Fig. 2.

NF-κB activation is necessary and sufficient to inhibit the adipogenic differentation of arthritic rFLS. (A) NF-κB is required for the antidifferentiating activity of IL-1 in the adipogenic differentiation of arthritic rFLS. Primary FLS from rats with PG-PS-induced arthritis were infected with a control, “empty” adenovirus (Control) (a–c) or with adenovirus expressing superrepressor IκBα (AdsrIκBα) (d). Cells were incubated in DMEM (a) or in an adipogenesis-inducing medium (MDI) (b), or in the MDI medium in the presence of IL-1β (100 units/ml) (c and d). After 21 days, differentiated cells were visualized by staining the accumulated lipids with an Oil red stain. Representative data of three experiments are shown. (Original magnification: ×100.) (B–D) Constitutive activation of NF-κB by retrovirus-mediated gene transfer of a dominant-positive IKKβ and RelA. Primary rFLS were transduced with a control RV Babe (control Babe) or with vectors that expressed cDNA of FLAG-tagged constitutively active IKKβ mutant [IKK(EE)], or FLAG-tagged RelA cDNA. After puromycin selection, NF-κB activation was assessed. (B) The expression of exogenous IKK(EE) and RelA proteins was assessed by immunoblotting with anti-FLAG Ab. (C) The expression of IKK(EE) results in accumulation of a phosphorylated form of IκBα. The control (Babe) and IKK(EE) cells were detected by using antiphospho IκBα Ab. Where indicated, cells were pretreated with a proteasomal inhibitor, MG132, to allow for accumulation of the phosphorylated IκBα. (D) Ectopic expression of IKK(EE) and RelA induces constitutive activation of NF-κB-driven gene expression. Transduced rFLS were transiently cotransfected with an NF-κB-inducible reporter vector expressing the firefly luciferase and a constitutively active reporter expressing the Renilla luciferase. Normalized values of NF-κB activation from duplicated samples are shown. Representative data of two experiments are shown. (E and F) Constitutive activation of NF-κB inhibits the adipogenic differentiation of arthritic rFLS. (E) RV-transduced cells (as in B) were incubated in a control medium (DMEM) or in an adipogenesis-inducing medium (MDI). After 21 days, the adipogenic differentiation was assessed by staining accumulated lipids with Nile red. The staining was visualized by fluorescent microscopy (Left). (Original magnification: ×100.) Cells were counterstained with the nuclear stain Hoechst 33342 (Right). (F) The expression of an adipogenic differentiation marker (PPARγ2) was assessed in cells shown in B by RT-PCR.