scholarly journals In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.

1995 ◽  
Vol 15 (3) ◽  
pp. 1294-1301 ◽  
Author(s):  
I Alkalay ◽  
A Yaron ◽  
A Hatzubai ◽  
S Jung ◽  
A Avraham ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
Extended Period ◽  
Activation Process ◽  
Nf Kappa B ◽  
Inflammatory Reactions ◽  
I Kappa B Alpha ◽  
Cytoplasmic Complex ◽  
Stimulation Of

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

scholarly journals Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation.

1993 ◽  
Vol 13 (6) ◽  
pp. 3301-3310 ◽  
Author(s):  
A A Beg ◽  
T S Finco ◽  
P V Nantermet ◽  
A S Baldwin
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Interleukin 1 ◽  
Activation Process ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Subsequent Loss ◽  
Autoregulatory Mechanism ◽  
Necrosis Factor

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.

scholarly journals Type I Interferon-mediated Stimulation of T Cells by CpG DNA

1998 ◽  
Vol 188 (12) ◽  
pp. 2335-2342 ◽  
Author(s):  
Siquan Sun ◽  
Xiaohong Zhang ◽  
David F. Tough ◽  
Jonathan Sprent
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Type I Interferons ◽  
Type I ◽  
Cpg Dna ◽  
Stimulation Of

Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs). We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro. Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs. Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I). First, T cell activation by CpG DNA was undetectable in IFN-IR−/− mice. Second, in contrast to normal T cells, the failure of purified IFN-IR−/− T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR+ APCs. Third, IFN-I (but not IFN-γ) caused the same pattern of partial T cell activation as CpG DNA. Significantly, T cell activation by IFN-I was APC independent. Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR. Functional studies suggested that activation of T cells by IFN-I was inhibitory. Thus, exposing normal (but not IFN-IR−/−) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro.

scholarly journals Inducible degradation of I kappa B alpha in vitro and in vivo requires the acidic C-terminal domain of the protein.

1995 ◽  
Vol 15 (5) ◽  
pp. 2413-2419 ◽  
Author(s):  
M S Rodriguez ◽  
I Michalopoulos ◽  
F Arenzana-Seisdedos ◽  
R T Hay
Keyword(s):  
Amino Acids ◽  
Proteolytic Activity ◽  
Cell Activation ◽  
Nf Kappa B ◽  
Free System ◽  
In Vitro System ◽  
I Kappa B Alpha ◽  
Terminal Domain

After exposure of cells to tumor necrosis factor (TNF), I kappa B alpha is rapidly degraded by a proteolytic activity that is required for nuclear localization and activation of transcription factor NF-kappa B. To investigate this problem, we have developed a cell-free system to study the degradation of I kappa B alpha initiated in vivo. In this in vitro system, characteristics of endogenous I kappa B alpha degradation were comparable to those observed in vivo. Recombinant I kappa B alpha, when added to lysates from cells exposed to TNF, was specifically degraded by a cellular proteolytic activity; however, it was stable in extracts from unstimulated cells. Inhibition characteristics of the proteolytic activity responsible for I kappa B alpha degradation suggest the involvement of a serine protease. Analysis of mutated forms of I kappa B alpha in the in vitro system demonstrated that an I kappa B alpha species which was unable to interact with NF-kappa B was still efficiently degraded. In contrast, deletion of the C-terminal 61 amino acids from I kappa B alpha rendered the protein resistant to proteolytic degradation. Expression of I kappa B alpha mutated forms in COS-7 cells confirmed the importance of the C-terminal domain for the degradation of the protein in vivo following cell activation. Thus, it is likely that the acidic, negatively charged region represented by the C-terminal 61 amino acids of the protein contains residues critical for TNF-inducible degradation of I kappa B alpha.

scholarly journals Inhibition of activation of transcription factor AP-1 by CD28 signalling in human T-cells

1994 ◽  
Vol 302 (1) ◽  
pp. 119-123 ◽  
Author(s):  
M Los ◽  
W Dröge ◽  
K Schulze-Osthoff
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Dna Binding ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Regulatory Region ◽  
Nf Kappa B ◽  
Stimulation Of

