Transcription of the RelB gene is regulated by NF-kappaB.

RelA and RelB are two members of the NF-kappaB family that differ structurally and functionally. While RelA is regulated through its cytosolic localization by inhibitor proteins or IkappaB and not through transcriptional mechanisms, the regulation of RelB is poorly understood. In this study we demonstrate that stimuli (TNF or LPS) lead within minutes to the nuclear translocation of RelA, but require hours to result in the nuclear translocation of RelB. The delayed nuclear translocation of RelB correlates with increases in its protein synthesis which are secondary to increases in RelB gene transcription. RelA is alone sufficient to induce RelB gene transcription and to mediate the stimuli-driven increase in RelB transcription. Cloning and characterization of the RelB 5' untranslated gene region indicates that RelB transcription is dependent on a TATA-less promoter containing two NF-kappaB binding sites. One of the NF-kappaB sites is primarily involved in the binding of p50 while the other one in the binding and transactivation by RelA and also RelB. Lastly, it is observed that p21, a protein involved in cell cycle control and oncogenesis known to be regulated by NF-kappaB, is upregulated at the transcriptional level by RelB. Thus, RelB is regulated at least at the level of transcription in a RelA and RelB dependent manner and may exert an important role in p21 regulation.

NF-kappaB cis-acting motifs of the human immunodeficiency virus (HIV) long terminal repeat regulate HIV transcription in human macrophages.

The role of NF-kappaB in the reactivation of human immunodeficiency virus (HIV) from latency in CD4 T lymphocytes is well documented. However, its role in driving HIV transcription in human macrophages, which contain a constitutive nuclear pool of NF-kappaB, is less well understood. In this study we have investigated the role that the constitutive pool of NF-kappaB and the NF-kappaB cis-acting motifs of the HIV long terminal repeat (LTR) play in regulating HIV transcription in human monocytic cells and primary macrophages. Inhibition of the constitutive nuclear pool of NF-kappaB (RelA and RelB) in the promonocytic U937 cell line using dominant-negative IkappaBalpha significantly decreases HIV replication. Moreover, it is demonstrated that in the differentiated monocytic cell line THP1, which contains a constitutive nuclear pool of NF-kappaB (RelB),an HIV provirus containing mutations of the kappaB cis-acting sites in the LTR is transcriptionally impaired. Reduction of the constitutive pool of NF-kappaB in human macrophages by an adenovirus vector expressing a dominant-negative IkappaBalpha also reduces HIV transcription. Lastly, mutation of the NF-kappaB cis-acting sites in the LTR of an R5 HIV provirus completely abrogates the first cycle of HIV transcription. These studies indicate that the cis-acting NF-kappaB motifs of the HIV LTR are critical in initiating HIV transcription in human macrophages and suggest that the constitutive nuclear pool of NF-kappaB is important in regulating HIV transcription in these cells.

Soluble recombinant neutral endopeptidase (CD10) as a potential antiinflammatory agent.

Several endogenous peptides, including bradykinin and substance P, have potent inflammatory effects in the joint. Levels of these peptides are regulated by plasma and cell-associated peptide degrading enzymes. One of these peptidases, neutral endopeptidase-24.11 (NEP-24.11), is expressed constitutively and in high density on human synovial cells and is presumed to play a critical role in local regulation of peptide levels in the joint. We examined the role of endogenous NEP-24.11 in regulating bradykinin-mediated effects in an articular model, and investigated the ability of soluble, recombinant human NEP-24.11 to augment the effects of the endogenous enzyme. Our studies demonstrate that endogenous synovial NEP-24.11 does not significantly modulate inflammatory peptide effects on cells when competing with colocalizing peptide receptors expressed in high density. Administration of excess, soluble recombinant NEP-24.11 can overcome this problem, however. Furthermore, the activity of the recombinant enzyme was not compromised in the presence of oxidants or inflammatory joint fluids. Recombinant NEP-24.11 holds promise as a novel therapeutic strategy for the treatment of inflammatory arthritis.

Mechanisms of prostanoid synthesis in human synovial cells: cytokine-peptide synergism.

Bradykinin (BK)2 and interleukin-1 (IL-1) interact synergistically to stimulate prostaglandin synthesis in human synovial fibroblast-like cells. The effect of BK is rapid and correlates with its capacity to elevate cytosolic levels of calcium ([Ca2+]i), while IL-1's effect is slow and s dependent upon de novo protein synthesis. The mechanism of this synergistic interaction was investigated. In the basal state, high levels of arachidonic acid (AA) were spontaneously released from synovial cells but near absent levels of cyclooxygenase activity prevented metabolism of AA to prostanoid. BK was a potent stimulus for elevating AA, but not prostaglandins, above basal levels. IL-1, in contrast, increased prostaglandins but not AA, above basal levels. IL-1 treatment was not associated with a loss or redistribution of AA among phospholipid classes. These results are consistent with high basal phospholipase activity in synovial cells and demonstrate the ability of BK, presumably via its ability to raise [Ca2+]i, to further elevate this activity(ies). Metabolism of AA to prostanoid is minimal in resting and BK-stimulated synovial cells, however, without the concomitant induction of cyclooxygenase activity by IL-1. These studies clarify the different, but synergistic, mechanisms of action of a peptide and cytokine in stimulating prostanoid synthesis in synovial cells. In addition, these data extend the results of previous investigations in demonstrating that basal phospholipase activity provides sufficient AA substrate for IL-1 induced prostanoid synthesis without invoking the concomitant induction of phospholipase activity by IL-1.