Monosodium Urate and Tumor Necrosis Factor-α Increase Apoptosis in Human Chondrocyte Cultures

Abstract

Monosodium urate and tumor necrosis factor-α, are two potent mediators of separate inflammatory response pathways in arthritic joints where inflammation may be accompanied by the loss of chondrocyte vitality via apoptosis. To address this possibility in vitro, chondrocyte cultures were employed to determine the extent to which monosodium urate and recombinant TNF-α altered the frequency of apoptotic chondrocytes. Apoptosis as a function of the activation of p38 kinase, C-Jun-terminal kinase, signal transducer and activator of transcription-3 and/or the activity of xanthine oxidase was also studied. Using normal human chondrocytes, monosodium urate or recombinant tumor necrosis factor-α increased the frequency of apoptosis and activity of xanthine oxidase. However, the xanthine oxidase-specific inhibitor, febuxostat, failed to blunt this response. Monosodium urate, tumor necrosis factor-α or the Janus kinase inhibitor, AG-490, increased the frequency of apoptotic nuclei in macroaggregate pellet cultures initiated from juvenile human chondrocytes, but not in pellet cultures derived from mesenchymal stem cells. In OA chondrocytes, activation of p38, C-Jun-NH2-kinase and signal transducer and activator of transcription-3 preceded apoptosis. Activation of signal transducer and activator of transcription-3 also was seen in pellet cultures initiated from juvenile chondrocytes and MSCs incubated with MSU, recombinant tumor necrosis factor-α or febuxostat, but apoptosis was increased only in the pellet cultures derived from juvenile chondrocytes. Although AG-490 or the combination of AG-490 and febuxostat inhibited signal transducer and activator of transcription-3 activation, apoptosis was unaffected. These results showed that recombinant tumor necrosis factor-α, monosodium urate and AG-490 increased apoptosis in normal human chondrocytes, OA chondrocytes and human juvenile chondrocyte pellet cultures, but not in chondrocyte pellet cultures initiated from MSCs. The increased frequency of apoptotic chondrocytes in response to recombinant tumor necrosis factor-α or monosodium urate was not dependent on either activation of STAT3 or the activity of XO.

Intracellular Signaling Pathways in Rheumatoid Arthritis

“…Thus, dysfunctional intracellular signaling induced by pro-inflammatory cytokines was shown to be responsible for aberrant immune-cell survival [31-33], articular chondrocyte apoptosis [33,34] and/or “apoptosis-resistance” of cells in RA synovial tissue [35-38]. Abnormalities in the aforementioned JAK/ STAT, SAPK/MAPK and PI-3K/AKT/mTOR pathways [26,27,39-41] as well as aberrant activities in spleen tyrosine kinase (Syk) [42-46], the sphingosine kinases, SphK1 and SphK2 [47-52], transforming growth factor β-activated kinase-1 (TAK1) [53], bone marrow kinase (BMX) [54] and nuclear factor-κB-inducing kinase (NIK) [55] have all been found in patients with active RA.…”

U0126, an Inhibitor of MEK1/2, Increases Tumor Necrosis Factor-?-Induced Apoptosis, but not Interleukin-6 Induced Apoptosis in C-28/I2 Human Chondrocytes

“…Our laboratory had previously shown that rhTNF-α was a potent pro-inflammatory cytokine inducer of apoptosis in human chondrocytes which had been enzymatically dissociated from OA knee cartilage [24]. This was indicated by the increased number of DAPI/TUNEL-positive chondrocytes in the rhTNF-α-treated group compared to a control group.…”

“…The number of apoptotic chondrocytes was determined by the 4', 6-DiAmidino-2-Phenyl/Indole/Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (DAPI/TUNEL) assay as previously described [24]. C-28/I2 chondrocytes were incubated with either DMEM/F12 (1:1) plus 0.5% FBS, DMEM/F12 (1:1) plus 10% FBS, DMEM/F12 (1:1) plus DMSO and 0.5% FBS, or rhTNF-α (20 ng/ml), rhIL-6 (50 ng/ml), or rhOSM (50 ng/ml) or various combinations of rhTNF-α (20 ng/ml) and U0126 (10 μM), rhIL-6 (50 ng/ml) and U0126 (10 μM), rhIL-6 plus tocilizumab (200 ng/ml) or rhOSM (50 ng/ml) which were all maintained in DMEM/F12 (1:1) plus 0.5% FBS for 24 hr.…”

STAT1 is Constitutively Activated in the T/C28a2 Immortalized Juvenile Human Chondrocyte Line and Stimulated by IL-6 Plus Soluble IL-6R

“…We employed the 30 min time point for analysis of STAT1 protein activation based primarily on our previously published western blot data [47] which showed that activated STAT proteins accumulated in human chondrocyte protein lysate in response to tumor necrosis factor-α for up to 30 min after which time the anti-p-STAT band was undetectable by western blotting.…”

“…Western blots were produced as previously described [47]. However, in this study we employed a sequential antibody-probing/stripping technique to measure either U-STAT1 or p-STAT1 after which the immunoblot was stripped and reprobed with the β-actin antibody.…”