CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line.
(25/2395)
Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicated in epithelial cell injury from oxidative and immunologic stress. The NOS2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytokines in medullary thick ascending limb of Henle's loop (MTAL) cells and other cell types. The 5'-flanking region of the NOS2 gene contains a consensus element for CCAAT/enhancer binding proteins (C/EBP) at -150 to -142 that we hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon-gamma (IFN-gamma) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN-gamma compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-gamma activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBPbeta stimulated basal NOS2 promoter activity. Thus C/EBPbeta appears to be an important trans-activator of the NOS2 gene in the MTAL. (+info)
ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.
(26/2395)
The transcription of genes encoding proteins involved in the hepatic synthesis of lipids from glucose is strongly stimulated by carbohydrate feeding. It is now well established that in the liver, glucose is the main activator of the expression of this group of genes, with insulin having only a permissive role. While ADD1/SREBP-1 has been implicated in lipogenic gene expression through temporal association with food intake and ectopic gain-of-function experiments, no genetic evidence for a requirement for this factor in glucose-mediated gene expression has been established. We show here that the transcription of ADD1/SREBP-1c in primary cultures of hepatocytes is controlled positively by insulin and negatively by glucagon and cyclic AMP, establishing a link between this transcription factor and carbohydrate availability. Using adenovirus-mediated transfection of a powerful dominant negative form of ADD1/SREBP-1c in rat hepatocytes, we demonstrate that this factor is absolutely necessary for the stimulation by glucose of L-pyruvate kinase, fatty acid synthase, S14, and acetyl coenzyme A carboxylase gene expression. These results demonstrate that ADD1/SREBP-1c plays a crucial role in mediating the expression of lipogenic genes induced by glucose and insulin. (+info)
Inhibition of tumor necrosis factor alpha-induced prostaglandin E2 production by the antiinflammatory cytokines interleukin-4, interleukin-10, and interleukin-13 in osteoarthritic synovial fibroblasts: distinct targeting in the signaling pathways.
(27/2395)
OBJECTIVE: To investigate the effects of the antiinflammatory cytokines interleukin-4 (IL-4), IL-10, and IL-13 on tumor necrosis factor alpha (TNFalpha)-induced prostaglandin E2 (PGE2) release in the cellular signaling cascade on human osteoarthritis (OA) synovial fibroblasts. METHODS: Human OA synovial fibroblasts were cultured to explore the impact of IL-4, IL-10, and IL-13 on TNFalpha binding to TNF receptors (TNFR), soluble TNFR (sTNFR), cytoplasmic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX-2) production, and on the binding activity of the transcription factors nuclear factor kappaB (NF-kappaB), CCAAT-enhancer binding protein (C/EBP), activator protein 2 (AP-2), and cyclic AMP response element-binding protein (CREB). RESULTS: IL-4, IL-10, and IL-13 at 5 ng/ml dramatically reduced TNFalpha-induced PGE2 release by approximately 90% (P < 0.0001). IL-4 up-regulated the level of TNFalpha-induced TNFR by 47% (P < 0.06), while IL-10 down-regulated it by 71% (P < 0.02); IL-13 had no effect. Although statistical significance was not reached, all 3 cytokines up-regulated the basal level of sTNFR-55. IL-4 and IL-10, while not altering the basal level of sTNFR-75, significantly increased the TNFalpha-stimulated release of sTNFR-75. IL-4, IL-10, and IL-13 reduced the TNFalpha-induced COX-2 level, and IL-4 and IL-10 reduced the cPLA2 level. IL-4 had no effect on TNFalpha up-regulation of NF-kappaB, and a slight decrease was noted with IL-10 and IL-13 at the highest concentration used (5 ng/ml). IL-4 and IL-13 decreased the TNFa-induced C/EBP accumulation in a dose-dependent manner, while IL-10 up-regulated its basal level. AP-2 and CREB were not induced by TNFalpha. CONCLUSION: The results indicate that these antiinflammatory cytokines reversed the TNFalpha-induced release of PGE2 by OA synovial fibroblasts, by acting at various levels of the TNFa-dependent signaling cascade. These data shed new light on the mechanisms by which these cytokines reduce inflammatory processes. (+info)
A composite enhancer element directing tissue-specific expression of mouse mammary tumor virus requires both ubiquitous and tissue-restricted factors.
