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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.6477 ◽

1996 ◽

Vol 16 (11) ◽

pp. 6477-6485 ◽

Cited By ~ 12

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.

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Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2166 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2166-2172 ◽

Cited By ~ 70

Author(s):

E Jaffray ◽

K M Wood ◽

R T Hay

Keyword(s):

Dna Binding ◽

Binding Activity ◽

Ankyrin Repeat ◽

Inhibitor Protein ◽

Nf Kappa B ◽

I Kappa B Alpha ◽

Terminal Domain ◽

Dna Binding Activity ◽

Protease Cleavage ◽

Flexible Region

The DNA-binding activity and cellular distribution of the transcription factor NF-kappa B are regulated by the inhibitor protein I kappa B alpha. I kappa B alpha belongs to a family of proteins that contain multiple repeats of a 30- to 35-amino-acid sequence that was initially recognized in the erythrocyte protein ankyrin. Partial proteolysis has been used to study the domain structure of I kappa B alpha and to determine the sites at which it interacts with NF-kappa B. The data reveal a tripartite structure for I kappa B alpha in which a central, protease-resistant domain composed of five ankyrin repeats is flanked by an unstructured N-terminal extension and a compact, highly acidic C-terminal domain that is connected to the core of the protein by a flexible linker. Functional analysis of V8 cleavage products indicates that I kappa B alpha molecules lacking the N-terminal region can interact with and inhibit the DNA-binding activity of the p65 subunit of NF-kappa B, whereas I kappa B alpha molecules which lack both the N- and C-terminal regions are incapable of doing so. Protease cleavage of the N terminus of I kappa B alpha was unaffected by the presence of the p65 subunit of NF-kappa B, whereas bound p65 blocked cleavage of the flexible linker connecting the C-terminal domain to the ankyrin repeat-containing core of the protein. This linker region is highly conserved within the human, rat, pig, and chicken homologs of I kappa B alpha, and while it has been suggested that it represents a sixth ankyrin repeat, it is also likely that this is a flexible region of the protein that interacts with NF-kappa B.

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Calorie restriction attenuates inflammatory responses to myocardial ischemia-reperfusion injury

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.5.h2094 ◽

2001 ◽

Vol 280 (5) ◽

pp. H2094-H2102 ◽

Cited By ~ 62

Author(s):

B. Chandrasekar ◽

J. F. Nelson ◽

J. T. Colston ◽

G. L. Freeman

Keyword(s):

Oxidative Stress ◽

Dna Binding ◽

Antioxidant Enzyme ◽

Calorie Restriction ◽

Inflammatory Responses ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Binding Activity ◽

Dna Binding Activity ◽

Myocardial Oxidative Stress

The life-prolonging effects of calorie restriction (CR) may be due to reduced damage from cumulative oxidative stress. Our goal was to determine the long-term effects of moderate dietary CR on the myocardial response to reperfusion after a single episode of sublethal ischemia. Male Fisher 344 rats were fed either an ad libitum (AL) or CR (40% less calories) diet. At age 12 mo the animals were anaesthetized and subjected to thoracotomy and a 15-min left-anterior descending coronary artery occlusion. The hearts were reperfused for various periods. GSH and GSSG levels, nuclear factor-κB (NF-κB) DNA binding activity, cytokine, and antioxidant enzyme expression were assessed in the ischemic zones. Sham-operated animals served as controls. Compared with the AL diet, chronic CR limited oxidative stress as seen by rapid recovery in GSH levels in previously ischemic myocardium. CR reduced DNA binding activity of NF-κB. The κB-responsive cytokines interleukin-1β and tumor necrosis factor-α were transiently expressed in the CR group but persisted longer in the AL group. Furthermore, expression of manganese superoxide dismutase, a key antioxidant enzyme, was significantly delayed in the AL group. Collectively these data indicate that CR significantly attenuates myocardial oxidative stress and the postischemic inflammatory response.

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

Biochemical Journal ◽

10.1042/bj3120833 ◽

1995 ◽

Vol 312 (3) ◽

pp. 833-838 ◽

Cited By ~ 19

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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Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4609-4617.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4609-4617

Author(s):

C K Galang ◽

C A Hauser

Keyword(s):

Dna Binding ◽

Redox Regulation ◽

Mutational Analysis ◽

Cysteine Residue ◽

Binding Activity ◽

Nf Kappa B ◽

Amino Terminal ◽

Dna Binding Activity ◽

Specific Oxidation

The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.

