STAT6 Transcription Factor Is a Facilitator of the Nuclear Receptor PPARγ-Regulated Gene Expression in Macrophages and Dendritic Cells

Summary Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.


Isolation and culture of human monocytes
Human monocytes were isolated from healthy volunteer's buffy coat obtained from the Regional Blood Bank. Separation was carried out according to the manufacturer's

Isolation and culture of mouse peritoneal and bone marrow cells
Thioglycolate-elicited macrophages were harvested from the peritoneal cavity 4 days after injection of 3 ml 3% thioglycolate solution. Cells were washed in saline and cultured in RPMI1640 supplemented with 10% FBS, 2 mM glutamine, penicillin and streptomycin for two days. Bone marrow cells were isolated from the femur of mice then were washed in saline; cultured in RPMI1640 supplemented with 10% FBS, 2mM glutamine, penicillin and streptomycin. Monocytes were isolated from bone marrow using negative selection method with magnetic separation (Miltenyi). Bone marrow cells were differentiated to macrophages by M-CSF (20 ng/ml) or to DCs by GM-CSF (20 ng/ml) and IL-4 (20 ng/ml) for 10 days. For activation of macrophages similar methods were used as in case of the human macrophages: IL-4 (20 ng/ml), IFN-γ (100 ng/ml), TNF (20 ng/ml), E. coli (O55:B5 serotype) LPS (100 ng/ml). Fresh medium containing cytokines and ligands were added every third day to complement the old medium.

RNA isolation and real-time quantitative RT PCR
Total RNA was isolated from cells using Trizol Reagent (Invitrogen) Table   4.

Western blotting
Cells were treated for two days as indicated and were washed in PBS then lysed in buffer Peroxidase-conjugated secondary antibody and ECL detection kit (Pierce) was used for signal detection.

Transient transfection
For

Pull-down assays
Human PPARγ1 cDNA fused to a streptavidin-binding protein and to a calmodulinbinding protein was cloned into a modified pET30a vector and was expressed in Rosetta BL21 (Novagen) after overnight induction with 40 μM isopropyl-Dthiogalactopyranoside (IPTG). Bacteria were lysed in non-denaturing lysis buffer with 3 freeze-thaw cycles and 5 minutes sonication. PPARγ1 was purified with streptavidinresin. Whole cell lysates of STAT6-or mock-transfected HEK293T cells were added to the resin and incubated overnight. After 3 washing steps samples were boiled in Laemmli buffer and analyzed by immunoblotting.

Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed as described earlier (Balint et al., 2005) with modifications. Briefly, cells were fixed with 1% formaldehyde for 10 minutes,

Mice
Mice carrying null or floxed alleles of Pparg were created as described previously (Barak et al., 1999;Hevener et al., 2003;Miles et al., 2000). These mice were contained sterilized bedding material. The room lightning was automatically switched on at 6:00 and off at 18:00. The room temperature was 20± 2 °C, the relative humidity was 50%.

Microarray analysis
Microarray analysis was performed on human and mouse macrophages. We used wildtype, Pparg +/-LysCre, Pparg fl/-LysCre and Stat6 -/mouse macrophages and analyzed them on Affymetrix microarrays. Total RNA was isolated using RNeasy kit (Qiagen). cRNA was generated from 5 μg of total RNA by using the SuperScript Choice Kit (

Bioinformatic analysis
The PhastCons conservation scores for placental mammalian species were obtained from the UCSC site. The scores were calculated from the MULTIZ (UCSC/Penn State Bioinformatics) 44 vertebrate species whole genome alignment. The graph in Figure 5G shows the 10kbp promoter region of the human FABP4 gene until the second exon. It corresponds to the hg18 chromosome 8 genomic position from 82555216 to 82568028 on the negative strand. The X-axis shows the (decreasing) chromosomal position on the negative strand. The Y-axis shows the PhastCons conservation scores (in the range 0-1) for each position. The wigFix PhastScore files were converted with a PERL script to a bed-file style comma-separated format suitable for spreadsheet processing. The empty (without PhastScore) regions were filled with zeros.
Pre-cleared extracts were incubated with 10 μg MacPPRE and 50 μl streptavidin-agarose for 2h at 4 °C, and then washed three times with OP buffer. Captured protein was resolved by SDS-PAGE (7.5%) and Western blotted with monoclonal anti-PPARγ antibody (E-8, Santa Cruz). The membrane was then stripped and re-probed with STAT6 antibody (M-20, Santa Cruz)

Statistical tests
All data are presented as means ±SD. In real-time quantitative PCR and reporter assays the mean and standard deviation were calculated for both the normalized and the normalizer values. To incorporate the random errors of the measurements we used the propagation of errors to determine the standard deviation of the normalized values. For all experiments we made at least three biological replicates and on the fold changes we performed an unpaired (two tail) t test and results were considered significant with p<0.01.