pubmed.ncbi.nlm.nih.gov

P53-derived hybrid peptides induce apoptosis of synovial fibroblasts in the rheumatoid joint - PubMed

  • ️Sun Jan 01 2017

P53-derived hybrid peptides induce apoptosis of synovial fibroblasts in the rheumatoid joint

Shih-Yao Chen et al. Oncotarget. 2017.

Abstract

Loss of p53-mediated suppression by its dominant-negative counterpart is commonly observed in human cancers, and activating p73 is a therapeutic strategy in p53-mutated oncological patients. In synovial fibroblasts (SFs) from rheumatoid arthritis (RA), mutant p53 can lead to the transformation-like features with resistance to the apoptosis induction. We examined whether intra-articular (i.a.) administration of p53-derived hybrid peptides to activate p73 can induce apoptosis of SFs by using adenoviral vectors encoding 37 amino acid (Ad37AA), a p53-derived hybrid peptide capable of activating p73, to transduce SFs in vitro and inject collagen-induced arthritis (CIA) joints in vivo. Increased p73 expression was found in synovial lining layers and SFs of RA patients and CIA rats. Higher expression of p53 up-regulated modulator of apoptosis (PUMA) and Bax with enhanced apoptosis were found in Ad37AA-transduced SFs, and silencing p73 abrogated the up-regulation of PUMA and Bax. Articular indexes and histologic scores were reduced in Ad37AA-injected joints with decreased SF densities, increased apoptotic cell numbers, and higher PUMA expression levels. We demonstrate that i.a. administration of p53-derived hybrid peptides can activate p73 to induce apoptosis of SFs and ameliorate the rheumatoid joint, implicating an enhancement of the p73-dependent apoptotic mechanism as a pharmacological strategy in the RA therapy.

Keywords: apoptosis; p53-derived hybrid peptide; p73; rheumatoid arthritis; synovial fibroblast.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST All authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1. Increased p73 levels with co-localized iASPP expression in the rheumatoid joint

(A) Immunofluorescence staining of p73 (green, FITC), iASPP (red, Texas red) and nucleus (blue, DAPI) in RA and OA synovial tissues (×400 magnification). The yellow-boxed areas in upper panels are magnified and shown in lower panels (×1,600 magnification). The blue-rectangled areas in the right upper corner of upper panels are isotype control IgG staining of p73 and iASPP. Immunofluorescence staining of p73 (green, Alexa Fluor 488), iASPP (red, Alexa Fluor 594) and nucleus (blue, DAPI) in RASF (×1,000 magnification, scale bar = 20 μm). (B) p73- or p73/iASPP double positive frequencies of DAPI-positive cells in synovial tissues. Each value represents the mean and SEM (n = 7). (C) Immunohistochemical detection of p73 and iASPP in synovial tissues of normal and CIA rats. Scale bars represent 500 and 50 μm in ×40 and ×400 magnifications, respectively. The boxed areas in left panels are magnified and shown in right panels. (D) Quantitative real-time PCR analysis of p73 levels from synovial tissues of normal and CIA rats. Each value represents the mean and SEM (n = 5). (E) Representative immunoblot photographs of p73 and cadherin-11 (Cad-11) expression in SFs from normal and CIA rats. The results in A to E are representative of at least two independent experiments with similar findings.

Figure 2
Figure 2. Induction of apoptosis in SFs by the activation of p73

(A) Immunoblot and immunofluorescence analyses reveal a high efficacy of Ad37AA transfection in CIASFs with expression of GFP protein and green fluorescence, respectively. (B) Cell viability is expressed as the percentages of surviving cells relative to those in mock cells. Each value shown in B represents the mean and SEM (n = 6). (C) TUNEL-positive cells were observed after the transfection of Ad37AA at a MOI of 10 for 7 days. DNase-treated cells were used as a positive control. Arrows indicate positive cells. Percentages were determined by averaging TUNEL-positive cells in 3 randomly selected fields at the ×100 magnification (n = 3). (D) Quantitative real-time PCR analysis demonstrates PUMA expression levels in Ad37AA-transduced CIASFs for 4 and 6 days. Each value shown represents the mean and SEM (n = 8). (E) Quantitative real-time PCR analysis reveals p73, PUMA and Bax expression levels in p73-silenced and control CIASF transfectants transduced with Ad37AA or AdLacZ for 6 days (n = 5). (F) Representative immunoblot photographs of iASPP-associated p73 expression in Ad37AA-treated CIASFs. Input represents the loading control. AdLacZ-transfected CIASFs were served as a control group in A to D. (G) A schematic diagram shows the dissociation of the binding with iASPP by overexpressing 37AA to activate p73 and induce apoptosis in SFs. The results in A to F are representative of at least two independent experiments with similar findings.

Figure 3
Figure 3. Amelioration of experimental arthritis by i.a. activation of p73

(A) Left, Reduced articular indexes in Ad37AA-injected CIA joints as compared with AdGFP (P < 0.0001) or PBS (P = 0.0047) control joints. Right, Representative GFP immunohistochemical photographs of CIA joints. (B) Representative H&E photographs and histologic scores in Ad37AA-injected CIA and control joints. (C) Left, Representative cadherin-11 (Cad-11) immunohistochemical photographs of CIA joints. Right, Immunoblot analysis of Cad-11 in CIA synovial extracts with quantification of the band intensity by comparing with β-actin. (D) Representative in situ apoptosis photographs and calculated TUNEL-positive cell numbers in CIA joints. (E) Immunohistochemical intensity with representative photographs of PUMA expression in CIA joints. CIA joints receiving different treatments were obtained on day 18 upon sacrifice. Scale bars represent 500, 200, and 50 μm in ×40, ×100 and ×400 magnifications, respectively. Each value in the graphs represents are mean and SEM (n = 8 in A and B, n = 3 in C, n = 5 in D and E). The results in A to E are representative of two independent experiments with similar findings.

Similar articles

Cited by

References

    1. Pfister NT, Prives C. Transcriptional regulation by wild-type and cancer-related mutant forms of p53. In: Lozano G, Levine AJ, editors. The p53 protein: from cell regulation to cancer. Cold Spring Harbor Laboratory Press; NY: 2016. pp. 69–94.
    1. Hientz K, Mohr A, Bhakta-Guha D, Efferth T. The role of p53 in cancer drug resistance and targeted chemotherapy. Oncotarget. 2017;8:8921–46. https://doi.org/10.18632/oncotarget.13475 - DOI - PMC - PubMed
    1. Maas AM, Bretz AC, Mack E, Stiewe T. Targeting p73 in cancer. Cancer Lett. 2013;332:229–36. - PubMed
    1. Billant O, Léon A, Le Guellec S, Friocourt G, Blondel M, Voisset C. The dominant-negative interplay between p53, p63 and p73: a family affair. Oncotarget. 2016;7:69549–64. https://doi.org/10.18632/oncotarget.11774 - DOI - PMC - PubMed
    1. Firestein GS. Biomedicine. Every joint has a silver lining. Science. 2007;315:952–3. - PubMed