Open AccessPUMA regulation and proapoptotic effects in fibroblast‐like synoviocytes
Author(s) -
Cha HoonSuk,
Rosengren Sanna,
Boyle David L.,
Firestein Gary S.
Publication year - 2006
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.21631
Subject(s) - puma , apoptosis , transfection , microbiology and biotechnology , western blot , dna fragmentation , biology , chemistry , programmed cell death , cell culture , gene , biochemistry , genetics
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 up‐regulated 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, full‐length 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.