Bcl-2 and Bcl-XL serve an anti-inflammatory function in endothelial cells through inhibition of NF-kappaB.
(1/2626)
To maintain the integrity of the vascular barrier, endothelial cells (EC) are resistant to cell death. The molecular basis of this resistance may be explained by the function of antiapoptotic genes such as bcl family members. Overexpression of Bcl-2 or Bcl-XL protects EC from tumor necrosis factor (TNF)-mediated apoptosis. In addition, Bcl-2 or Bcl-XL inhibits activation of NF-kappaB and thus upregulation of proinflammatory genes. Bcl-2-mediated inhibition of NF-kappaB in EC occurs upstream of IkappaBalpha degradation without affecting p65-mediated transactivation. Overexpression of bcl genes in EC does not affect other transcription factors. Using deletion mutants of Bcl-2, the NF-kappaB inhibitory function of Bcl-2 was mapped to bcl homology domains BH2 and BH4, whereas all BH domains were required for the antiapoptotic function. These data suggest that Bcl-2 and Bcl-XL belong to a cytoprotective response that counteracts proapoptotic and proinflammatory insults and restores the physiological anti-inflammatory phenotype to the EC. By inhibiting NF-kappaB without sensitizing the cells (as with IkappaBalpha) to TNF-mediated apoptosis, Bcl-2 and Bcl-XL are prime candidates for genetic engineering of EC in pathological conditions where EC loss and unfettered activation are undesirable. (+info)
Sequence analysis and expression of a mouse homolog of human IkappaBL gene.
(2/2626)
The family of transcriptional inhibitors, IkappaBLs, are critical to the regulation of cytokine and chemokine production. We have identified the complete cDNA sequence of the mouse IkappabL gene. The predicted 381-amino-acid sequence showed evidence of two ankyrin repeats characteristic of Ikappab family proteins and 92% identity to the IkappaBL human homolog. Although human IkappaBL has been reported to be ubiquitously expressed, here we show that mouse IkappaBL is transcribed in a more tissue-specific manner. (+info)
Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed-Sternberg cells.
(3/2626)
A common characteristic of malignant cells derived from patients with Hodgkin's disease (HD) is a high level of constitutive nuclear NF-kappaB/Rel activity, which stimulates proliferation and confers resistance to apoptosis. We have analysed the mechanisms that account for NF-kappaB activation in a panel of Hodgkin/Reed-Sternberg (H-RS) cell lines. Whereas two cell lines (L428 and KMH-2) expressed inactive IkappaBalpha, no significant changes in NF-kappaB or IkappaB expression were seen in other H-RS cells (L591, L1236 and HDLM-2). Constitutive NF-kappaB was susceptible to inhibition by recombinant IkappaBalpha, suggesting that neither mutations in the NF-kappaB genes nor posttranslational modifications of NF-kappaB were involved. Endogenous IkappaBalpha was bound to p65 and displayed a very short half-life. IkappaBalpha degradation could be blocked by inhibitors of the NF-kappaB activating pathway. Proteasomal inhibition caused an accumulation of phosphorylated IkappaBalpha and a reduction of NF-kappaB activity in HDLM-2 and L1236 cells. By in vitro kinase assays we demonstrate constitutive IkappaB kinase (IKK) activity in H-RS cells, indicating ongoing signal transduction. Furthermore, H-RS cells secrete one or more factor(s) that were able to trigger NF-kappaB activation. We conclude that aberrant activation of IKK's, and in some cases defective IkappaBs, lead to constitutive nuclear NF-kappaB activity, which in turn results in a growth advantage of Hodgkin's disease tumor cells. (+info)
Physical interaction of the bHLH LYL1 protein and NF-kappaB1 p105.
(4/2626)
The LYL1 gene was first identified upon the molecular characterization of the t(7;9)(q35;p13) translocation associated with some human T-cell acute leukemias (T-ALLs). In adult tissues, LYL1 expression is restricted to hematopoietic cells with the notable exclusion of the T cell lineage. LYL1 encodes a basic helix-loop-helix (bHLH) protein highly related to TAL-1, whose activation is also associated with a high proportion of human T-ALLs. A yeast two-hybrid system was used to identify proteins that specifically interact with LYL1 and might mediate its activities. We found that p105, the precursor of NF-kappaB1 p50, was the major LYL1-interacting protein in this system. The association between LYL1 and p105 was confirmed both in vitro and in vivo in mammalian cells. Biochemical studies indicated that the interaction was mediated by the bHLH motif of LYL1 and the ankyrin-like motifs of p105. Ectopic expression of LYL1 in a human T cell line caused a significant decrease in NF-kappaB-dependent transcription, associated with a reduced level of NF-kappaB1 proteins. (+info)
IkappaB-alpha enhances transactivation by the HOXB7 homeodomain-containing protein.
