RESUMO
Objective • To investigate the role and the regulation mechanism of SUMOylation of peroxisome proliferator activated receptor γ1 (PPARγ1) in macrophage M2 polarization induced by interleukin-4 (IL-4). Methods • To investigate the SUMOylation of PPARγ1 and identify its SUMOylated site, immunoprecipitation (IP) with anti-FLAG/HA antibody and Western blotting were used after plasmids FLAG-PPARγ1-WT/mutant and HA-SUMO1 being co-transfected into HEK293T cells. To determine SENP1 can de-SUMOylate PPARγ1, IP was used when HEK293T cells were co-transfected by FLAG-PPARγ1-WT, HA-SUMO1 and RGS-SENP1-WT, or SENP1 mutant plasmids. The change of the endogenous SUMOylation of PPARγ1 during M2 polarization was checked by IP and Western blotting by using PPARγ or SUMO1 antibodies in cell lysates of RAW264.7 cells and primary peritoneal macrophages induced by IL-4. The expression of some M2 related marker genes were detected by real-time quantitative polymerase chain reaction in PPARγ1-WT/mutants stably-overexpressed RAW264.7 cells. Chromatin immunoprecipitation (ChIP) experiment was used to confirm the different ability of binding to the promoter of arginase (Arg1) between PPARγ1-WT and PPARγ1-K77R. Results • It has been identified that the major SUMOylated site of PPARγ1 was Lys77, which could be de-SUMOylated by SENP1. The endogenous SUMOylation of PPARγ1 decreased when macrophage polarized to M2 macrophage induced by IL-4. The expression of Arg1 increased in PPARγ1-K77R stably-overexpressed RAW264.7 cells. PPARγ1-K77R easily bound to the promoter of Arg1 gene, showing more transcription activity. Conclusion • De-SUMOylation of PPARγ1 at Lys77 can enhance its transcription activity by promoting the expression of Arg1 gene, which is involved in the regulation of macrophage M2 polarization.
RESUMO
Objective To observe the protective effect of high expression of mouse proxisome proliferator activated receptor γ1（PPARγ1）on free fatty acid （FFA）-induced βTC3 cell impairment. Methods The recombinant plasmid pcDNA3.1/PPARγ1 was generated with cloning and was stably transfected into pancreatic β TC3 cells. The expression was detected with semi-quantitative RT-PCR. Then the cell viability of wild βTC3 cells was compared with that of the βTC3 cells with high expressed PPARγ1 by MTT viability assay after they were exposed to high-level FFA for 48 hours. Results The sequencing results for amplified target gene showed that the sequence of PPARγ1 from Chinese Kunming mouse is similar to that of mouse PPARγ1 in Genebank, only the codon coding Asp at the site of 421 amino acid changed from AAU to AAC. PPARγ1 was efficiently expressed in βTC3 cells in vitro. The cell viability of wild βTC3 cells reduced after being exposed to high-level FFA for 48 hours（P0.05）. Conclusion The high expression of PPARγ1 could protect βTC3 cells from FFA-induced impairment