Koichi Tomaru

Angiotensin II and mechanical stretch induce production of tumor necrosis factor in cardiac fibroblasts

To determine whether ANG II as well as mechanical stress affect the production of tumor necrosis factor (TNF) in the heart, neonatal rat cardiac myocytes and fibroblasts were cultured separately and treated for 6 h with ANG II, lipopolysaccharide (LPS), or cyclic mechanical stretch. LPS induced the production of TNF in cardiac myocytes and fibroblasts. However, TNF synthesis in fibroblasts was 20- to 40-fold higher than in myocytes. ANG II (≥10-8 M) and mechanical stretch stimulated the production of TNF in cardiac fibroblasts but not in myocytes. Furthermore, both ANG II and LPS increased the expression of TNF-α mRNA in cardiac fibroblasts. Isoproterenol inhibited both LPS- and ANG II-induced production of TNF in cardiac fibroblasts with increasing intracellular cAMP level. Moreover, both isoproterenol and dibutyryl cAMP inhibited LPS-induced TNF-α mRNA expression. Thus activation of the renin-angiotensin system, as well as mechanical stress, can stimulate production of TNF in cardiac fibroblasts. Furthermore, β-adrenergic receptors may be responsible for the regulation of TNF synthesis at the transcriptional level by elevating intracellular cAMP.To determine whether ANG II as well as mechanical stress affect the production of tumor necrosis factor (TNF) in the heart, neonatal rat cardiac myocytes and fibroblasts were cultured separately and treated for 6 h with ANG II, lipopolysaccharide (LPS), or cyclic mechanical stretch. LPS induced the production of TNF in cardiac myocytes and fibroblasts. However, TNF synthesis in fibroblasts was 20- to 40-fold higher than in myocytes. ANG II (>/=10(-8) M) and mechanical stretch stimulated the production of TNF in cardiac fibroblasts but not in myocytes. Furthermore, both ANG II and LPS increased the expression of TNF-alpha mRNA in cardiac fibroblasts. Isoproterenol inhibited both LPS- and ANG II-induced production of TNF in cardiac fibroblasts with increasing intracellular cAMP level. Moreover, both isoproterenol and dibutyryl cAMP inhibited LPS-induced TNF-alpha mRNA expression. Thus activation of the renin-angiotensin system, as well as mechanical stress, can stimulate production of TNF in cardiac fibroblasts. Furthermore, beta-adrenergic receptors may be responsible for the regulation of TNF synthesis at the transcriptional level by elevating intracellular cAMP.

Transcriptional stimulation of the eNOS gene by the stable prostacyclin analogue beraprost is mediated through cAMP-responsive element in vascular endothelial cells: Close link between PGI2 signal and NO pathways

Abstract— Beraprost sodium (BPS), an orally active prostacyclin analogue, has been reported to be beneficial in the treatment of primary pulmonary hypertension and obstructive peripheral arterial disease. Although BPS was originally described for its effects on platelet aggregation and vasodilatory response, the effect on endothelial cells has been poorly understood. In this study, we examined the effects of BPS on the eNOS gene expression in mouse aorta and cultured human and bovine aortic endothelial cells. Treatment of these cells with BPS increased the eNOS expression as assessed by Northern blots, Western blots, and NO production by NO-specific fluorescence (DAF2-DA) and by the Griess method. Standard mRNA decay assays showed that BPS increases the stability of eNOS mRNA. In addition, BPS increased the promoter activity of the human eNOS gene, as determined by luciferase assays of the eNOS promoter gene. Progressive 5′-deletion and site-specific mutation analyses defined the BPS-responsive sequences as cAMP-responsive elements (CRE) located at −733 and −603. By using the oligonucleotide probe containing this CRE sequence in electrophoretic mobility shift assays, we showed that the phosphorylated form of CRE-binding protein is a major constituent of the complex in BPS-treated cells. Western blot analyses indicate that BPS but not endogenous prostacyclin phosphorylates CRE-binding protein. The presence of functional CRE sites within human eNOS promoter may represent a novel mechanism for regulating eNOS gene expression.

Transcription factor Sp1 regulates SERCA2 gene expression in pressure-overloaded hearts: a study using in vivo direct gene transfer into living myocardium.

Pressure-overload hypertrophy results in downregulation of the sarcoplasmic reticulum Ca(2+)-ATPase pump encoding SERCA2 gene that regulates Ca(2+) uptake and myocardial relaxation. We previously characterized a proximal promoter region containing four Sp1 element consensus sequences (-284 to -72 base pairs (bp)) that was responsible for pressure-overload-induced transcriptional regulation. The purpose of the present study was to determine which of the Sp1 sites was responsible for the downregulation of SERCA2 gene transcription under pressure overload. Using an in vivo direct gene transfer assay, SERCA2 gene transcriptional activity was measured under pressure overload. Site-directed mutagenesis of the four Sp1 sites (I-IV) in the SERCA2 gene promoter (-284 to -72 bp) was performed. Wild-type and Sp1 mutant-luciferase reporter constructs were injected into the left-ventricular apices of pressure overload or sham-operated rats, and Sp1 mRNA and SERCA2 gene-luciferase activity was measured sequentially from 3 to 14 d after surgery. At 5 d, Sp1 mRNA in the pressure-overload rats increased to 124 +/- 7% of sham group levels, and pressure-overload-induced SERCA2 transcriptional activity was 15 +/- 4% of sham group when all four Sp1 sites remained intact. Mutation of the Sp1 mutant sites I (-196 to -191 bp) and III (-118 to -113 bp) blocked the inhibitory effect of pressure overload and resulted in SERCA2 gene transcriptional activity of 54 +/- 15% and 56 +/- 7% of sham group, respectively. We conclude that the pressure-overload-induced decrease in SERCA2 mRNA is mediated by Sp1 sites I and III.