(5/2626)
Combinatorial interactions between distinct transcription factors generate specificity in the controlled expression of target genes. In this report, we demonstrated that the HOXB7 homeodomain-containing protein, which plays a key role in development and differentiation, physically interacted in vitro with IkappaB-alpha, an inhibitor of NF-kappaB activity. This interaction was mediated by the IkappaB-alpha ankyrin repeats and C-terminal domain as well as by the HOXB7 N-terminal domain. In transient transfection experiments, IkappaB-alpha markedly increased HOXB7-dependent transcription from a reporter plasmid containing a homeodomain consensus-binding sequence. This report therefore showed a novel function for IkappaB-alpha, namely a positive regulation of transcriptional activation by homeodomain-containing proteins. (+info)
Nuclear translocation of green fluorescent protein-nuclear factor kappaB with a distinct lag time in living cells.
(6/2626)
A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor kappaB (NF-kappaB) p50 and p105 (p50/IkappaB gamma), a precursor protein of NF-kappaB p50. GFP-p50 and GFP-p105 were expressed in monkey COS-7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP-p50 (without IkappaB gamma) in the transfected cells resided in the nucleus. On the other hand, GFP-p105 (GFP-p50 with IkappaB gamma) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF-kappaB/IkappaB. In addition, the translocation of NF-kappaB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10-20 min after stimulation with hydrogen peroxide or tumor necrosis factor alpha. It was suggested that this might be due to the existence of a limiting step where NF-kappaB is released from NF-kappaB/IkappaB by the proteasome. (+info)
Characterization of IkappaBalpha nuclear import pathway.
(7/2626)
IkappaBalpha controls the transcriptional activity of nuclear factor (NF)-kappaB by retaining it in the cytoplasm; but, when expressed in the nucleus, it can also inhibit the interaction of NF-kappaB with DNA and promote the export of NF-kappaB from the nucleus to the cytoplasm. Here, we report that IkappaBalpha, when not bound to NF-kappaB, is constitutively transported to the nucleus, and we confirm that the interaction of IkappaBalpha with NF-kappaB retains IkappaBalpha in the cytoplasm. Nuclear import of IkappaBalpha does not result from passive diffusion but from a specific energy-dependent transport process that requires the ankyrin repeats of IkappaBalpha. Nuclear accumulation of IkappaBalpha is dependent on importins alpha and beta as well as the small GTPase Ran, which are also responsible for the nuclear import mediated by basic nuclear localization sequences (NLS). However, these proteins are not sufficient to promote IkappaBalpha nuclear translocation. Factor(s) can be removed selectively from cell extracts with ankyrin repeats of IkappaBalpha which strongly reduce import of IkappaBalpha but not of proteins containing basic NLS. These findings indicate that IkappaBalpha is imported in the nucleus by a piggy-back mechanism that involves additional protein(s) containing a basic NLS and able to interact with ankyrin repeats of IkappaBalpha. (+info)
UV-A-induced decrease in nuclear factor-kappaB activity in human keratinocytes.
(8/2626)
Previous reports have demonstrated an increase in nuclear factor-kappaB (NF-kappaB) activity in response to UV radiation. These studies have essentially focused on the DNA-damaging fraction of solar UV radiation (UV-B and UV-C). In contrast, the effects of UV-A radiation (320-400 nm) on NF-kappaB are not well known. In this study, we present evidence that UV-A radiation induces a marked decrease in NF-kappaB DNA-binding activity in NCTC 2544 human keratinocytes. In addition, NCTC 2544 keratinocytes pretreated with UV-A fail to respond to NF-kappaB inducers. Moreover, UV-A radiation induces a decrease in NF-kappaB-driven luciferase reporter gene expression in NCTC 2544 keratinocytes. The expression of the gene encoding IkappaBalpha (IkappaB is the NF-kappaB inhibitor), which is closely associated with NF-kappaB activity, is also reduced (3-fold) upon UV-A treatment. Our results indicate that the UV-A-induced decrease in NF-kappaB DNA-binding activity is associated with a decrease in the levels of the p50 and p65 protein subunits. This is the first evidence that an oxidative stress, such as UV-A radiation, may induce a specific decrease in NF-kappaB activity in mammalian cells, probably through degradation of NF-kappaB protein subunits. These findings suggest that UV-A could modulate the NF-kappaB-dependent gene expression. (+info)