Figure 1.
Inhibition of cPLA2α reduces TNF-induced expression of PLA2G4A, COX2, MMP3 and IL8.
Fibroblast-like synoviocytes were treated with AVX002 (2 hrs) in indicated concentrations (A, B), 10 µM (C), or 5 µM (D), prior to TNF stimulation (10 ng/mL, 24 hrs). Total RNA was isolated and transcription of MMP3 (A) and IL8 (B), PLA2G4A and COX2 (D), was analyzed by real-time PCR as described in the Methods section. Amplification efficiency of all primer pairs were calculated by the LinRegPCR software and fold-change in gene expression compared to untreated samples was calculated by the REST 2009 software with GAPDH as reference gene. Supernatants were collected and analyzed by ELISA for MMP3 and IL8 protein (C) as described in the Method section (note starting point of Y-axis at 0.8). Data shown in all graphs are mean ± SEM (A, B, D) or mean ± SD (C) fold change compared to untreated samples for one representative of at least three independent experiments performed in duplicates. Significance is indicated as follows: A) #p ≤ 0.01 vs control; *p, **p ≤ 0.01 vs control and TNF-treated cells. B) #p ≤ 0.01 vs control; *p, **p, ***p ≤ 0.03 vs control and TNF-treated cells. C) IL8: #p ≤ 0.01 vs control; *p ≤ 0.01 vs control and TNF-treated cells. MMP3: ##p ≤ 0.01 vs control, **p ≤ 0.01 vs control and TNF-treated cells. D) COX2: #p ≤ 0.01 vs control; *p ≤ 0.03 vs control and TNF-treated cells. PLA2G4A: ##p ≤ 0.02 vs control; **p ≤ 0.03 vs TNF-treated cells.
Figure 2.
cPLA2α regulates TNF-induced AA release and PGE2 synthesis.
SW982 synoviocytes were treated with either AVX002 or ATK (2 hrs) in indicated concentrations (A, C) or 5 µM (B) prior to stimulation with TNF (10 ng/mL) for 24 hrs (A, C) or indicated times (B). Analysis of released [3H]-AA (A, B) or PGE2 (C) was performed as described in the Methods section. Presented values are mean ± SD of TNF-induced release compared to untreated control samples in one representative of at least three independent experiments performed in duplicates (PGE2) or triplicates ([3H]-AA). AA release levels for untreated samples are indicated by a dash line in (B). Synoviocytes were stimulated with TNF (10 ng/mL) for 6 hours prior to lysis and detection of in vitro cPLA2α enzyme activity as described in the Methods section (D). Presented values are mean ± SD of in vitro cPLA2α activity (%) in one representative of at least three independent experiments performed in duplicates. Statistical significance is indicated as follows: A: #p≤ 0.01 when compared to untreated control values, *p ≤ 0.01 (AVX002) and ¤p ≤ 0.01 (ATK) when compared to TNF-stimulated control values. B: #p ≤ 0.05 when compared to untreated control values, *p ≤ 0.05 when compared to TNF-stimulated control values. C: #p ≤ 0.01, ##p ≤ 0.02 when compared to untreated samples, and *p ≤ 0.01 when compared to TNF-stimulated and control values. D: #p ≤ 0.03 when compared to untreated cell lysates, *p ≤ 0.02 when compared to untreated or TNF-stimulated cell lysates without inhibitor.
Figure 3.
Proposed TNF-induced and cPLA2α dependent signaling in synoviocytes.
TNF is a powerful inducer of inflammation and joint destruction in RA. In the SW982 synoviocyte model system, TNF induces activation of the cPLA2α enzyme to release AA from membranes, presumably through binding to its receptors TNF receptor 1 and TNF receptor 2 (TNFR1/TNFR2). AA released by cPLA2α is metabolized by COX2 to PGE2, a commonly recognized inducer of inflammation. The TNF-induced AA cascade can be self-reinforced by transcriptional regulation of COX2 and PLA2G4A genes. MMP3 and IL8 are central effectors in RA through cartilage destruction (MMP3), angiogenesis and attraction of immune cells (IL8). cPLA2α regulates TNF-induced expression of MMP3 and IL8 on transcriptional and protein levels. Consequently, cPLA2α functions to coordinate joint destructive and inflammatory processes in synoviocytes.