Peroxisome proliferator-activated receptor gamma overexpression and knockdown: impact on human B cell lymphoma proliferation and survival

Abstract

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a multifunctional transcription factor that regulates adipogenesis, immunity and inflammation. Our laboratory previously demonstrated that PPARgamma ligands induce apoptosis in malignant B cells. While malignant B lineage cells such as B cell lymphoma express PPARgamma, its physiological function remains unknown. Herein, we demonstrate that silencing PPARgamma expression by RNAi in human Burkitt's type B lymphoma cells increased basal and mitogen-induced proliferation and survival, which was accompanied by enhanced NF-kappaB activity and increased expression of Bcl-2. These cells also had increased survival upon exposure to PPARgamma ligands and exhibited a less differentiated phenotype. In contrast, PPARgamma overexpression in B lymphoma cells inhibited cell growth and decreased their proliferative response to mitogenic stimuli. These cells were also more sensitive to PPARgamma-ligand induced growth arrest and displayed a more differentiated phenotype. Collectively, these findings support a regulatory role for PPARgamma in the proliferation, survival and differentiation of malignant B cells. These findings further suggest the potential of PPARgamma as a therapeutic target for B cell malignancy.

Fig. 1

Construction of a lentiviral vector for delivering human PPARγ siRNA. a Schematic diagram of the siRNA-expressing lentiviral vector. The short hairpin form of siRNA is expressed under the control of a human U6-RNA Pol III promoter (Pol III). The vector contains a GFP marker under the UbiC promoter for tracking transduced cells b HEK 293 cells were mock transfected (lane 1) or were co-transfected with a FLAG-tagged-PPARγ-WT vector (Flag-PPARγ) and with either an empty DNA vector, (pcDNA3.1, lane 2), increasing DNA concentrations of empty parental FG12 vector (lanes 3, 4) or increasing DNA concentrations of the FG12 vector expressing siRNA against PPARγ (lane 5, 6). Numbers represent microgram amounts of plasmid DNA. All transfections included equivalent concentrations of DNA, which were normalized with the empty DNA vector, pcDNA3.1. c Ramos cells were infected at an MOI of 5 and the GFP-positive cells were sorted by FACS. After 5 days, cells were analyzed by flow cytometry to determine the purity. LV-control and LV-PPARγ-siRNA infected cells showed >95% GFP-positive cells. d Reduction of PPARγ protein levels in Ramos cells transduced with LV-PPARγ-siRNA (siRNA). Total actin was used as a loading control

Fig. 2

Reduction of PPARγ expression in Ramos B lymphoma cells results in enhanced proliferation and reduced sensitivity to PPARγ ligand induced cell death. a LV-control and LV-PPARγ-siRNA transduced Ramos B lymphoma cells were untreated or treated with 1:1,000 Pansorbin (fixed S. aureus), human CD40L, 10 μg/ml anti-IgM, a combination of CD40L + anti-IgM, and CD40L + Pansorbin for 24 h. Proliferation was measured by [³H] thymidine incorporation. b LV-Control and LV-PPARγ-siRNA infected Ramos cells were exposed to increasing concentrations of the PPARγ ligand CDDO. Viability was measured by MTT assay. PPARγ-knockdown Ramos cells have increased survival in comparison to control cells when exposed to the PPARγ agonist CDDO. (*P < 0.05), ns not significant

Fig. 3

PPARγ-knockdown Ramos human B lymphoma cells have a less differentiated phenotype. a Ramos B lymphoma cells stably transduced with LV-control or LV-PPARγ-siRNA were analyzed for the expression of CD20, CD19 and CD38. Cells that express PPARγ-siRNA showed increased levels of CD20 and CD19 and decreased levels of CD38. MFI’s are shown in the histogram. This experiment is representative of three separate experiments. b LV-control and LV-PPARγ-siRNA infected cells were lysed and expression of PAX-5 and BLIMP-1 were analyzed by western blot as indicated. Total actin was used to normalize protein loading

