OBJECTIVES: Activating transcription factor-3 (ATF3) is a key regulator of inflammatory responses. We aimed to investigate the effects and mechanisms of ATF3 on the inflammatory cytokines are induced by Mycoplasma pneumonia (MP). STUDY DESIGN: RAW264.7 and mouse peritoneal macrophages were exposed to various time with or without MP infection (3, 6, 12, 24, and 48 h), and detect the expression of ATF3. Adenovirus-expression of ATF3 (Ad/ATF3) or Ad/βgal was transfected into cells which were exposed to MP for 48 h, RT-PCR and ELISA was used to evaluate the expression and secretion of TNF-α, IL-1β, IL-6, and IL-18. In addition, intravenous administration Ad/ATF3 or Ad/βgal into the mice, the secretion of inflammatory cytokines were detected using ELISA. ChIP assay was used to determine whether ATF3 can bind to the promoter of Early growth response protein 1 (Egr-1). Western blot was used to detect the expression of Egr-1 and Fyn. RESULTS: ATF3 was increased at 3, 6, 12, and 24 h and the highest expression levels occurs in 6 h, there is no significant differences at 24 and 48 h compared with 0 h or CON group in RAW 264.7. Similar results were seen in mouse peritoneal macrophages. Overexpression of ATF3 resulted in the reduction of inflammatory cytokines. ChIP assay revealed that ATF3 can bind to the promoter of Egr-1. Overexpression of ATF3 inhibited the protein expression of Egr-1 and Fyn; conversely, ATF3-deficiency promoted the expression of Egr-1 and Fyn. Overexpression of Egr-1 reduced the anti-inflammatory action of ATF3. CONCLUSIONS: ATF3 inhibit the expression and release of TNF-α, IL-1β, IL-6, and IL-18 induced by MP in vitro and in vivo, which is associated with its negative regulation of Egr-1/Fyn signaling pathway.
OBJECTIVES:Activating transcription factor-3 (ATF3) is a key regulator of inflammatory responses. We aimed to investigate the effects and mechanisms of ATF3 on the inflammatory cytokines are induced by Mycoplasma pneumonia (MP). STUDY DESIGN: RAW264.7 and mouse peritoneal macrophages were exposed to various time with or without MP infection (3, 6, 12, 24, and 48 h), and detect the expression of ATF3. Adenovirus-expression of ATF3 (Ad/ATF3) or Ad/βgal was transfected into cells which were exposed to MP for 48 h, RT-PCR and ELISA was used to evaluate the expression and secretion of TNF-α, IL-1β, IL-6, and IL-18. In addition, intravenous administration Ad/ATF3 or Ad/βgal into the mice, the secretion of inflammatory cytokines were detected using ELISA. ChIP assay was used to determine whether ATF3 can bind to the promoter of Early growth response protein 1 (Egr-1). Western blot was used to detect the expression of Egr-1 and Fyn. RESULTS:ATF3 was increased at 3, 6, 12, and 24 h and the highest expression levels occurs in 6 h, there is no significant differences at 24 and 48 h compared with 0 h or CON group in RAW 264.7. Similar results were seen in mouse peritoneal macrophages. Overexpression of ATF3 resulted in the reduction of inflammatory cytokines. ChIP assay revealed that ATF3 can bind to the promoter of Egr-1. Overexpression of ATF3 inhibited the protein expression of Egr-1 and Fyn; conversely, ATF3-deficiency promoted the expression of Egr-1 and Fyn. Overexpression of Egr-1 reduced the anti-inflammatory action of ATF3. CONCLUSIONS:ATF3 inhibit the expression and release of TNF-α, IL-1β, IL-6, and IL-18 induced by MP in vitro and in vivo, which is associated with its negative regulation of Egr-1/Fyn signaling pathway.