(28/2395)
Mouse mammary tumor virus (MMTV) expression is restricted primarily to mammary epithelial cells. Sequences responsible for both the mammary-specific expression of MMTV and the activation of cellular oncogenes are located within two enhancer elements at the 5'-end of the long terminal repeat. Whereas the Ban2 enhancer (-1075 to -978) has been well characterized, the mammary-specific enhancer of MMTV from -956 to -862 has only recently been recognized as a key determinant of mammary-specific oncogene activation by MMTV. The present study identifies and characterizes three binding sites located within this element. Transient transfection of deletion and mutation constructs shows that all three sites contribute to the basal expression of MMTV in mammary cells. One of the binding activities (footprint I) is restricted to mammary cells, whereas the other two sites bind factors found in both mammary and nonmammary cells. The multimerized mammary-specific enhancer of MMTV on its own can enhance a minimal promoter in a mammary-specific fashion. However, the FpI binding site alone cannot mediate this effect. Thus, it is the binding of multiple factors in a combinatorial fashion that mediates the mammary-restricted expression of MMTV. (+info)
The transcription factor nuclear factor I mediates repression of the GLUT4 promoter by insulin.
(29/2395)
Insulin represses GLUT4 expression in 3T3-L1 adipocytes through an insulin response element located at bases -706 to -676 in the 5'-flanking sequence. Nuclear proteins related to the nuclear factor I (NF1) family of transcription factors bind to this insulin response element. Mutations that disrupt binding of NF1 proteins to the insulin response element impair the insulin response in reporter gene assays. Insulin treatment of 3T3-L1 adipocytes induces a rapid change in the level of phosphorylation of NF1 proteins, providing a potential mechanism for insulin's ability to regulate gene expression through NF1. Another as yet unidentified protein, not related to NF1, also binds to the GLUT4 insulin response element and is able to mediate partial repression of the GLUT4 promoter in reporter gene assays. (+info)
CCAAT/enhancer binding protein epsilon is critical for effective neutrophil-mediated response to inflammatory challenge.
(30/2395)
Targeted mutation of CCAAT/enhancer binding protein (C/EBP) epsilon in mice results in early death, primarily due to spontaneous infection with Pseudomonas aeruginosa. Functional analysis of C/EBPepsilon-deficient neutrophils, in an in vivo model of peritoneal inflammation, shows multiple defects. Reduction of phagocytotic killing by C/EBPepsilon-deficient neutrophils is a result of decreased uptake of opsonized bacteria as well as little to no expression of secondary granule proteins. Abnormalities in neutrophil migration detected in a chemical peritonitis model are likely secondary to abnormal CD11b integrin and L-selectin expression on C/EBPepsilon-deficient neutrophils. Alterations in neutrophil cytokine expression in response to inflammation show decreased levels of interleukin-1 receptor antagonist (IL-1Ra) and increased levels of tumor necrosis factor-alpha (TNF-alpha) expression by C/EBPepsilon-deficient neutrophils. Additionally, TNF-alpha expression is increased in nonactivated, circulating C/EBPepsilon-deficient neutrophils. Overall, C/EBPepsilon-deficient neutrophils are severely functionally impaired, evoking an abnormal microenvironment, which may contribute to the loss of normal responses to inflammatory stimuli. Similarities between the C/EBPepsilon-deficient mouse model and the human disease, specific granule deficiency, will be discussed. (+info)
Amino acid limitation regulates CHOP expression through a specific pathway independent of the unfolded protein response.
(31/2395)
The gene encoding CHOP (C/EBP-homologous protein) is transcriptionally activated by many stimuli and by amino acid deprivation. CHOP induction was considered to be due to an accumulation of unfolded protein into the ER (unfolded protein response (UPR)). We investigate the role of the UPR in the induction of CHOP by amino acid deprivation and show that this induction is not correlated with BiP expression (an UPR marker). Moreover, amino acid deprivation and UPR inducers regulate the CHOP promoter activity using distinct cis elements. We conclude that amino acid deprivation does not activate the UPR and regulates CHOP expression through a pathway that is independent of the UPR. (+info)
Induction of a secreted protein by the myxoid liposarcoma oncogene.
(32/2395)
The TLS-CHOP oncoprotein, found in the majority of human myxoid liposarcomas, consists of a fusion between the transcription factor CHOP/GADD153 and the N terminus of an RNA-binding protein TLS/FUS. Clinical correlation and in vitro transformation assays indicate that the N terminus of TLS plays an important role in oncogenesis by TLS-CHOP. Until now, however, the only activity attributed to the oncoprotein is that of inhibiting the binding of transcription factors of the C/EBP class to certain adipogenic target genes, a function that TLS-CHOP shares with the nononcogenic CHOP protein. Here we report the isolation of a gene, DOL54, that is activated in primary fibroblasts by the expression of TLS-CHOP. DOL54 is expressed in the neoplastic component of human myxoid liposarcomas and increases the tumorigenicity of cells injected in nude mice. Activation of DOL54 requires an intact DNA-binding and dimerization domain in TLS-CHOP, a suitable cellular dimerization partner, and depends on the TLS N terminus. Normal adipocytic differentiation is associated with an early and transient expression of DOL54, and the gene encodes a secreted protein that is tightly associated with the cell surface or extracellular matrix. TLS-CHOP thus leads to the unscheduled expression of a gene that is normally associated with adipocytic differentiation. (+info)