Prothymosin α Lacking the Nuclear Localization Signal as an Effective Gene Therapeutic Strategy in Collagen-Induced Arthritis

Abstract

Prothymosin α (ProT) is regulated by c-Myc, an oncoprotein overexpressed in synovium of rheumatoid arthritis, and is associated with cell proliferation. However, ProT also exerts immunomodulatory activities. The growth-promoting activity of ProT can be abolished by deleting its nuclear localization signal (NLS). In this study, we showed that AdProTΔNLS, an adenoviral vector encoding ProT lacking the NLS, did not enhance the proliferation of synovial fibroblasts. AdProTΔNLS treatment abolished the up-regulation of the MIP-1α promoter activity induced by TNF-α in synovial fibroblasts. AdProTΔNLS suppressed macrophage chemotaxis and reduced macrophage infiltration into the ankle joints in rats with collagen-induced arthritis (CIA). Neutralization test confirmed the involvement of MIP-1α in macrophage chemotaxis. Administration of AdProTΔNLS reduced the severity of CIA in the clinical, radiographic, and histological aspects. The levels of TNF-α (mean ± SEM, 1261.9 ± 107.9 vs 2880.1 ± 561.4 pg/mg total protein; p < 0.05), IL-1β (56.8 ± 8.0 vs 109.2 ± 4.9 pg/mg total protein; p < 0.01), and MIP-1α (41.7 ± 3.6 vs 55.2 ± 1.1 pg/mg total protein; p < 0.05) in the ankle joints were lower in the AdProTΔNLS-treated rats with CIA than those in their control counterparts. In the AdProTΔNLS-treated ankle joints, matrix metalloproteinase-9 expression was decreased by 40% and infiltrating macrophages reduced by 50%. Our results demonstrate that intra-articular delivery of AdProTΔNLS significantly ameliorated the clinical course of CIA in rats. This study is the first to suggest that ProT lacking the NLS may have therapeutic potential for the management of rheumatoid arthritis.

Intra-articular lentivirus-mediated delivery of galectin-3 shRNA and galectin-1 gene ameliorates collagen-induced arthritis

“…Sections of paraffin-embedded joint specimens were stained with hematoxylin and eosin and immunostained with rabbit anti-von Willebrand factor (Dako, Carpinteria, CA, USA) or anti-CD3 (BD Biosciences, San Diego, CA, USA) antibodies as described previously. [6][7][8][9] Assay of antigen-mediated activation-induced T-cell death in the lymph node cells transduced with lentiviral vectors Rats with CIA were sacrificed on day 18. Cells obtained from the inguinal lymph nodes were infected with Lt.Gal1, Lt.GFP or Lt.shGal3 at an multiplicity of infection of 10 in the presence of polybrene (8 mg ml À1 ) and cultured for 72 h. Cells were then treated with denatured type II collagen (100 mg ml À1 ) for 24 h. They were stained with propidium iodide and analyzed for the proportions of dead cells by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA, USA).…”

“…injection into ankle joints of rats with CIA. [6][7][8][9] Such proof of concept in animal models of RA provides important validation of the pre-clinical miniature, and has led to the elucidation of putative roles for certain molecules in the pathogenesis of inflamed joints.…”

Suppression of collagen-induced arthritis by intra-articular lentiviral vector-mediated delivery of Toll-like receptor 7 short hairpin RNA gene

“…After removal of sodium dodecyl sulfate with 1% Triton X-100 for 1 h, the gels were incubated in 40 mM Tris-HCl (pH 7.5) containing 10 mM CaCl 2 , then stained with 7.5% acetic acid/10% propanol-2 containing 0.5% Coomassive Brilliant Blue G250, and further destained in water as described previously. 39 The TLR7 expression in the synovial tissues of ankles of CIA rats receiving Lt.shTLR7 or Lt.scramble treatment for 48 h were examined by the reverse transcription-PCR. Total RNA was isolated by the TRIzol reagent (Invitrogen).…”

“…Freshly prepared synovial tissues from ankle joints of CIA rats were cultured as described previously. 39 After the adherence for 48 h, non-adherent cells were removed and adherent cells were cultured continuously until confluence. The CIASFs became a homogeneous population after 44 passages, and the immunohistochemical staining of TLR7 was performed in these cultured SFs.…”

Progress and Prospects: genetic treatments for disorders of bones and joints

“…Those encoding IL-13, 15 modified prothymosin 16 and an IL-18-binding protein:IL-4 fusion protein 17 were effective, whereas IGF-1 18 was not. Unlike the other immunomodulatory products tested, IGF-1 was evaluated on the basis of its potential to protect and restore articular cartilage.…”