scholarly journals Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B.

1995 ◽  
Vol 15 (5) ◽  
pp. 2689-2696 ◽  
Author(s):  
F Arenzana-Seisdedos ◽  
J Thompson ◽  
M S Rodriguez ◽  
F Bachelerie ◽  
D Thomas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Regulatory Function ◽  
Primary Role ◽  
Nf Kappa B ◽  
Nuclear Expression ◽  
I Kappa B Alpha

The transcription factor NF-kappa B is exploited by many viruses, including the human immunodeficiency virus, for expression of viral genes, but its primary role appears to be in the rapid induction of cellular genes during immune and inflammatory responses. The inhibitor protein I kappa B alpha maintains NF-kappa B in an inactive form in the cytoplasms of unstimulated cells, but upon cell activation, I kappa B alpha is rapidly degraded, leading to nuclear translocation of free NF-kappa B. However, NF-kappa B-dependent transcription of the I kappa B alpha gene leads to rapid resynthesis of the I kappa B alpha protein and inhibition of NF-kappa B-dependent transcription. Here we demonstrate a new regulatory function of I kappa B alpha exerted on NF-kappa B in the nuclear compartment. Although normally found in the cytoplasm, I kappa B alpha, newly synthesized in response to tumor necrosis factor or interleukin I, is transported to the nucleus. In the nucleus I kappa B alpha associates with the p50 and p65 subunits of NF-kappa B, inhibiting DNA binding of the transcription factor. Furthermore, nuclear expression of I kappa B alpha correlates with transcription termination of transfected NF-kappa B-dependent luciferase genes. Following the appearance of I kappa B alpha in the nuclei of activated cells, a dramatic reduction in the amount of nuclear p50 occurs, suggesting that NF-kappa B-I kappa B alpha complexes are cleared from the nucleus.

scholarly journals Hyaluronan fragments activate an NF-kappa B/I-kappa B alpha autoregulatory loop in murine macrophages.

1996 ◽  
Vol 183 (5) ◽  
pp. 2373-2378 ◽  
Author(s):  
P W Noble ◽  
C M McKee ◽  
M Cowman ◽  
H S Shin
Keyword(s):  
Molecular Weight ◽  
Dna Binding ◽  
Inflammatory Response ◽  
Time Course ◽  
Inflammatory Responses ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Murine Macrophages ◽  
I Kappa B Alpha ◽  
Dna Binding Activity

Macrophages play an important role in the acute tissue inflammatory response through the release of cytokines and growth factors in response to stimuli such as lipopolysaccharide (LPS). Macrophage inflammatory effector functions are also influenced by interactions with the extracellular matrix (ECM). Such macrophage-ECM interactions may be important in regulating chronic inflammatory responses. Recent evidence has suggested that hyaluronan (HA), a glycosaminoglycan (GAG) component of ECM can induce inflammatory gene expression in murine macrophages. HA exists in its native form as a large polymer, but is found as smaller fragments under inflammatory conditions. The NF-kappa B/I-kappa B transcriptional regulatory system has been shown to be a critical component of the host inflammatory response. We examined the effects of high molecular weight HA and lower molecular weight HA fragments on NF-kappa B activation in mouse macrophages. Only the smaller HA fragments were found to activate NF-kappa B DNA binding activity. After HA stimulation, I-kappa B alpha mRNA was induced and I-kappa B alpha protein levels, which initially decreased, were restored. The induction of I-kappa Balpha expression was not observed for other GAGs. The time course of I-kappa B alpha protein regeneration in response to HA fragments was consistent with an autoregulatory mechanism. In support of this mechanism, in vitro translated murine I-kappa B alpha inhibited HA fragment-induced NF-kappa B DNA binding activity. The NF-kappa B DNA binding complex in HA-stimulated extracts was found to contain p50 and p65 subunits. Activation of the NF-kappa B/I-kappa B system in macrophages by ECM fragments may be an important mechanism for propagating the tissue inflammatory response.

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 Both amino- and carboxyl-terminal sequences within I kappa B alpha regulate its inducible degradation.

1996 ◽  
Vol 16 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
S Sun ◽  
J Elwood ◽  
W C Greene
Keyword(s):  
Transcription Factor ◽  
Nuclear Translocation ◽  
Terminal Region ◽  
Dominant Negative ◽  
Tnf Alpha ◽  
Human Immune Deficiency Virus ◽  
Nf Kappa B ◽  
Inhibitory Protein ◽  
I Kappa B Alpha ◽  
Transcription Factor Complex

