Abstract

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.

Highlights

  • Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and invasion into cartilage and bone

  • Wild-type and p53-/- murine fibroblast-like synoviocytes (FLS) were examined to evaluate whether p53 is required. p53deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were determined by Western blot analysis

  • In addition to RA FLS, we examined FLS derived from osteoarthritis FLS in most experiments

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Summary

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and invasion into cartilage and bone. Several genes have been evaluated as potential gene therapy targets, including Fas [2], TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) [3], p53 [4], and PUMA (p53 up-regulated modulator of apoptosis) [5]. The latter is an especially interesting target because it rapidly induces apoptosis in cultured synoviocytes [5]. Our previous studies showed that p53 is only a weak inducer of PUMA in FLS, which could account for the variable pro-apoptotic effect of p53 in this cell lineage, with no significant apoptosis induced by p53 overexpression in some studies [9,10]

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