Transcriptional Regulation of Inducible Nitric Oxide Synthase in Cultured Neonatal Rat Cardiac Myocytes

Abstract

Previous work has demonstrated that inducible NO synthase (iNOS) can be expressed in cardiac myocytes. In this study, we investigated transcriptional regulation of the iNOS gene in these cells. Lipopolysaccharide (LPS) induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. H-89, dexamethasone, herbimycin, genistein, staurosporine, or pyrrolidine dithiocarbamate (PDTC) attenuated the iNOS induction by LPS. Forskolin, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma enhanced the LPS-induced iNOS expression. Combined stimulation of IL-6 and TNF-alpha also induced iNOS. The 5'-upstream sequence of the rat iNOS gene contains the nuclear factor-kappa B (NF-kappa B) site, CAAT box, IFN-gamma activation site (GAS), and IFN regulatory factor (IRF) site. DNase I footprinting assay revealed that the nuclear factors binding to these elements were increased by LPS exposure. Transient transfection assay suggested that these elements were indispensable for transcriptional regulation of the iNOS induction. Electrophoretic mobility shift assay revealed that LPS or TNF-alpha increased binding activity for the NF-kappa B site. A slower-migrating complex binding to the CAAT box gave rise after exposure to LPS or forskolin. Competition assay suggested that this slower-migrating complex consisted of a heterodimer between a member of CAAT box/enhancer binding (C/EBP) protein family and cAMP responsive element binding protein (CREB). LPS or IL-6 increased binding complexes for the IRF site, which was compatible with induction of IRF-1. LPS, IL-6, or IFN-gamma induced a novel binding complex for GAS, which also existed in the 5'-flanking region of the IRF-1 gene. These data suggest that (1) iNOS induction simultaneously requires both NF-kappa B activation and IRF-1 induction, and (2) the heterodimer between C/EBP and CREB has synergistic effects on the iNOS induction via the CAAT box.

Novel Role of Pin1 Induction in Type II Collagen-Mediated Rheumatoid Arthritis

“…COX-2 and iNOS expression are also transcriptionally regulated by C/EBP and CREB as well as NF-B, and these transcription factors may be synergistically or independently involved in the expression of this gene expression (26,43,44). In this study, CRE and C/EBPs were consistently activated by Pin1, and these transcription factors are active to increase COX-2 expression in Pin1-overexpressed chondrocytes.…”

Modular Activation of Nuclear Factor-κB Transcriptional Programs in Human Diabetic Nephropathy

“…Ohmori and Hamilton (19) demonstrated the cooperative interaction between transcription factors binding to an interferon-stimulated response element, a promoter sequence found in several interferon-inducible genes, with NF-B binding at a proximal site that leads to synergistic induction of the CXCL10/IP-10 gene. Later complementary data regarding synergistic effects of IRF and NF-B were described in the induction of B2M, CCL5/RANTES (20), EDN1 (21), IFNB1 (22), NOS2A (23), and VCAM1 (24).…”

Protein kinase A mediates microglial activation induced by plasminogen and gangliosides

“…Plasm inogen regulates DNA binding activities of CREB and NF-κB via protein kinase A cAMP response element (CRE) and NF-κB binding sites exist in the promoter regions of genes encoding IL-1β, TNF-α and iNOS (Collart et al, 1990;Hiscott et al, 1993;Wong et al, 1996;Kinugawa et al, 1997;Stylianou and Saklatvala, 1998;Tsai et al, 2000;Chang et al, 2004). Furthermore, plasminogen increases the DNA binding activities of CRE binding protein (CREB) and NF-κB (Min et al, 2003).…”