IFN-γ + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38 mapk in a mouse macrophage cell line

2001 ◽  
Vol 280 (3) ◽  
pp. C441-C450 ◽  
Author(s):  
Edward D. Chan ◽  
David W. H. Riches
Keyword(s):  
Nitric Oxide ◽  
Cell Line ◽  
Inhibitory Effect ◽  
Interferon Γ ◽  
Dominant Negative ◽  
Jnk Pathway ◽  
Mutant Proteins ◽  
Inos Promoter ◽  
Ifn Γ ◽  
Inos Induction

Nitric oxide (NO·) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO· by interferon-γ (IFN-γ) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-γ in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-γ alone with respect to iNOS induction. In this RAW 264.7γNO(−) subclone, IFN-γ and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38 mapk inhibited IFN-γ + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-γ + LPS, also implicating the c-Jun NH2-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-γ + LPS-induced tumor necrosis factor-α protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38 mapk appears more complex and may be due to the ability of p38 mapk to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO· expression by IFN-γ + LPS.

scholarly journals Acquired interferon γ responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression

Gut ◽  
1999 ◽  
Vol 44 (5) ◽  
pp. 659-665 ◽  
Author(s):  
A M Chavez ◽  
M J Morin ◽  
N Unno ◽  
M P Fink ◽  
R A Hodin
Keyword(s):  
Nitric Oxide ◽  
Cell Differentiation ◽  
Interferon Γ ◽  
Intestinal Barrier Function ◽  
Inos Mrna ◽  
Pyrrolidine Dithiocarbamate ◽  
No Production ◽  
Mrna Induction ◽  
Ifn Γ ◽  
Inos Induction

BACKGROUNDImpairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon γ (IFN-γ) induced nitric oxide (NO) production. Previous in vivo studies have shown that systemic lipopolysaccharide treatment caused an induction of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in villus cells, rather than in undifferentiated crypt cells.AIMSTo examine iNOS induction by IFN-γ in vitro as a function of enterocyte differentiation.METHODSPreconfluent and postconfluent Caco-2 cells were treated with IFN-γ in the presence or absence of various inhibitors. Northern analyses were performed to assess the magnitude of iNOS mRNA induction. IFN-γ receptor mRNA and protein levels were determined.RESULTSiNOS mRNA induction by IFN-γ occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response. iNOS induction and nitrite/nitrate increases were inhibited by dexamethasone and pyrrolidine dithiocarbamate, supporting an important role for the NF-κB transcription factor in this process. The stimulated iNOS induction was seen almost exclusively under conditions of cellular differentiation—that is, in postconfluent Caco-2 cells. This increased IFN-γ responsiveness seen in postconfluent Caco-2 cells correlated with an increased expression of IFN-γ receptor, whereas T84 and HT-29 cells did not show any significant alterations in either iNOS induction or IFN-γ receptor levels as a function of postconfluent growth.CONCLUSIONSWith regard to iNOS mRNA induction, IFN-γ responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increase in the numbers of IFN-γ receptors.

scholarly journals Inducible Nitric Oxide Synthase Can Be Induced in the Absence of Active Nuclear Factor-κB in Rat Mesangial Cells

1999 ◽  
Vol 10 (4) ◽  
pp. 721-729 ◽  
Author(s):  
OSAMU NAKASHIMA ◽  
YOSHIO TERADA ◽  
SEIJI INOSHITA ◽  
MICHIO KUWAHARA ◽  
SEI SASAKI ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mesangial Cells ◽  
Nuclear Factor Κb ◽  
Nitrite Production ◽  
Tnf Α ◽  
Inos Promoter ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide ◽  
Inos Induction

Abstract. The contribution of nuclear factor-κB (NF-κB) and interferon-γ (IFN-γ) signaling to nitric oxide generation is not completely understood. The effect of NF-κB release and its inhibition on nitrite production and the involvement of Janus kinase 2 (JAK2) in inducible nitric oxide synthase (iNOS) induction were investigated. The following assays were performed. (1) Nitrite produced by rat mesangial cells in primary culture was measured in incubations with tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), with or without IFN-γ. Cells were stimulated with TNF-α or LPS plus IFN-γ in the presence of NF-κB inhibitors, herbimycin A (HerA), or the more specific JAK2 inhibitor AG490. (2) Immunoblotting was performed against the p65 and p50 subunits of NF-κB and iNOS. (3) Electrophoretic mobility shift assays were performed against NF-κB in the presence of NF-κB inhibitors or AG490. (4) iNOS promoter activity was measured in the presence of AG490 or JAK2 antisense oligonucleotides. TNF-α or LPS alone did not induce nitrite production, but with IFN-γ these compounds did induce nitrite production. Pyrrolidine dithiocarbamate (PDTC),N-acetyl-L-cysteine, dexamethasone (Dex), HerA, and AG490 partially inhibited LPS/IFN-γ- or TNF-α/IFN-γ-induced nitrite production. p65 was inhibited by the three NF-κB inhibitors described above, whereas p50 was not. PDTC and Dex completely inhibited the p65/p50 heterodimer, but HerA and AG490 had little effect on p65/p50. AG490 and JAK2 antisense oligonucleotides suppressed iNOS promoter activity. It can be concluded that (1) iNOS can be induced without active NF-κB; (2) Dex, acetylsalicylic acid, and PDTC inhibit only p65; and (3) JAK2 is involved in iNOS induction, and the contribution of JAK2 to nitrite production is greater than that of NF-κB.

