Hic-5 binds and coactivates PPARγ in a ligand-depended manner (A) Bosc cells were transiently cotransfected with 50 ng of expression vector for PPARγ and 50 ng of a luciferase reporter plasmid with three PPARγ response elements (DR1-sites), in the presence of 250 ng Hic-5 or 100 ng PGC1α expression vectors. Empty vector was used as a control. The following day cells were treated with 1 μM pioglitazone (Pio). Cell lysates were then analyzed for relative transcriptional activity, and data were normalized to β-gal activity, derived from a cotransfected β-gal expressing vector. Values were expressed relative to activation of empty vector and presented as mean ± SE of three different experiments. (B) Coimmunoprecipitation assay. Bosc cells were transfected with either control vector or vector expressing Flag-PPARγ. Cell lysates were then incubated with beads bound to an antibody directed against the Flag epitope. The interaction between Flag-PPARγ and endogenous Hic-5 was tested in the presence and absence of rosiglitazone. Ectopic expression of gfp-PGC1α was used as a positive control for binding to PPARγ. (C) Bosc cells were transiently transfected with various gfp-Hic-5 alleles. The cells were harvested, and extracts were incubated with immobilized GSTPPARγ, which was purified from Sf21 baculovirus cells in the presence of PPARγ ligand troglitazone (Tro). After washing, bound proteins were eluted and separated by SDS-PAGE, followed by Western blotting using an anti-gfp antibody. (D) In vitro interaction assay. Either GST or GST-PPARγ was incubated with ³⁵S-labeled Hic-5 alleles (shown on the right panel). SRC-1 was used as positive control to show in vitro binding to PPARγ.

Hic-5 and PPARγ are coexpressed in normal and malignant gut development. (A) Small intestines from mouse embryos were dissected at different stages of embryonic development, and total RNA was extracted. The level of expression of PPARγ, Hic-5, keratin 19, keratin 18, and L-FABP was analyzed by using real-time RT-PCR assay. (B) Immunohistochemistry of PPARγ and Hic-5 in sections of embryonic and adult mouse small intestines. Brown color indicates positive staining; blue denotes nuclear counter-staining. (C) Mice were treated with AOM as described earlier (Girnun et al. 2002). Colonic tumors and adjacent tissue were harvested, and RNA was extracted. Levels of PPARγ, Hic-5, paxillin, keratin 20 KLF4, and PGC1α were analyzed by real-time RT-PCR. The levels of RNA expression were normalized with TBP and are mean±SD. (n = 5). Statistical analysis was done by using Student's t-test. (^★) p < 0.01

Ectopic expression of Hic-5 enhances PPARγ-mediated induction of several epithelial markers in colon cancer cells. (A) Human colon cancer cells (Moser) were infected with a control retrovirus or a retrovirus expressing Hic-5. Cells were treated with or without 1 μM rosiglitazone and were then harvested, and RNA was extracted. Levels of mRNA for keratin 20, L-FABP, KLF4, and ADRP were quantified by real-time RT-PCR. Statistical analysis was done using Student's t-test. (^★) p < 0.05; (NS) not significant. (B) Cells were treated as described in A. Levels of mRNA for BPG, CEA, annexin A1, and annexin A2 were determined by real-time RT-PCR analysis. Statistical analysis was done using Student's t-test. (NS) Not significant. (C) Moser cells were infected with either gfp or PGC1α expressing adenovirus. Cells were treated with or without 1 μM rosiglitazone and were then harvested, and RNA was extracted. Levels of mRNA for keratin 20, L-FABP, KLF4, and ADRP were quantified by real-time RT-PCR. Statistical analysis was done using Student's t-test. (^★) p < 0.05; (NS) not significant.

TGFβ enhances PPARγ induction of keratin 20, while dominant-negative Hic-5 blocks it. (A) Cos-1 cells were transiently cotransfected with 50 ng expression vector for PPARγ, 50 ng luciferase reporter plasmid with three PPARγ response elements (DR1-luc) in the presence of 250 ng gfp-Hic-5, 400 ng gfp-C terminus Hic-5(209-444), gfp-N terminus Hic-5(1-208) expression vectors, or gfp expressing vector as a control. Cells were treated with increasing concentrations of pioglitazone (Pio) as shown. Cell lysate were then analyzed for relative transcriptional activity, as described in Figure 1A. (B) Cos-1 cells were transiently tranfected and treated as described in A. To determine potential dominant-negative activities, Hic-5 expressing vector (100 ng) was cotransfected with increasing amount of either gfp-N terminus Hic-5 or gfp-C terminus Hic-5(100-400 ng). (C) Moser cells transduced with retroviral expressing Hic-5, dominant-negative Hic-5 (gfp-N terminus), or gfp were incubated with or without TGFβ or PPARγ ligand (pioglitazone). After 2 d, total RNA and protein were extracted. (Top) Relative keratin 20 and PPARγ mRNA levels were analyzed by real-time RT-PCR. Values of keratin 20 and PPARγ were normalized to expression level without treatment in control cells, corrected to β-actin and were mean ± SE of three different experiments. (Bottom) Protein lysates were extracted and separated by SDS-PAGE gels. Keratin 20 and GPDH levels were analyzed by Western blot using keratin 20 and GPDH antibodies respectively. GPDH levels are shown as a loading control.