Nuclear expression and consequent biological action of the eukaryotic NF-kappa B transcription factor complex are tightly regulated through its cytoplasmic retention by an ankyrin-rich inhibitory protein termed I kappa B alpha. I kappa B alpha specifically binds to and masks the nuclear localization signal of the RelA subunit of NF-kappa B, thereby effectively sequestering this transcription factor complex in the cytoplasm. Specific cellular activation signals lead to the rapid proteolytic degradation of I kappa B alpha and the concomitant nuclear translocation of NF-kappa B. However, the precise biochemical mechanisms underlying the inhibitory effects of I kappa B alpha on RelA and its inducible pattern of degradation remain unclear. By using HeLa cells transfected with various cDNAs end-coding epitope-tagged mutants of I kappa B alpha, our studies demonstrate the following: (i) sequences within the 72-amino-acid N-terminal region of I kappa B alpha are required for tumor necrosis factor alpha (TNF-alpha)-induced degradation but are fully dispensable for I kappa B alpha binding to and inhibition of RelA; (ii) serine residues located at positions 32 and 36 within the N-terminal region of I kappa B alpha represent major sites of induced phosphorylation (substitution of these serine residues with alanine abrogates TNF-alpha-induced degradation of I kappa B alpha); (iii) the C-terminal 40 residues of I kappa B alpha (amino acids 277 to 317), which include a PEST-like domain, are entirely dispensable for TNF-alpha-induced degradation and inhibition of RelA; (iv) a glutamine- and leucine-rich (QL) region of I kappa B alpha located between residues 263 and 277 and overlapping with the sixth ankyrin repeat is required for both inducible degradation and inhibition of RelA function; (v) regulation of I kappa B alpha degradation by this QL-rich region appears to occur independently of phosphorylation at serines 32 and 36. These findings thus indicate that I kappa B alpha is generally organized within distinct modular domains displaying different functional and regulatory properties. These studies have also led to the identification of a novel class of dominant-negative I kappa B alpha molecules that retain full inhibitory function on NF-kappa B yet fail to undergo stimulus-induced degradation. These molecules, which lack N-terminal sequences, potently inhibit TNF-alpha-induced activation of the human immune deficiency virus type 1 kappa B enhancer, thus indicating their possible use as general inhibitors of NF-kappa B.

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 Identification of a Portable Repression Domain and an E1A-Responsive Activation Domain in Pax4: a Possible Role of Pax4 as a Transcriptional Repressor in the Pancreas

1999 ◽  
Vol 19 (12) ◽  
pp. 8281-8291 ◽  
Author(s):  
Yoshio Fujitani ◽  
Yoshitaka Kajimoto ◽  
Tetsuyuki Yasuda ◽  
Taka-Aki Matsuoka ◽  
Hideaki Kaneto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Repressor ◽  
Pancreas Development ◽  
Primary Role ◽  
Transactivation Domain ◽  
Activation Domain ◽  
Pax Family ◽  
Repression Domain

ABSTRACT Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic β/δ-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic β- and α-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development.

scholarly journals Interaction of the v-Rel oncoprotein with NF-kappaB and IkappaB proteins: heterodimers of a transformation-defective v-Rel mutant and NF-2 are functional in vitro and in vivo.

1996 ◽  
Vol 16 (3) ◽  
pp. 1169-1178 ◽  
Author(s):  
D W White ◽  
G A Pitoc ◽  
T D Gilmore
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Cellular Protein ◽  
Lymphoid Cells ◽  
Spleen Cells ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Chicken Spleen

The v-Rel oncoprotein of the avian Rev-T retrovirus is a member of the Rel/NF-kappa B family of transcription factors. The mechanism by which v-Rel malignantly transforms chicken spleen cells is not precisely known. To gain a better understanding of functions needed for transformation by v-Rel, we have now characterized the activities of mutant v-Rel proteins that are defective for specific protein-protein interactions. Mutant v-delta NLS, which has a deletion of the primary v-Rel nuclear localizing sequence, does not interact efficiently with I kappa B-alpha but still transforms chicken spleen cells approximately as well as wild-type v-Rel, indicating that interaction with I kappa B-alpha is not essential for the v-Rel transforming function. A second v-Rel mutant, v-SPW, has been shown to be defective for the formation of homodimers, DNA binding, and transformation. However, we now find that v-SPW can form functional DNA-binding heterodimers in vitro and in vivo with the cellular protein NF-kappa B p-52. Most strikingly, coexpression of v-SPW and p52 from a retroviral vector can induce the malignant transformation of chicken spleen cells, whereas expression of either protein alone cannot. Our results are most consistent with a model wherein Rel homodimers or heterodimers must bind DNA and alter gene expression in order to transform lymphoid cells.

scholarly journals I(kappa)B(gamma) inhibits DNA binding of NF-kappaB p50 homodimers by interacting with residues that contact DNA.

