Peroxisome proliferator-activated receptor-γ1 is dephosphorylated and degraded during BAY 11-7085-induced synovial fibroblast apoptosis

Abstract

Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-γ has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-γ agonist 15d-PGJ2; the expression of PPAR-γ in these cells was studied. Both PPAR-γ1 and PPAR-γ2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-γ1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-γ1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-γ1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-γ1 protein degradation. PPAR-γ1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-γ1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-γ1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-γ dephosphorylation and degradation. Furthermore, PPAR-γ antagonist BADGE induced PPAR-γ1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-γ1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-γ1 dephosphorylation but before PPAR-γ1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.