Session Title: Rheumatoid Arthritis - Animal Models I
Session Type: Abstract Submissions (ACR)
Our previous studies have demonstrated p53 mutations competent for the inactivation of wild type p53 in synovial fibroblasts (SF) from either rheumatoid arthritis patients or collagen-induced arthritis (CIA) rats; however, the pathogenic role of p73 (a transcription factor belonging to the p53 family) remains to be explored in rheumatoid joints. The 37 amino acid (37AA) peptide, a hybrid small peptide corresponding to p53 residues, inhibits the binding of p73 with inhibitory apoptosis stimulating protein of p53 (iASPP), thus activating p73 and further inducing apoptosis in p53-null cells.
Male Sprague-Dawley rats were immunized with bovine type II collagen with Freund’s adjuvant on days 0 and 7 to induce arthritis, and SF were isolated from CIA rats. CIASF were transfected with adenoviral vectors encoding 37AA gene (Ad37AA), and then subjected to TUNEL, colorimetric WST-8 and real-time RT-PCR analyses to examine apoptotic status, cell viability and expression levels of p53 upregulated modulator of apoptosis (PUMA, a downstream target gene of p73), respectively. The association status of iASPP with p73 in Ad37AA-transfected CIASF was identified by immunoprecipitation with anti-iASPP, followed by immunoblot with anti-p73 antibodies. Serial expression levels of iASPP on synovium from rats were investigated by immunohistochemical (IHC) staining before and after the induction of arthritis. The therapeutic effect of Ad37AA on CIA rats were examined by intra-articular (i.a.) injection of 5×107 plaque-forming units into ankle joints on day 12, and evaluated by articular index and histological analyses including H&E staining and a specific surface marker of SF. Furthermore, expression levels of PUMA and IL-6 on CIA synovium were examined by immunoblot and enzyme-linked immunosorbent assay, respectively.
Increased apoptosis status with reduced cell viability was found in Ad37AA-transfected CIASF as compared with control vector or mock-treated counterpart. There were significantly increased expression levels of PUMA on days 4 and 6 after the Ad37AA transfection. Decreased levels of associated p73 with iASPP were identified in Ad37AA-trasfected CIASF as compared with control vector-treated cells. Serial synovium sections revealed higher expression of iASPP in synovial lining layer after the induction of arthritis. Articular indexes of Ad37AA-injected joints were significantly smaller as compared with control vector or phosphate-buffered saline-treated ankles. There were significantly lower histological scores with milder synovial hyperplasia and bone erosion in Ad37AA-injected joints as compared with control groups. Notably, Ad37AA-treated synovium revealed up-regulation of PUMA, lower numbers of SF and decreased concentrations of IL-6.
These data demonstrate amelioration of CIA in rats by i.a. gene transfer of a small peptide corresponding to p53 residues via induction of apoptosis in CIASF through p73 activation. Such findings implicate a pathogenic role of p73 and the enhancement of p73-dependent pathway as a potential therapeutic strategy in rheumatoid joints.
C. R. Wang,
S. Y. Chen,
A. L. Shiau,
Y. T. Li,
C. T. Weng,
I. M. Jou,
M. F. Liu,
C. L. Wu,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/amelioration-of-collagen-induced-arthritis-by-modulation-of-inhibitory-apoptosis-stimulating-protein-of-p53-to-activate-transcription-factor-p73/