PUMA regulation and proapoptotic effects in fibroblast‐like synoviocytes

Abstract

Objective. Although p53 is overexpressed in rheumatoid arthritis (RA) synovial tissue (ST), few synoviocytes undergo apoptosis. This could be partly due to low expression of proapoptotic genes. Deficient p53 upregulated modulator of apoptosis (PUMA), which is a major effector of p53-mediated cell death, could contribute to this phenomenon. To evaluate a method to induce apoptosis, the expression and function of PUMA was investigated in ST and cultured fibroblast-like synoviocytes (FLS).Methods. PUMA expression in ST was measured by immunohistochemistry, Western blot analysis, and quantitative polymerase chain reaction analysis. Ad-p53 and plasmids encoding hemagglutinin-tagged, fulllength PUMA expression vector (HA-PUMA), PUMA lacking the Bcl-2 homology 3 domain, or pCEP4 were used to transfect FLS. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation, and caspase 3 activation.Results. PUMA protein was detected in RA ST, although most of the immunoreactive protein was localized to sublining cells rather than the intimal lining synoviocytes. Western blot analysis showed no difference between RA ST and osteoarthritis (OA) ST. PUMA messenger RNA was detected in RA and OA ST, although the amounts were markedly lower than in the spleen and FLS. To determine if PUMA was inducible, FLS were transduced with Ad-p53. Even though p53 protein was produced and p21 expression was increased, PUMA expression was not enhanced. Consistent with this observation, Ad-p53 did not induce apoptosis in FLS. However, HA-PUMA transfection into FLS resulted in rapid apoptosis with the activation of caspase 3.Conclusion. PUMA can induce apoptosis by FLS and represents a potential target in RA.

Regulation of apoptosis in fibroblast‐like synoviocytes by the hypoxia‐induced Bcl‐2 family member Bcl‐2/adenovirus E1B 19‐kd protein–interacting protein 3

“…It has been shown that the levels of p45 up-regulated modulator of apoptosis (PUMA; or, Bcl-2-binding component 3 ), which is a downstream effector of p53-mediated apoptosis, are low in FLS, and this has been proposed to contribute to the low apoptosis in the synovial intimal lining (36). As with BNIP-3 (29), PUMA is also a BH3 proapoptotic Bcl-2 family member that interacts with the antiapoptotic Bcl-2/Bcl-x L , this in turn, promoting mitochondrial dysfunction and cell death through Bax/Bak.…”

Aryl Hydrocarbon Receptor Antagonism Attenuates Growth Factor Expression, Proliferation, and Migration in Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis

“…Repression of AHR expression was achieved using small interfering RNA (siRNA) (Dharmacon siRNA, control oligo D-001810-0X, AHR oligo J-004990-07; Pittsburgh, PA). Cells were electroporated with siRNA using the U-020 program of Amaxa nucleofection system (Lonza, Walkersville, MD) as previously described (Cha et al, 2006;Dinatale and Perdew, 2010). Transfected cells were seeded into six-well plates at 2 Â 10 6 cells/well synoviocyte growth media.…”

Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide

“…Exogenous p53 overexpression induced p21, but not Puma in FLS and this was associated with resistance of FLS to apoptosis. However, transfection of plasmid encoding Puma expression vector lead to rapid and profound apoptosis with caspase-3 activation in RA FLS (Cha et al, 2006). Tissue-and cell type-specific selectivity of p53 in transactivation of downstream genes has been reported (Fei et al, 2002).…”