Constitutive expression of p50 homodimer in freshly isolated human monocytes decreases with in vitro and in vivo differentiation: a possible mechanism influencing human immunodeficiency virus replication in monocytes and mature macrophages

Abstract

Human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in vitro in differentiated macrophages than in freshly isolated monocytes. We investigated whether this may be partly explained by changes in expression of NF-B with monocyte differentiation. We demonstrated that constitutive expression of NF-B in primary human monocytes changed significantly with differentiation in vitro to monocyte-derived macrophages (MDMs) and differentiation in vivo to alveolar macrophages (AMs). Freshly isolated monocytes constitutively expressed high levels of transcriptionally inactive p50 homodimer which decreased with time in culture in favor of the transcriptionally active p50/p65 and p50/RelB heterodimers. As in MDMs, AMs constitutively expressed p50/p65 and p50/RelB although at lower levels. HIV infection of fresh monocytes failed to induce p50/p65 as seen in MDMs. The replacement of p50 homodimers with transcriptionally active heterodimers following time in culture may partially explain the progressive increase in susceptibility of monocytes to HIV infection during in vitro culture. The change in NF-B components with monocyte differentiation in vivo may also explain the different transcriptional activities of these cell populations in HIVinfected individuals. MATERIALS AND METHODSNuclear extracts. Nuclear extracts were prepared by a high-salt extraction method (4a) using some modifications described by Bellas et al. (4). This method allowed the use of small numbers of cells and minimized proteolysis, a common problem in the manipulation of myelomonocytic cells. Briefly, 5 ϫ 10 6 to 10 ϫ 10 6 cells were collected, washed in ice-cold phosphate-buffered saline (Cytosystems, Sidney, Australia), and resuspended in 2 ml of buffer A (10 mM Tris-HCl [pH 7.5], 2 mM MgCl 2 , 5 mM KCl, 10% glycerol, 1 mM EDTA), which was then

Fold-change detection of NF-κB at target genes with different transcript outputs

“…4a). NF-B p50, NF-B p52, and BCL3 are potential candidates for this competitor protein, because they are induced by RelA (19), and they suppress transcription of B site-containing genes, including IL6 and the HIV long terminal repeat (20,21). This model accurately described our observations in HeLa cells for RelA-target genes with high TNF-inducible transcription (7).…”

Down-Regulation of Procollagen α1[I] Messenger RNA by Titanium Particles Correlates with Nuclear Factor κB (NF-κB) Activation and Increased Rel A and NF-κB1 Binding to the Collagen Promoter

“…Of note, this NF‐κB binding site is a variant κB element that binds Rel A and NF‐κB1 (p50) subunits, binding characteristics it shares with the procollagen α 1 [I] gene promoter (Table 1). NF‐κB1 lacks an activation domain and homodimers of NF‐κB1 have been reported to act as a negative transcription factor capable of down‐regulating gene expression from NF‐κB binding sites (40) . Along these lines, we identified three variant NF‐κB binding sites within the distal region of the human procollagen α 1 [I] gene promoter.…”

Particulate Wear Debris Activates Protein Tyrosine Kinases and Nuclear Factor κB, Which Down-Regulates Type I Collagen Synthesis in Human Osteoblasts

“…NF-B1 lacks an activation do-main and homodimers of NF-B1 have been reported to act as a negative transcription factor capable of downregulating gene expression from NF-B binding sites. (40) It is possible that titanium down-regulates procollagen ␣1[I] gene expression via these upstream NF-B binding sites in a manner analogous to the TNF-␣ down-regulation of procollagen ␣2[I] gene expression. Indeed, recently, it was shown that the murine procollagen ␣1[I] gene is downregulated by Rel A subunit of NF-B via an interaction with the transcription factor Sp1.…”