Biomedical subjects

A B Agranoff

Publications and source records attributed to A B Agranoff.

6 recordsLinked to original sources

PubMed1995

Cytokine induction of nuclear factor kappa B in cycling and growth-arrested cells. Evidence for cell cycle-independent activation.

Nuclear factor kappa B (NF-kappa B) is a pleiotropic transcription factor which regulates the expression of a large number of cellular and viral genes. Induction of NF-kappa B has been shown previously to occur during cell cycle transition from G0 to G1, but the relationship of cytokine induction of this transcription factor to cell cycling has not been directly addressed. Here we examine the inductions of NF-kappa B in serum-deprived and cycling cells in response to tumor necrosis factor-alpha (TNF-alpha). In 3T3 fibroblasts deprived of serum, and in the temperature-sensitive G2 phase mutant carcinoma line FT210, we find that NF-kappa B DNA binding activity is rapidly induced upon addition of TNF-alpha. In addition, NF-kappa B induction in cycling cells occurs without a significant change in cell cycle distribution. These data reveal that NF-kappa B is rapidly induced by TNF-alpha in both proliferating and arrested cells and suggest that distinct activation pathways can lead to cell cycle-dependent or -independent induction of NF-kappa B.

3T3 Cells↗

PubMed1994

Dissociation of endogenous cellular ceramide from NF-kappa B activation.

The participation of cell ceramide in tumor necrosis factor (TNF)-alpha-stimulated NF-kappa B activation in Jurkat T cells and HL-60 cells was studied. TNF-alpha readily stimulated NF-kappa B activity in both cell lines as assayed by electrophoretic mobility shift assay and the use of a human immunodeficiency virus-chloramphenicol acetyltransferase reporter construct. However, TNF-alpha stimulation did not increase cell ceramide levels in either cell line. The exogenous addition of a short chain ceramide, N-acetylsphingosine, to Jurkat cells had no effect on NF-kappa B activity. When Jurkat T cells were exposed to the glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, endogenous ceramide levels increased 4-fold. The increase in ceramide, however, did not result in NF-kappa B activation nor did it potentiate TNF-alpha or phorbol ester-stimulated activity. We conclude that TNF-alpha-induced NF-kappa B activation occurs in Jurkat and HL-60 cell lines that do not demonstrate an increase in TNF-alpha-induced ceramide. Increasing ceramide levels by the addition of short chain ceramides or the use of a glucosylceramide synthase inhibitor can be dissociated from activation of NF-kappa B by TNF-alpha.

Antineoplastic Agents↗

PubMed1994

Transcription factor AP-2 regulates human immunodeficiency virus type 1 gene expression.

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by an enhancer region composed of multiple potential cis-acting regulatory sites. Here, we describe binding sites for the transcription factor AP-2 in the HIV-1 long terminal repeat which modulate HIV enhancer function. One site is embedded within the two previously described kappa B elements, and a second site is detected further downstream. DNase I footprinting and electrophoretic mobility shift assay experiments demonstrated that AP-2 binds to the site between the kappa B elements. Interestingly, AP-2 and NF-kappa B bind to this region in a mutually exclusive manner. Mutations which disrupt this AP-2-binding site lower basal levels of transcription but do not affect NF-kappa B-mediated induction by tumor necrosis factor alpha in Jurkat T leukemia cells.

Base Sequence↗

PubMed1994

An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation.

Induction of human immunodeficiency virus type 1 (HIV-1) gene expression in stimulated T cells has been attributed to the activation of the transcription factor NF-kappa B. The twice-repeated kappa B sites within the HIV-1 long terminal repeat are in close proximity to three binding sites for Sp1. We have previously shown that a cooperative interaction of NF-kappa B with Sp1 is required for the efficient stimulation of HIV-1 transcription. In this report, we define the domains of each protein responsible for this effect. Although the transactivation domains seemed likely to mediate this interaction, we find, surprisingly, that this interaction occurs through the putative DNA-binding domains of both proteins. Sp1 specifically interacted with the amino-terminal region of RelA(p65). Similarly, RelA bound directly to the zinc finger region of Sp1. This interaction was specific and resulted in cooperative DNA binding to the kappa B and Sp1 sites in the HIV-1 long terminal repeat. Furthermore, the amino-terminal region of RelA did not associate with several other transcription factors, including MyoD, E12, or Kox15, another zinc finger protein. These findings suggest that the juxtaposition of DNA-binding sites promotes a specific protein interaction between the DNA-binding regions of these transcription factors. This interaction is required for HIV transcriptional activation and may provide a mechanism to allow for selective activation of kappa B-regulated genes.

Animals↗

PubMed1993

A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation.

The human immunodeficiency virus (HIV-1) long terminal repeat (LTR) contains two binding sites for NF-kappa B in close proximity to three binding sites for the constitutive transcription factor, Sp1. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF-kappa B to the viral enhancer. In this report, we show that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, a protein-protein interaction must occur between NF-kappa B and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF-kappa B and Sp1 was confirmed by electrophoretic mobility shift gel analysis, DNase footprinting, chemical cross-linking and transfection studies in vivo. With a heterologous promoter, we find that the interaction of NF-kappa B with Sp1 is dependent on orientation and position, and is not observed with other elements, including GATA, CCAAT or octamer. An increase in the spacing between the kappa B and Sp1 elements virtually abolishes this functional interaction, which is not restored when these sites are brought back into the same helical position. Several other promoters regulated by NF-kappa B also contain kappa B in proximity to Sp1 binding sites. These findings suggest that an interaction between NF-kappa B and Sp1 is required for inducible HIV-1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes.

Avian Sarcoma Viruses↗

PubMed1993

Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-kappa B subunits in endothelial and epithelial cells.

Vascular cell adhesion molecule 1 (VCAM-1) is expressed in both endothelial and epithelial cell types, where it contributes to lymphocyte migration to sites of inflammation. Its expression is regulated by cytokines, in part through two kappa B-like regulatory elements. Because NF-kappa B can be composed of multiple alternative subunits with differential effects on gene expression, the role of different specific NF-kappa B family members subunits in VCAM-1 regulation is unknown. In this report, we define the contribution of different NF-kappa B family members to VCAM-1 gene regulation. We show that both kappa B sites in the VCAM-1 enhancer are required to optimally stimulate gene expression, but the enhancer is differentially regulated by specific combinations of NF-kappa B subunits. At low concentrations, RelA(p65) acted in concert with the approximately 50-kDa product of p105 NF-kappa B, NF-kappa B1(p50), to stimulate transcription, and at high concentrations, RelA(p65) alone stimulated the VCAM-1 promoter. In contrast, NF-kappa B2 inhibited functional activation of the VCAM reporter by p65. Consistent with this finding, an additional binding complex was detected by using recombinant NF-kappa B2(p49)/RelA(p65) with radiolabeled VCAM kappa B site probes. Interestingly, the human immunodeficiency virus enhancer responded differently to stimulation by NF-kappa B subunits, with optimal response to p49(100)/p65. Analysis of NF-kappa B mRNA in human umbilical vein endothelial cells revealed that nfkb1, nfkb2, and relA NF-kappa B but not c-rel were induced by tumor necrosis factor alpha and lipopolysaccharide, which also induce VCAM-1. These data suggest that specific subunits of NF-kappa B regulate VCAM-1 and differentially activate other genes in these cells.

Animals↗