siRNA against Hic-5 inhibits PPARγ induction of certain epithelial genes. (A) Moser cells infected with siRNA expressing vector (pSuper-neo) directed against Hic-5 (siRNA-5) mRNA or scrambled oligonucleotides as a control. After antibiotic selection cells were treated with and without 1 μM rosiglitazone. (Inset) RNA and proteins were isolated, and Hic-5 mRNA levels were determined by real-time RT-PCR and Western blot analysis. The levels of RNA expression were normalized with TBP and are mean ± SD of three different experiments. Statistical analysis was done using Student's t-test. (^★) p < 0.01. (B) Cells were treated as described in A. RNA was isolated, and levels of mRNA for, KLF4, L-FABP, keratin 20, and ADRP were analyzed by real-time RT-PCR. Statistical analysis was done using Student's t-test. (^★) p < 0.05. (C) Moser cells infected with adenovirus expressing either siRNA directed against PGC1α mRNA (siRNA-α) (Koo et al. 2004), or scrambled oligonucleotide as a control. After 48 h cells were harvested, and levels of PGC1α and its target gene ERRα were measured by real-time PCR. Analysis was done as described in A. (D) Moser cells were infected as described in C. After infection cells were treated with or without rosiglitazone (1 μM). Levels of KLF4, keratin 20, L-FABP, and ADRP were analyzed as described in B.

Modulation of PPARγ and Hic-5 expression in epithelial gut development. (A) Small intestines were isolated from PPARγ^+/+ and PPARγ^+/- mice (Akiyama et al. 2002). After RNA isolation levels of PPARγ keratin 20, keratin 19, keratin 18, KLF4, and L-FABP were determined using real-time RT-PCR analysis, as described in Figure 5 (n = 6). (B) Small-intestines were isolated from mice embryos between E12 and E13 and were injected with either siRNA directed against Hic-5 (siRNA-5-I, siRNA-5-II) or scrambled oligonucleotide expressing vector, followed by electroporation to express the plasmids in the epithelial layer. Embryonic small-intestines were then grown for 2 d ex vivo under differentiation condition, and protein lysates were harvested as described in Tou et al. (2004). Hic-5, keratin 19, and L-FABP protein levels were analyzed by Western blot analysis, using GPDH as loading control. Relative protein expression was determined by using densitometer analysis of the Western blot corrected for GPDH expression levels. This is a representative experiment (n = 5). (C) Embryonic small intestines were isolated and treated as described in B, only Hic-5 expressing vector and an empty vector as a control were used. RNA was isolated, and Hic-5, keratin 20, and KLF4 mRNA levels were analyzed using real-time RT-PCR as described in Figure 5.

Ectopic expression of Hic-5 in 3T3-L1 preadipocytes inhibits adipocyte differentiation while inducing markers of epithelial gut differentiation. (A) Hic-5 mRNA levels during 3T3-L1 adipogenesis were analyzed using real-time RT-PCR. (B) 3T3-L1 preadipocytes were infected with either retrovirus expressing Hic-5 or an empty retrovirus. Cells were then differentiated using a standard protocol with addition of rosiglitazone. After 4 d cells were fixed, and phase-contrast pictures were taken (top panel) or stained for lipid droplet formation by Oil-Red-O (bottom panel). (C) 3T3-L1 preadipocytes were treated as described in B. After 4 d of differentiation with or without rosiglitazone, cells were harvested and RNA was extracted. Levels of mRNA for adipocyte markers adipsin, acrp30, and aP2 and gut epithelial markers KLF4 L-FABP and keratin 20 were determined by real-time quantitative RT-PCR. Statistical analysis was done using Student's t-test. (^★) p < 0.05. (D) Illustration of Hic-5 and PPARγ combination in epithelial differentiation.