scholarly journals CCAAT/enhancer binding protein-βtrans-activates murine nitric oxide synthase 2 gene in an MTAL cell line

1999 ◽  
Vol 276 (4) ◽  
pp. F599-F605 ◽  
Author(s):  
Ashish K. Gupta ◽  
Bruce C. Kone
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Cell Line ◽  
Cell Injury ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Thick Ascending Limb ◽  
Ifn Γ ◽  
Nitric Oxide Synthase 2 ◽  
Oxide Synthase

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 NOS2gene 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-γ (IFN-γ) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBPβ, but not C/EBPα, C/EBPδ, or C/EBPε. NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN-γ compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-γ activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBPβ stimulated basal NOS2 promoter activity. Thus C/EBPβ appears to be an important trans-activator of the NOS2 gene in the MTAL.

Prolonged survival of mice with glioma injected intracerebrally with double cytokine—secreting cells

1995 ◽  
Vol 83 (6) ◽  
pp. 1038-1044 ◽  
Author(s):  
Terry Lichtor ◽  
Roberta P. Glick ◽  
Tae Sung Kim ◽  
Roger Hand ◽  
Edward P. Cohen
Keyword(s):  
Cell Line ◽  
Interleukin 2 ◽  
Glioma Cells ◽  
Fibroblast Cell ◽  
Interferon Γ ◽  
Glioma Cell Line ◽  
Mouse Fibroblast ◽  
Spleen Cells ◽  
Content Type ◽  
Ifn Γ

✓ A novel approach toward the treatment of glioma was developed in a murine model. The genes for both interleukin-2 (IL-2) and interferon-γ (IFN-γ) were first transfected into a mouse fibroblast cell line that expresses defined major histocompatibility complex (MHC) determinants (H—2k). The double cytokine—secreting cells were then cotransplanted intracerebrally with the Gl261 murine glioma cell line into syngeneic C57BL/6 mice (H—2b) whose cells differed at the MHC from the cellular immunogen. The results indicate that the survival of mice with glioma injected with the cytokine-secreting allogeneic cells was significantly prolonged, relative to the survival of mice receiving equivalent numbers of glioma cells alone. Using a standard 51Cr-release assay, the specific release of isotope from labeled Gl261 cells coincubated with spleen cells from mice injected intracerebrally with the glioma cells and the cytokine-secreting fibroblasts was significantly higher than the release of isotope from glioma cells coincubated with spleen cells from nonimmunized mice. The cellular antiglioma response was mediated by natural killer/lymphokine-activated killer and Lyt-2.2+ (CD8+) cells. The increased survival of mice with glioma and the specific immunocytotoxic responses after immunization with fibroblasts modified to secrete both IL-2 and IFN-γ indicate the potential of an immunotherapeutic approach to gliomas with cytokine-secreting cells.

Nitric oxide activates PKCα and inhibits Na+-K+-ATPase in opossum kidney cells

1999 ◽  
Vol 277 (6) ◽  
pp. F859-F865 ◽  
Author(s):  
Mingyu Liang ◽  
Franklyn G. Knox
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Proximal Tubule ◽  
Cell Line ◽  
Soluble Guanylate Cyclase ◽  
Inhibitory Effect ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Opossum Kidney ◽  
Ok Cells

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCα from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCα in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCα in OK, but not in LLC-PK1, cells. The activation of PKCα in OK cells by NO is associated with inhibition of Na+-K+-ATPase.

scholarly journals Inactivation of the Fanconi Anemia Group C Gene Augments Interferon-γ–Induced Apoptotic Responses in Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 974-985 ◽  
Author(s):  
R. Keaney Rathbun ◽  
Gregory R. Faulkner ◽  
Marika H. Ostroski ◽  
Tracy A. Christianson ◽  
Grant Hughes ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Inhibitory Effect ◽  
Interferon Γ ◽  
Interferon Response ◽  
Low Doses ◽  
Order Of Magnitude ◽  
Ifn Γ ◽  
Fas Pathway ◽  
Anemia Group ◽  
Induced Apoptosis

