Activating transcription factor 3 (ATF3) represses the expression of CCL4 in murine macrophages

Acute expression of macrophage inflammatory protein-1 beta (also known as CCL4) promotes beneficial leukocyte recruitment to infected tissues, but chronic expression of this chemokine contributes to inflammatory disease. CCL4 expression is controlled largely at the transcriptional level and an ATF/CRE sequence located in the promoter (-104 to -97bp, relative to the transcriptional start site) has been identified as a critical cis-acting element. The trans-acting binding proteins that influence CCL4 transcription via this site are largely unknown. We investigated whether activating transcription factor 3 (ATF3), a member of the ATF/CREB family of transcription factors, binds to the CCL4 ATF/CRE site in macrophages. Using the electrophoretic mobility shift assay and the chromatin immunoprecipitation assay, we found that ATF3 binds to the ATF/CRE site within the CCL4 promoter in untreated and lipopolysaccharide (LPS)-stimulated macrophages. Quantitative RT-PCR analysis showed that CCL4 mRNA levels in elicited peritoneal macrophages from ATF3(-/-) mice are significantly higher than in congenic ATF3(+/+) macrophages under both unstimulated and LPS-stimulated conditions, suggesting that ATF3 represses transcription of the CCL4 gene. Consistent with the higher gene expression, ATF3-deficient macrophages secreted more CCL4 protein than ATF3(+/+) macrophages. Similar results were obtained in bone-marrow-derived macrophages treated with Toll-like receptor 2, 3, 4 and 5 agonists. Thus, we conclude that ATF3 constitutively binds to the ATF/CRE site in the CCL4 promoter where it represses basal and pathogen-associated molecular pattern (PAMP)-stimulated transcription. Consequently, ATF3 appears to be part of a control mechanism that limits the amount of CCL4 released by macrophages, preventing excessive inflammation.