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DNA-binding properties of the E1A-associated 300-kilodalton protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2826-2836.1992 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2826-2836

Author(s):

Y Rikitake ◽

E Moran

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Biochemical Properties ◽

Binding Activity ◽

Physical Interaction ◽

Nf Kappa B ◽

Biochemical Basis ◽

Dna Binding Activity ◽

P300 Binding ◽

Dna Binding Properties

One of the major E1A-associated cellular proteins is a 300-kDa product (p300) that binds to the N-terminal region of the E1A products. The p300 binding site is distinct from sequences involved in binding the retinoblastoma product and other E1A-associated cellular products such as p60-cyclin A and p107. p300 binding to E1A is linked genetically to the enhancer repression function of E1A and the other E1A-mediated gene-regulating functions as well as to the transforming functions of E1A. However, the biochemical properties of p300 have not yet been characterized. We report here that p300 has an intrinsic DNA-binding activity and shows a preferential affinity for specific DNA sequences. The sequences selectively bound by p300 are related to those of a series of enhancer elements that are recognized by NF-kappa B. The direct physical interaction of p300 with enhancer elements provides a biochemical basis for the genetic evidence linking the E1A-mediated enhancer repression function with the p300-binding activity of E1A.

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Constitutive nuclear NF-κB in cells of the monocyte lineage

Biochemical Journal ◽

10.1042/bj3040087 ◽

1994 ◽

Vol 304 (1) ◽

pp. 87-94 ◽

Cited By ~ 71

Author(s):

M Frankenberger ◽

A Pforte ◽

T Sternsdorf ◽

B Passlick ◽

P A Baeuerle ◽

...

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Binding Activity ◽

Pyrrolidine Dithiocarbamate ◽

Colony Stimulating Factor ◽

Nf Kappa B ◽

Dna Binding Activity ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

In monocytes, the nuclear factor NF-kappa B has been invoked as an important transcription factor in the expression of cytokine genes, of cell-surface receptors and in the expression of human immunodeficiency virus. In such cells, DNA binding activity of NF-kappa B can be detected without intentional stimulation. In our studies, cells of the human monocytic line Mono Mac 6, cultured in medium containing fetal-calf serum and low levels of lipopolysaccharide (LPS), also exhibit such ‘constitutive’ NF-kappa B, as demonstrated by mobility-shift analysis of nuclear extracts. This nuclear NF-kappa B was still present when contaminant LPS was removed by ultrafiltration and when serum was omitted. Protein-DNA complexes of constitutive NF-kappa B are similar in mobility to the LPS-induced NF-kappa B and both are recognized by an antibody specific to the p50 subunit of NF-kappa B. By contrast, treatment of cells with pyrrolidine dithiocarbamate (PDTC) will only block LPS-induced NF-kappa B, but not the constitutive binding protein. Using LPS-free and serum-free conditions, constitutive NF-kappa B can be detected in different cell lines of the monocytic lineage (HL60, U937, THP-1, Mono Mac 1 and Mono Mac 6), but not in Molt 4 T cells or K562 stem cells. When ordered according to stage of maturation, the amount of constitutive NF-kappa B was not increased in more mature cell lines. Furthermore, when inducing differentiation in Mono Mac 6 cells, with vitamin D3, no change in constitutive or inducible NF-kappa B can be detected. Analysis of primary cells revealed substantial constitutive NF-kappa B-binding activity in blood monocytes, pleural macrophages and alveolar macrophages. The constitutive NF-kappa B appears to be functionally active, since a low level of tumour necrosis factor (TNF) transcript is detectable in monocytes, and this level can be increased by blocking transcript degradation using cycloheximide. The level of constitutive NF-kappa B in these cells is variable and is frequently found to be lower in the more mature macrophages. Constitutive NF-kappa B was not maintained by autocrine action of cytokines TNF, interleukin 6, interleukin 10, granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor, since neutralizing antibodies did not reduce constitutive DNA-binding activity. Furthermore, blockade of prostaglandin or leukotriene biosynthesis did not affect constitutive NF-kappa B.(ABSTRACT TRUNCATED AT 400 WORDS)

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NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6089 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6089-6101 ◽

Cited By ~ 64

Author(s):