1996 ◽  
Vol 16 (11) ◽  
pp. 6477-6485 ◽  
Author(s):  
S Bell ◽  
J R Matthews ◽  
E Jaffray ◽  
R T Hay
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Protein Footprinting ◽  
I Kappa B Alpha ◽  
Dna Binding Activity ◽  
Cellular Genes ◽  
Phosphate Backbone ◽  
Nf Kappab ◽  
Contact Dna

NF-(kappa)B is an inducible transcription factor that activates many cellular genes involved in stress and immune response and whose DNA binding activity and cellular distribution are regulated by I(kappa)B inhibitor proteins. The interaction between NF-(kappa)B p50 and DNA was investigated by protein footprinting using chemical modification and partial proteolysis. Both methods confirmed lysine-DNA contacts already found in the crystal structure (K-147, K-149, K-244, K-275, and K-278) but also revealed an additional contact in the lysine cluster K-77-K-78-K-80 which was made on an extended DNA. Molecular modelling of such a DNA-protein complex revealed that lysine 80 is ideally placed to make phosphate backbone contacts in the extended DNA. Thus, it seems likely that the entire AB loop, containing lysines 77, 78, and 80, forms a C-shaped clamp that closes around the DNA recognition site. The same protein footprinting approaches were used to probe the interaction of p50 with the ankyrin repeat containing proteins I(kappa)B(gamma) and I(kappa)B(alpha). Lysine residues in p50 that were protected from modification by DNA were also protected from modification by I(kappa)B(gamma) but not I(kappa)B(alpha). Similarly, proteolytic cleavage at p50 residues which contact DNA was inhibited by bound I(kappa)B(gamma) but was enhanced by the presence of I(kappa)B(alpha). Thus, I(kappa)B(gamma) inhibits the DNA binding activity of p50 by direct interactions with residues contacting DNA, whereas the same residues remain exposed in the presence of I(kappa)B(alpha), which binds to p50 but does not block DNA binding.

A prospective longitudinal study of cancer-related fatigue in patients undergoing breast-conserving surgery and radiation with or without chemotherapy for breast cancer.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 9122-9122
Author(s):  
Mylin Ann Torres ◽  
Thaddeus Pace ◽  
Jennifer Felger ◽  
Tian Liu ◽  
Karen D. Godette ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Binding ◽  
Inflammatory Mediators ◽  
Inflammatory Responses ◽  
Binding Motif ◽  
Nf Kappa B ◽  
Prospective Longitudinal Study ◽  
Cancer Related Fatigue ◽  
Whole Breast Radiotherapy ◽  
Prospective Longitudinal

9122 Background: We prospectively evaluated risk factors for persistent cancer-related fatigue in women with breast cancer undergoing lumpectomy with or without chemotherapy (CTX) prior to whole breast radiotherapy (XRT). We assessed the potential role of inflammatory mediators, demographic characteristics, and treatment history including CTX. Methods: Following lumpectomy, 60 women received a definitive course of whole breast XRT (50 Gy plus a 10 Gy boost). Prior to XRT, at week 6 of XRT, and 6 weeks post XRT, subjects completed the Multidimensional Fatigue Inventory (MFI) and underwent blood draws for inflammatory mediators (protein and mRNA). Results: Independent multivariate analyses of clinical and demographic factors revealed that CTX (p<.001) , given neoadjuvantly or adjuvantly, and age <50 (p=.03) were significant predictors of higher fatigue scores post XRT. Mean MFI scores in patients treated with CTX (n=24) were 20 points higher than patients not treated with CTX (p<.001) with a clinically meaningful difference in scores being 10 points on the MFI. Gene ontology analysis of differentially expressed genes indicated increased activation of genes involved in immune and inflammatory responses in fatigued vs. non-fatigued patients (p<.001). Of the inflammatory mediators, plasma IL-6 prior to XRT was the strongest predictor of post XRT fatigue (p=.02). Moreover, plasma IL-6 concentrations prior to XRT were significantly higher in patients who received CTX (mean 4.96 vs. 2.53, p=.01). Patients who received CTX also had significantly higher levels of NF Kappa B DNA binding 6 weeks post XRT (p<.001), and transcription factor binding analysis revealed a greater representation of genes with the NF Kappa B DNA binding motif in fatigued vs. non-fatigued patients (p =.05). Conclusions: Collectively, these data suggest an interaction between CTX and XRT leading to inflammation and fatigue several weeks post XRT. This relationship was independent of whether CTX was given pre or post-operatively. Treatments targeting inflammation before XRT may reduce fatigue post therapy, particularly in patients previously treated with CTX.

scholarly journals Constitutive nuclear NFκB/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate

1995 ◽  
Vol 312 (3) ◽  
pp. 833-838 ◽  
Author(s):  
A F G Slater ◽  
M Kimland ◽  
S A Jiang ◽  
S Orrenius
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Gradient Centrifugation ◽  
Binding Activity ◽  
Pyrrolidine Dithiocarbamate ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Apoptotic Activity

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.

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