A lipopolysaccharide-induced DNA-binding protein for a class II gene in B cells is distinct from NF-kappa B.

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant decrease in mRNA encoding the Ia proteins which persists for at least 18 h. Within the upstream regulatory region of A alpha k, an NF-kappa B-like binding site is present. We have identified an LPS-induced DNA-binding protein in extracts from athymic mice whose spleens consist predominantly of B cells. Binding activity is present in low levels in unstimulated spleen cells and is increased by LPS treatment. This protein binds to two sites in a regulatory region of the Ia A alpha k gene, one of which contains the NF-kappa B-like binding site. DNA fragments containing these sites cross-compete for protein binding. Analysis by DNase I footprinting identified a target binding sequence, named the LPS-responsive element. Although this target sequence contains an NF-kappa B-like binding site, competition with a mutant oligonucleotide demonstrated that bases critical for NF-kappa B binding are not required for binding of the LPS-inducible protein. Therefore, we hypothesized that this inducible protein represents a new mediator of LPS action, distinct from NF-kappa B, and may be one mechanism to account for the decrease in mRNA encoding the Ia proteins.     

Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.     

Identification of a negative element in the human vimentin promoter: modulation by the human T-cell leukemia virus type I Tax protein.

The vimentin gene is a member of the intermediate filament multigene family and encodes a protein expressed, in vivo, in all mesenchymal derivatives and, in vitro, in cell types of various origin. We have previously demonstrated that the expression of this growth-regulated gene could be trans activated by the 40-kDa Tax protein of HTLV-I (human T-cell leukemia virus type I) and that responsiveness to this viral protein was mediated by the presence of an NF-kappa B binding site located between -241 and -210 bp upstream of the mRNA cap site (A. Lilienbaum, M. Duc Dodon, C. Alexandre, L. Gazzolo, and D. Paulin, J. Virol. 64:256-263, 1990). These previous assays, performed with deletion mutants of the vimentin promoter linked to the chloramphenicol acetyltransferase gene, also revealed the presence of an upstream negative region between -529 and -241 bp. Interestingly, the inhibitory activity exerted by this negative region was overcome after cotransfection of a Tax-expressing plasmid. In this study, we further characterize the vimentin negative element and define the effect of the Tax protein on the inhibitory activity of this element. We first demonstrate that a 187-bp domain (-424 to -237 bp) behaves as a negative region when placed upstream either of the NF-kappa B binding site of vimentin or of a heterologous enhancer such as that present in the desmin gene promoter. The negative effect can be further assigned to a 32-bp element which is indeed shown to repress the basal or induced activity of the NF-kappa B binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of NF-kappa B complexes in Theileria parva-transformedT cells

Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells.     

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt''s lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.     

Induction of NF-kappa B DNA-binding activity during the G0-to-G1 transition in mouse fibroblasts.

A DNA-binding factor with properties of NF-kappa B and another similar activity are rapidly induced when growth-arrested BALB/c 3T3 cells are stimulated with serum growth factors. Induction of these DNA-binding activities is not inhibited by pretreatment of quiescent cells with the protein synthesis inhibitor cycloheximide. Interestingly, the major NF-kappa B-like activity is not detected in nuclear extracts of proliferating cells, and thus its expression appears to be limited to the G0-to-G1 transition in 3T3 cells. These DNA-binding activities bind many of the expected NF-kappa B target sequences, including elements in the class I major histocompatibility complex and human immunodeficiency virus enhancers, as well as a recently identified NF-kappa B binding site upstream of the c-myc gene. Furthermore, both the class I major histocompatibility complex and c-myc NF-kappa B binding sites confer inducibility on a minimal promoter in 3T3 cells stimulated with serum growth factors. The results demonstrate that NF-kappa B-like activities are immediate-early response proteins in 3T3 cells and suggest a role for these factors in the G0-to-G1 transition.     

Two distinct NF-kappa B complexes differing in their larger subunit bind the E-selectin promoter kappa B element.

We have investigated the proteins binding the E-selectin promoter NF-kappa B element in its natural DNA context, using probes extending beyond the NF-kappa B recognition decamer. In band shift assays, we detected two distinct NF-kappa B complexes using nuclear extracts from several cytokine-induced cells. Subunit-specific antisera as blockers of complex formation plus DNA-protein cross-linking experiments revealed the faster migrating form to contain the NF-kappa B p50 plus p65 subunits. In contrast, the slower migrating form is composed of p50 plus the p65-related p75 protein. We show as the crucial determinant in generation of the larger complex the presence of more than five basepairs extra DNA sequence downstream of the NF-kappa B-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappa B binding site is. This may be explained by a requirement for activated p50 as part of this complex. The potential for a regulatory role for the p75 containing complex on the E-selectin promoter is discussed.     

Activation of the human immunodeficiency virus long terminal repeat by herpes simplex virus type 1 is associated with induction of a nuclear factor that binds to the NF-kappa B/core enhancer sequence.

It has been previously shown that herpes simplex virus type 1 (HSV-1) infection of HeLa cells results in augmentation of gene expression directed by the human immunodeficiency virus (HIV) long terminal repeat (LTR). This effect is presumably mediated by protein interactions with the LTR. We have used two different assays of DNA-protein interactions to study the HSV-induced activation of the HIV LTR. Activation of the HIV LTR is associated with increased protein binding to LTR sequences in a region including the NF-kappa B/core enhancer and the Sp1 binding sequences as monitored by an exonuclease protection assay. Gel retardation assays demonstrated that HSV-1 infection resulted in the induction of a nuclear factor(s) that binds to the NF-kappa B/core enhancer sequence. In addition to the activation of the HIV LTR, HSV induction of NF-kappa B activity may be important for the regulation of HSV gene expression during a herpesvirus infection.