Co-stimulation of T-lymphocytes by T-cell receptor (TcR) occupancy and activation of the CD28 surface molecule results in enhanced proliferation and interleukin 2 (IL-2) production. The increase in IL-2 gene expression triggered by CD28 involves a kappa B-like sequence in the 5′-regulatory region of the IL-2 promoter, called CD28-responsive element. Stimulation of T-cells by agonistic anti-CD28 antibodies in conjunction with phorbol 12-myristate 13-acetate (PMA)- or TcR-derived signals induces the enhanced activation of the transcription factor NF-kappa B. Here we report that CD28 engagement, however, exerts opposite effects on the transcription factor AP-1. Whereas anti-CD28 together with PMA increased the DNA binding and trans-activation activity of NF-kappa B, PMA-induced activation of AP-1 was significantly suppressed. The inhibitory effect exerted by anti-CD28 was observed at the level of DNA binding as well as in functional reporter-gene assays. These results suggest that the two transcription factors are independently regulated and may perform different functions during T-cell activation.

scholarly journals CD4 T cells integrate signals delivered during successive DC encounters in vivo

2005 ◽  
Vol 202 (9) ◽  
pp. 1271-1278 ◽  
Author(s):  
Susanna Celli ◽  
Zacarias Garcia ◽  
Philippe Bousso
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Late Phase ◽  
Activation Process ◽  
Two Photon ◽  
History Of

The cellular mode of T cell priming in vivo remains to be characterized fully. We investigated the fate of T cell–dendritic cell (DC) interactions in the late phase of T cell activation in the lymph node. In general, CD4 T cells detach from DCs before undergoing cell division. Using a new approach to track the history of antigen (Ag)-recognition events, we demonstrated that activated/divided T cells reengage different DCs in an Ag-specific manner. Two-photon imaging of intact lymph nodes suggested that T cells could establish prolonged interactions with DCs at multiple stages during the activation process. Importantly, signals that are delivered during subsequent DC contacts are integrated by the T cell and promote sustained IL-2Rα expression and IFN-γ production. Thus, repeated encounters with Ag-bearing DCs can occur in vivo and modulate CD4 T cell differentiation programs.

scholarly journals RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter.

1995 ◽  
Vol 15 (8) ◽  
pp. 4260-4271 ◽  
Author(s):  
J H Lai ◽  
G Horvath ◽  
J Subleski ◽  
J Bruder ◽  
P Ghosh ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Specific Antigen ◽  
Transcriptional Activator ◽  
Nf Kappa B ◽  
Enhancer Activity ◽  
Response Element

T-cell activation requires two different signals. The T-cell receptor's recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors.

scholarly journals Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation

1993 ◽  
Vol 13 (6) ◽  
pp. 3301-3310 ◽  
Author(s):  
A A Beg ◽  
T S Finco ◽  
P V Nantermet ◽  
A S Baldwin
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Interleukin 1 ◽  
Activation Process ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Subsequent Loss ◽  
Autoregulatory Mechanism ◽  
Necrosis Factor

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.

scholarly journals Overexpression of RelA in transgenic mouse thymocytes: specific increase in levels of the inhibitor protein I kappa B alpha.

1995 ◽  
Vol 15 (7) ◽  
pp. 3523-3530 ◽  
Author(s):  
P Perez ◽  
S A Lira ◽  
R Bravo
Keyword(s):  
Transgenic Mice ◽  
Binding Activity ◽  
Mrna Levels ◽  
Inhibitor Protein ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Specific Increase ◽  
Stimulation Of

RelA (p65) is one of the strongest activators of the Rel/NF-kappa B family. As a first step to elucidate the mechanisms that regulate its activity in vivo, we have generated transgenic mice overexpressing RelA in the thymus. Although the levels of RelA were significantly increased in thymocytes of transgenic mice, the overall NF-kappa B-binding activity in unstimulated cells was not augmented compared with that in control thymocytes. This could be explained by the dramatic increase of endogenous I kappa B alpha levels observed in RelA-overexpressing cells in both cytoplasmic and nuclear compartments. The ikba mRNA levels were not augmented by overexpressed RelA, but I kappa B alpha inhibitor was found to be stabilized through association with RelA. Although a fraction of RelA was associated with cytoplasmic p105, no changes in the precursor levels were observed. Upon stimulation of RelA-overexpressing thymocytes with phorbol 12-myristate 13-acetate and lectin (phytohemaglutinin), different kappa B-binding complexes, including RelA homodimers, were partially released from I kappa B alpha. Association of RelA with I kappa B alpha prevented complete degradation of the inhibitor. No effect of phorbol 12-myristate 13-acetate-lectin treatment was detected on RelA associated with p105. Our data indicate that cytoplasmic retention of overexpressed RelA by I kappa B alpha is the major in vivo mechanism controlling the potential excess of NF-kappa B activity in long-term RelA-overexpressing thymocytes.

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