Regulation of the human tumor necrosis factor-α promoter by angiotensin II and lipopolysaccharide in cardiac fibroblasts: different cis-acting promoter sequences and transcriptional factors

We recently showed that angiotensin (ANG) II as well as mechanical stretch stimulated production of tumor necrosis factor (TNF) in cardiac fibroblasts. Presently, we examined the molecular mechanisms by which ANGII and lipopolysaccharide (LPS) upregulate TNF-α gene expression. In neonatal rat cardiac fibroblasts, increased transcription of TNF-α mRNA was detected as luciferase activity associated with activity of the TNF-α promoter. Progressive deletion from this promoter located the LPS-responsive region between –200 and –120 bp from the transcription initiation site, while the sequence between –120 and –70 bp was required for ANGII-induced expression. Next, we examined which cis–acting sequences in the TNF-α promoter region were essential for induction of TNF-α transcription. Competition analysis by electrophoretic mobility shift assay with and without specific antibodies showed that LPS increased binding of Sp1 and Sp3 to the Sp1–binding site, while Egr-1 was unimportant. With ANGII, binding of ATF-2/c–jun to the CRE site was required for TNF-α gene induction; neither Ets nor NF-κB was essential. Mutation analysis confirmed that response to LPS relied upon the Sp1 site in the TNF-α promoter, while the CRE-binding site was essential for stimulation by ANGII. We concluded that since TNF-α gene expression is transcriptionally activated by ANGII or LPS in cardiac fibroblasts via different cis–acting sequences in the TNF-α promoter and different transcriptional factors, mechanisms inducing TNF production differ between heart failure or cardiac hypertrophy and infectious disease.