Fig. 4

PPARγ knockdown Ramos B lymphoma cells have enhanced NF-κB activity and express higher levels of the NF-κB-dependent pro-survival gene Bcl-2. a Nuclear and cytoplasmic extracts from uninfected, LV-control and LV-PPARγ-siRNA transduced Ramos cells were collected and NF-κB p65 levels analyzed by western blot. Total actin was used as a loading control. Graph shows representative densitometry. b Nuclear extracts were incubated with a radiolabeled DNA binding sequence for NF-κB and a gel shift assay was performed. c Bcl-2 protein expression was analyzed by western blot in whole cell lysates from uninfected, LV-control and LV-PPARγ-siRNA transduced Ramos B cells. Graph shows representative densitometry

Fig. 5

Design of a lentiviral vector for PPARγ overexpression in human B cell lymphoma. a Schematic diagram of the PPARγ1-expressing lentiviral vector. The Flag-PPARγ1 cDNA is expressed under the control of a CMV promoter. The vector also contains an elongated factor 1α (EF-1α) promoter driving the GFP marker gene for tracking transduced cells. 5′LTR, HIV-1 5′LTR; Δ3′LTR, HIV-1 self-inactivating 3′LTR; WPRE woodchuck hepatitis B virus RNA regulatory element. b Ramos B lymphoma cells were transduced with LV-empty and LV-PPARγ (MOI = 5). At 48 h post-transduction, cells were analyzed for GFP expression using flow cytometry. GFP-positive cells were then sorted on the basis of GFP expression. GFP expression was monitored over time (one day and one week after sorting) to determine purity and the stability of gene expression c Exogenous PPARγ1 expression was evaluated by western blotting using an anti-FLAG antibody. After sorting, the FLAG-PPARγ protein was detected in the cells infected with LV-PPARγ, but not in the LV-empty infected cells. β-Tubulin levels were assayed to normalize protein loading. d Nuclear extracts from Ramos cells stably transduced with either LV-empty or LV-PPARγ were collected and transactivation of PPARγ was analyzed by ELISA using TransAm^® technology (*P < 0.05)

Fig. 6

Ramos B lymphoma cells transduced with LV-PPARγ have decreased basal and stimulatory proliferative responses. a Ramos B lymphoma cells stably transfected with LV-Empty and LV-PPARγ were left untreated or were treated with human CD40L, 10 μg/ml anti-IgM, a combination of CD40L plus anti-IgM, 1:1,000 Pansorbin (fixed S. aureus), and CD40L plus Pansorbin for 24 h. Proliferation was measure by [³H] thymidine incorporation. (*P < 0.05), (**P < 0.001) b Cell cycle analysis: Ramos cells transduced with LV-empty and LV-PPARγ were incubated for 30 min with BrdU and labelled with anti-BrdU and 7-AAD (gated on GFP-positive cells). Cells that overexpressed PPARγ showed a reduction in the percentage of cells entering the S-phase of the cycle and a G2/M cell cycle arrest. This profile is representative of three separate experiments. c Raji B lymphoma cells were infected with either LV-empty or LV-PPARγ at an MOI of 5 and sorted by GFP expression. Cells were left untreated or were treated with 5 μM ciglitazone (+ Cig) for 48 h. Cells were pulsed with BrdU for 30 min and intracellularly stained with an anti-BrdU antibody. Cells that were infected with LV-PPARγ had a slight reduction in BrdU incorpotation in comparison to LV-empty infected cells. When cells were treated with ciglitazone, a dramatic reduction in BrdU incorporation was observed in the cells that overexpress PPARγ (LV-PPARγ), but not in the LV-empty infected cells. These results are representative of three separate experiments

Fig. 7

Ramos B lymphoma cells overexpressing PPARγ showed a more differentiated phenotype. a Ramos B lymphoma cells stably transduced with LV-Empty or LV-PPARγ were analyzed for the expression of CD20, CD19, CD38 and CD40. Cells that overexpress PPARγ showed a slight decrease on CD19 expression and a dramatic decrease on CD20 and CD40, but no changes in CD38 expression. b LV-empty and LV-PPARγ infected cells were lysed and expression of PAX-5 and BLIMP-1 were analyzed by western blot as indicated. Total actin was used to normalize protein loading