartilage obtained from joint replacement surgery, and in cartilage of a spontaneous osteoarthritis mouse model (STR/ORT), streptococcal cell wall-, antigen-induced arthritis, and of young (14 weeks) and old (>12 months) mice by Western blotting, immunohistochemistry or RT-qPCR. To study the functional consequences of decreased level of NF-kappaB-P65 in chondrocytes the murine H4 chondrocyte-cell line was stably transduced with a lentivirus expressing a short-hairpin RNA against NF-kappaB-P65. To study the biological consequences, conditioned medium of OA synovium was added to the murine H4 chondrocyte cell line with the lowest NF-kappaB-P65 level.
High NF-kappaB-P65 levels were detected by immunohistochemistry and Western blot in chondrocytes of RA patients, whereas in a subset of OA cartilage samples the levels were unexpected low (6 out of 12). In mouse models the level of NF-kappaB-P65 showed the same regulation. NF-kappaB-P65 levels in cartilage from murine arthritis models was increased up to 250% at day 2 after induction of streptococcal cell wall- or antigen-induced arthritis and at day 7 returned to the basal level of naive knee joints, whereas in STR/ORT mice levels were diminished more than 75% when joints became affected, as determined by immunohistochemistry. Levels of NF-kappaB-P65 in young and old mice were equal, but the older groups showed more variation, detected by immunohistochemistry. In vitro, we selected chondrocyte cell-lines with different levels of NF-kappaB-P65. By adding TNFalpha, cell death was only induced in the cells with low levels of NF-kappaB-P65, as detected by 7-AAD staining. A clear negative correlation between TNFalpha induced cell death and the levels of NF-kappaB-P65 in chondrocyte cell-lines was found. Adding conditioned medium of synovial explants from different OA patients to the murine chondrocyte cell-line with the lowest NF-kappaB-P65 level, resulted in more than 60% chondrocyte death in 3 of the 5 conditioned medium samples tested which could be prevented by preincubation of these media with soluble-TNFR1 (Enbrel). TNFalpha was detected using a Luminex assay in the same samples that caused cell death.
This study clearly demonstrated that lower levels of NF-kappaB-P65 makes chondrocytes more vulnerable for TNFalpha, a cytokine which can be produced during OA, and that this anti-apoptotic transcription factor is downregulated in chondrocytes in murine OA and in 50% of OA patients.
To cite this abstract, please use the following information:
A. J. van de Loo, Fons, J. Arntz, Onno, B. Bennink, Miranda, Blaney-Davidson, Esmeralda, M. van der Kraan, Peter, B. van den Berg, Wim; The Expression of the Anti-Apoptotic Transcription Factor NFkappaB-P65 Is Markedly Diminished in Chondrocytes of Murine Osteoarthritic Cartilage and in a Subset of Human Osteoarthritic Cartilage Samples. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1532