Abstract Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus (FAC −/−) are hypersensitive to the mitotic inhibitory effects of interferon (IFN-γ). We tested the hypothesis that HPC from the bone marrow of Fanconi group C children are similarly hypersensitive and that the fas pathway is involved in affecting programmed cell death in response to low doses of IFN-γ. In normal human and murine HPC, IFN-γ primed the fas pathway and induced both fas and interferon response factor-1 (IRF-1) gene expression. These IFN-γ-induced apoptotic responses in HPC from the marrow of a child with FA of the C group (FA-C) and in FAC −/− mice occurred at significantly lower IFN doses (by an order of magnitude) than did the apoptotic responses of normal HPC. Treatment of FA-C CD34+ cells with low doses of recombinant IFN-γ, inhibited growth of colony forming unit granulocyte-macrophage and burst-forming unit erythroid, while treatment with blocking antibodies to fas augmented clonal growth and abrogated the clonal inhibitory effect of IFN-γ. Transfer of the normal FAC gene into FA-C B-cell lines prevented mitomycin C–induced apoptosis, but did not suppress fas expression or inhibit the primed fas pathway. However, the kinetics of Stat1-phosphate decay in IFN-γ–treated cells was prolonged in mutant cells and was normalized by transduction of the normal FAC gene. Therefore, the normal FAC protein serves, in part, to modulate IFN-γ signals. HPC bearing inactivating mutations of FAC fail to normally modulate IFN-γ signals and, as a result, undergo apoptosis executed through the fas pathway.

scholarly journals Bcl-2 and Bcl-XL Block Thapsigargin-Induced Nitric Oxide Generation, c-Jun NH2-Terminal Kinase Activity, and Apoptosis

1999 ◽  
Vol 19 (8) ◽  
pp. 5659-5674 ◽  
Author(s):  
Rakesh K. Srivastava ◽  
Steven J. Sollott ◽  
Leila Khan ◽  
Richard Hansford ◽  
Edward G. Lakatta ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Caspase 3 ◽  
No Synthase ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
No Production ◽  
Acetoxymethyl Ester ◽  
Jnk Pathway ◽  
Induced Apoptosis ◽  
Jnk Activation

ABSTRACT The proteins Bcl-2 and Bcl-XL prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-XL in the regulation of cytosolic Ca2+, nitric oxide production (NO), c-Jun NH2-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca2+ATPase, was used to disrupt Ca2+ homeostasis. TG acutely elevated intracellular free Ca2+ and mitochondrial Ca2+ levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca2+ response with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N G-nitro-l-arginine methyl ester hydrochloride (l-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca2+ level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-XL (JT/Bcl-2 or JT/Bcl-XL), NO production, late (36-h) Ca2+ accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-XL cells to the NO donor,S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by l-NAME. Transient expression of a dominant negative mutant SEK1 (Lys→Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-XL inhibited TG-induced loss in mitochondrial membrane potential, release of cytochromec, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca2+ release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca2+-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-XL cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-XL protect the cells against TG-induced apoptosis by negatively regulating Ca2+-sensitive NO synthase activity or expression.

scholarly journals Oxidized Lipoproteins Suppress Nitric Oxide Synthase in Macrophages: Study of Glucocorticoid Receptor Involvement

1994 ◽  
Vol 3 (5) ◽  
pp. 323-327 ◽  
Author(s):  
K. E. Matthys ◽  
P. G. Jorens ◽  
B. Marescau ◽  
M. Rosseneu ◽  
H. Bult ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Glucocorticoid Receptor ◽  
Suppressive Effect ◽  
Inhibitory Effect ◽  
Interferon Γ ◽  
Inos Activity ◽  
Atherosclerotic Lesions ◽  
Oxidized Lipoproteins ◽  
Oxide Synthase

Activated cholesterol-laden macrophages in atherosclerotic lesions are believed to influence the progression of this disease. The induction of nitric oxide synthase (iNOS) activity was investigated in control and cholesterol-laden J774 macrophages, obtained by pre-incubation with oxidized or acetylated low density lipoproteins (oxLDL, acLDL). Loading with oxLDL caused a small induction of NOS activity in unstimulated cells, as indicated by nitrite and citrulline accumulation in the supernatant. However, it suppressed the iNOS activity resulting from stimulation of the cells with lipopolysaccharide with or without interferon-γ. AcLDL had no inhibitory effect, indicating that cholesterol accumulation as such was not responsible. Since the induction of NOS in macrophages is inhibited by glucocorticoids, the possibility that a glucocorticoid-like factor, formed during oxidation of LDL, may cause the inhibition, was investigated. However, addition of the glucocorticoid receptor antagonist mifepristone did not prevent the oxLDL-dependent NOS inhibition, indicating that the glucocorticoid receptor is not involved in the suppressive effect of oxLDL.

Sign in / Sign up

Export Citation Format

Share Document