Abstract:

To explore the role of transcriptional coactivatoractivating transcription factor 3（ATF3) in the nuclear factor of activated Tcells cytoplasmic 1 (NFATc1) induced podocyte injury.
Ｍｅｔｈｏｄｏｌｏｇｙ：(1) The  expression of ATF3 in the glomeruli of proteinuric patients (MCD, FSGS or DN)  were observed by  laser confocal microscopy and Western blotting; (2) The conditionally immortalized mouse podocyte cell line was cultured in vitro and exposed to LPS (100 μg/ml) or ionomycin (2 μmol/L) for different times. Realtime quantity PCR and Western blotting were used to analyze  the expression of ATF3; (3) Cell apoptosis in ATF3 knockdown podocytes was observed by flow cytometry. Realtime quantity PCR and Western blotting were used to analyze  the expression of BAX and Bcl2, and podocin expression in ATF3 knockdown podocytes was analyzed using Western blotting; (4) Western blotting and immunofluorescent staining were used to evaluate the change of nuclear localization of ATF3; (5) A chromatin immunoprecipitation assay (ChIP assay) was performed to confirm the potential ATF3 binding sites in the NFATc1 promoter region.
Ｒｅｓｕｌｔｓ：(1) The expression of ATF3 was all elevated in podocytes from MCD patients, FSGS or DN. (2) ATF3 mRNA and protein increased in LPS or Ionomycintreated podocytes for 1,2,4 hours. Western blot analysis demonstrated that ATF3 activation peaked at 2 hours and diminished  6 hours after LPS or ionomycin treatment. (3) After the knockdown of ATF3, the apoptosis ratio  reduced, BAX mRNA and protein was downregulated, Bcl2 mRNA and protein was upregulated, podocin protein  increased and recovered to a nearly normal expression. (4) After injurystimulation, nuclear localization of ATF3  increased. (5) ChIP assay demonstrated ATF3 were binding to the NFATc1 promoter region. When ionomycintreated, more chromatin immunoprecipitated by ATF3 antibodies was observed. In ATF3 knockdown podocytes, reduced NFATc1 mRNA and protein expression were observed by Realtime quantity PCR and Western blotting.
Ｃｏｎｃｌｕｓｉｏｎ：
ATF3, a transcriptional coactivator, promotes the transcription of NFATc1 through binding to NFATc1 promoter region and thus contributes to NFATc1mediated podocyte injury.

Key words: podocytes, activating transcription factor 3, nuclear factor of activated T-cells cytoplasmic 1