Levels of PPARγ protein are elevated in the APC^Min mouse. (A) Immunohistochemical analysis depicting levels of PPARγ protein in sections from the distal colon of wild-type C57BL/6(Left) and C57BL/6J-APC^Min (Right) mice by using a PPARγ-specific antibody. (B) Western blot analysis of protein extracts of colonic epithelial cells from wild-type and APC^Min mice, sequentially probed for PPARγ and actin. (Right) A graphical representation of comparative PPARγ levels in relation to actin levels.

β-Catenin activates a PPRE-driven reporter gene. PPARγ activity was monitored in HEK293 cells, transiently transfected with a PPRE-containing luciferase reporter, in combination with either PPARγ expression vector or vectors expressing a variety of β-catenin constructs. Cells were harvested 48 h posttransfection and assayed for luciferase activity. Each transient transfection was performed in doublets of three independent experiments. The result depicts one such experiment. Luciferase readings were correlated to total protein content. The values show fold luciferase activation of each transfection relative to the activity of the PPRE reporter gene alone. (A) Cells were cotransfected with the PPRE, in the presence or absence of the PPARγ, β-catenin, a mutated β-catenin, or the β-catenin DP expression vectors, as indicated. Selected transfections were also treated with the PPARγ synthetic ligand rosiglitazone. (B) PPRE cotransfected with β-catenin DP and/or a dominant negative mutant of PPARγ, PPARγ-AF2.

β-Catenin-driven transactivation of other nuclear receptor response elements. Transient transfections were performed in HEK293 cells with luciferase reporter plasmids specific for a number of different nuclear receptors. Transfections and subsequent reporter gene assays were performed as described in the legend for Fig. 4. The amounts of expression vectors transfected are indicated. For each response element, the luciferase values reflect the fold luciferase activation relative to the activity of reporter gene alone. (A) RXR-dependent reporter (RXR-RE) was transfected in combination with either the β-catenin or the RXRα expression vectors in the presence or absence of 1 μM LG100268, a synthetic ligand for RXR. (B) ER-dependent reporter (ER-RE) was cotransfected with either the ERα or β-catenin expression plasmids. (C) DR-dependent reporter (DR-RE) was transfected together with either a plasmid expressing β-catenin or one expressing a constitutively active dioxin receptor (ca-DR).

Physical interaction of PPARγ with β-catenin and Tcf-4. (A) Western blot analysis of β-catenin immunoprecipitations (IP) from the SW480 cell line. Whole-cell extracts were obtained from nontransfected cells (lanes 1 and 2), mock-transfected cells (lane 3), or cells transfected with PPARγ, FLAG-PPARγ, and/or β-catenin-DP as indicated (lanes 4-9). Lane 1 shows β-catenin coimmunoprecipitated by using an anti-Tcf-4 antibody (denoted T), whereas lane 2 shows β-catenin coimmunoprecipitated using an anti-PPARγ antibody. Lane 3 shows the endogenous β-catenin levels in SW480 cell lysates, whereas lane 4 represents a control IP with no antibody, only beads. In transfected cells, protein complexes containing β-catenin were immunoprecipitated (IP) with either a PPARγ-specific antibody (P, lanes 5, 6, and 8) or an anti-FLAG antibody (F, lanes 7 and 9). (B) Coimmunoprecipitations of endogenous Tcf-4 from the SW480 cell line. Lane 1 shows endogenous Tcf-4 levels in 25 μg of whole-cell extracts from SW480 cells, whereas lane 2 represents a mock-IP with no antibody, but with Sepharose A beads. Tcf-4 precipitated (IP) with a PPARγ-specific antibody (P) is shown in lane 3. Conditions for Tcf-4 IP in lane 4 are essentially as for lane 3, but with the addition of 10 μg/ml ethidium bromide during IP. (C) EMSA with nuclear extracts from the SW480 cells stimulated for 6 h with 20 mM LiCl. The extracts were incubated with different antibodies, as indicated, before addition of a radiolabeled PPRE probe. The major PPARγ-containing complex is indicated by the arrow.

PPARγ protein levels are increased in β-catenin-transfected cells. (A) Western blot analysis of nuclear extracts from SW480 cells (lane 1) and cells transfected with constitutively active β-catenin (DP) (lane 2), sequentially probed with anti-PPARγ, anti-β-catenin, and anti-actin antibodies. (B and C) PPARγ immunostaining of SW480 cells in the presence (C) or absence (B) of transfected β-catenin-DP. The cells were stained with a primary monoclonal antibody directed against PPARγ and a secondary anti-mouse antibody conjugated to the fluorescent Cy3. (D) β-catenin-augmented PPARγ protein levels led to an increase in PPARγ-DNA complexes. Shown are EMSA with nuclear extracts from the SW480 cells transfected with 2 μg of either pcDNA3 or the β-catenin-DP expression vector. The extracts were incubated with the indicated antibodies before addition of a radiolabeled PPRE probe. The major PPARγ-containing complex is indicated by the arrow. (E) RNase protection assay showing the mRNA levels of PPARγ in SW480, after transfection with increasing amounts of β-catenin-DP (lane 1, 2 μg; lane 2, 1 μg; lane 3, nontransfected control SW480 cells). The transcript for human γ-actin was used as an internal control. (F) Western blot analysis of nuclear extracts from HEK293 cotransfected with PPARγ and either mock DNA (lane 1) or β-catenin-DP (lane 2). The membrane was sequentially probed with anti-PPARγ, anti-β-catenin, and anti-actin antibodies. (G and H) PPARγ immunostaining of HEK293 cells in the presence (H) or absence (G) of transfected β-catenin-DP.

LiCl treatment modulates protein levels as well as PPARγ transcriptional activity. LiCl, a Wnt-signaling agonist is able to transactivate PPRE in the HEK293 cells. (A) Stimulation of HEK293 cells with either rosiglitazone in the presence of a transfected PPARγ expression vector or with increasing amounts of LiCl in cells transiently transfected with PPRE. Rosiglitazone stimulation was performed for 24 h and LiCl for 6 h at indicated concentrations. (B) Real-time PCR analysis of PPARγ target gene expression in SW480 cells treated for 24 h with DMSO, 10 μM rosiglitazone, 20 mM NaCl, and 20 mM LiCl, or left untreated. RT-PCR was performed, as described in Materials and Methods,to determine relative mRNA levels of ADFP, FABP2, and Keratin 20 in comparison with β-actin expression. Three independent experiments were performed, and data from one representative analysis are depicted here. LiCl treatment of SW480 cells leads to an increase in PPARγ protein levels. Depicted is immunocytochemistry, showing PPARγ protein levels in SW480 cells treated with 20 mM LiCl for 24 h (D) or in untreated cells (C). (E) Western blot analysis of nuclear extracts from SW480 cells, comparing levels of PPARγ in cells untreated and treated with 20 mM of LiCl for 6 h. The blot was subsequently reprobed for Ku70 as a loading control.