ATF4 is constitutively expressed in rheumatoid arthritis (RA) synovium, but translocated into the nucleus by C/EBPβ-LIP.
(A) Expression and distribution of ATF4 in RA synovium by immunofluorescence staining. Original magnification, 200×. (B) Expression of ATF4 protein levels from fibroblast-like synoviocytes from the synovium of RA patients (RA-FLS) by western blotting. ATF4 protein in HeLa cells was used as a positive control. (C) Expression of ATF4 mRNA of RA-FLS overexpressed C/EBPβ by quantitative RT-PCR. Isolated RA-FLS were transfected with adenovirus expression vectors for C/EBPβ-LAP, −LIP or LacZ control. The level of ATF4 mRNA relative to LacZ control is shown. (D) Synovial tissue from RA patients was cultured in serum-free medium for 24 hours and then replaced with fresh medium containing adenovirus expression vectors for C/EBPβ-LIP or LacZ control for a further 72 hours. Immunostaining for ATF4 was performed. Original magnification, 400 × .