R I Scheinman ◽

A A Beg ◽

A S Baldwin

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Phorbol Esters ◽

Interleukin 1 ◽

Binding Activity ◽

Proteolytic Processing ◽

Cellular Growth ◽

Nf Kappa B ◽

Dna Binding Activity ◽

Multiple Copies

NF-kappa B is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappa B activity is induced by numerous stimuli, such as phorbol esters, B- and T-cell mitogens, the cytokines tumor necrosis factor and interleukin-1, and serum growth factors. The standard model for the induction of NF-kappa B activity involves the release of the transcription factor from a cytoplasmic inhibitor termed I kappa B, allowing translocation of NF-kappa B to the nucleus. I kappa B contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappa B and related binding activities are members of the Rel multigene family. Two such subunits, p50 and p52 (also called p50B), are proteolytically processed from precursors of 105 kDa (also called p105 and NFKB1) and 100 kDa (also called p100, NFKB2, and Lyt-10), respectively. Both contain N-terminal Rel-homologous domains as well as multiple copies of C-terminal ankyrin repeats. We show here that NF-kappa B p100 is a component of the previously identified DNA-binding activity H2TF1. In addition, we show that p100 is localized in the cytoplasm in HeLa cells, where it is associated with c-Rel, p50, or p65 (RelA). In transient-transfection assays, p100 represses the ability of NF-kappa B p65 to activate a kappa B-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappa B probe, as measured by an electrophoretic mobility shift assay, and a loss of nuclear p65 immunoreactivity, as measured by immunoblotting. This loss of nuclear p65 is paralleled by a gain of p65 DNA-binding activity and immunoreactivity in the cytoplasm. We interpret these data as demonstrating that p100 functions as an I kappa B-like molecule to sequester Rel family members in the cytoplasm. Proteolytic processing of p100 to the activator p52 is predicted to generate several new forms of Rel family heterodimers and therefore represents a form of regulation of NF-kappa B activity distinct from the classic I kappa B pathway.

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Basal phosphorylation of the PEST domain in the I(kappa)B(beta) regulates its functional interaction with the c-rel proto-oncogene product.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.5974 ◽

1996 ◽

Vol 16 (11) ◽

pp. 5974-5984 ◽

Cited By ~ 58

Author(s):

Z L Chu ◽

T A McKinsey ◽

L Liu ◽

X Qi ◽

D W Ballard

Keyword(s):

Dna Binding ◽

Mammalian Cells ◽

Peptide Mapping ◽

Binding Activity ◽

Phosphoryl Group ◽

I Kappa B Alpha ◽

Dna Binding Activity ◽

Antigenic Properties

The product of the c-rel proto-oncogene (c-Rel) belongs to the NF-kappaB/Rel family of polypeptides and has been implicated in the transcriptional control of cell proliferation and immune function. In human T lymphocytes, c-Rel is sequestered in the cytoplasmic compartment by constitutively phosphorylated inhibitors, including I(kappa)B(alpha) and I(kappa)B(beta). Studies with bacterially expressed forms of these inhibitory proteins revealed that unphosphorylated I(kappa)B(alpha) but not I(kappa)B(beta) assembles with c-Rel and inhibits its DNA binding activity. Furthermore, latent I(kappa)B(beta)-c-Rel complexes derived from mammalian cells were sensitive to phosphatase treatment, whereas I(kappa)B(alpha)-c-Rel complexes were resistant. We have identified a constitutive protein kinase in unstimulated T cells that associates with and phosphorylates I(kappa)B(beta) in vitro. The substrate specificity, electrophoretic mobility, and antigenic properties of this I(kappa)B(beta)-associated kinase (BAK) suggest identity with casein kinase II (CKII), an enzyme known to mediate basal phosphorylation of I(kappa)B(alpha). Phosphorylation of recombinant I(kappa)B(beta) by either BAK or CKII restored the capacity of this inhibitor to antagonize the DNA binding activity of c-Rel. Peptide mapping and mutational analyses localized the bulk of the basal phosphorylation sites in I(kappa)B(beta) to the C-terminal PEST domain, which contains two potential acceptors for CKII-mediated phosphoryl group transfer (Ser-313 and Ser-315). Point mutations introduced into the full-length inhibitor at Ser-313 and Ser-315 led to a significant reduction in the phosphorylation of I(kappa)B(beta) and severely impaired its c-Rel inhibitory function in vivo. Taken together, these findings strongly suggest that basal phosphorylation of the PEST domain of I(kappa)B(beta) at consensus CKII sites is required for the efficient formation of latent I(kappa)B(beta)-c-Rel complexes.

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