Regulation of RelB Expression during the Initiation of Dendritic Cell Differentiation

Abstract

The transcription factor RelB is required for proper development and function of dendritic cells (DCs), and its expression is upregulated early during differentiation from a variety of progenitors. We explored this mechanism of upregulation in the KG1 cell line model of a DC progenitor and in the differentiation-resistant KG1a subline. RelB expression is relatively higher in untreated KG1a cells but is upregulated only during differentiation of KG1 by an early enhancement of transcriptional elongation, followed by an increase in transcription initiation. Restoration of protein kinase C␤II (PKC␤II) expression in KG1a cells allows them to differentiate into DCs. We show that PKC␤II also downregulated constitutive expression of NF-B in KG1a-transfected cells and restores the upregulation of RelB during differentiation by increased transcriptional initiation and elongation. The two mechanisms are independent and sensitive to PKC signaling levels. Conversely, RelB upregulation was inhibited in primary human monocytes where PKC␤II expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKC␤II-mediated regulation of transcriptional initiation and elongation.Dendritic cells (DCs) constitute a heterogeneous population of professional antigen-presenting cells with a unique role in the activation of naive T lymphocytes and the establishment of immunological tolerance and memory (2, 3). Their central role as regulators of the immune response has led to the examination of the underlying intracellular signaling pathways and gene expression that may regulate the differentiation of precursors into DCs (21, 60), most notably, the Rel/NF-B family of transcription factors (41, 61).NF-B exists in mammals as a homodimer or heterodimer of the Rel proteins NFB1 (p50/p105), NFB2 (p52/p100), c-Rel, RelA (p65), and RelB. The dimer can be sequestered in the cytoplasm by the inhibitory IBs, which in the canonical activation pathway can be phosphorylated, ubiquitinated, and degraded by the proteasome to allow the nuclear translocation of NF-B and activate transcription (63). On the other hand, the NF-B2 precursor form p100 can inhibit directly the nuclear translocation of heterodimers due to their IB-like ankyrin repeats and is activated by an alternative pathway that requires the NF-B-inducing kinase (NIK) and IKK␣ (62). In this pathway, the bound precursor p100 is processed in a regulated fashion to the p52 form to allow translocation to the nucleus. The p105 subunit also contains IB-like ankyrin repeats that could sequester NF-B dimers in the cytoplasm. However, its processing to the p50 form is constitutive (22,30).Overexpression of IB in mature DCs downregulates major histocompatibility complex (MHC) class II; the costimulatory molecules CD80, CD86, and CD40; and the proinflammatory cytokine tumor necrosis factor alpha (TNF-␣), indicating that antigen presentation is dependent on NF-B function (64). Likewise, the inhibitory p100 precursor ...

Interleukin-18 directly activates T-bet expression and function via p38 mitogen-activated protein kinase and nuclear factor-κB in acute myeloid leukemia–derived predendritic KG-1 cells

“…Incubation of KG-1 cells with IL-18 (10 ng/mL) for up to 32 h only modestly affected cell viability (12.0% F 2.1% reduction of viability by IL-18 versus unstimulated control after 32 h, n = 4, this trend did not reach statistical significance). We also observed the induction of the transcription factor RelB by IL-18 (10 ng/mL, 4 h; data not shown) confirming that IL-18 is able to initiate processes that are associated with KG-1 cell differentiation towards a dendritic phenotype (24). To investigate a potential role for endogenous IL-18, Western blot analysis was done and revealed that KG-1 cells express clearly detectable levels of pro-IL-18, which are at least similar to those seen in monocytic THP-1 cells (Fig.…”

IL-3 Inhibits Human Osteoclastogenesis and Bone Resorption through Downregulation of c-Fms and Diverts the Cells to Dendritic Cell Lineage

“…RelB is critical for osteoclastogenesis, because bone marrow macrophages lacking RelB fail to undergo RANKL-mediated osteoclastogenesis in vitro (51). Also, RelB is required for development and function of dendritic cells, and its expression is upregulated during early differentiation (52). RelB was downregulated by IL-3 only on day 7 and not on day 3, suggesting that RelB is required for dendritic cell differentiation on day 3, whereas it is more critical for osteoclast differentiation on day 7.…”

miR-146a Is Differentially Expressed by Myeloid Dendritic Cell Subsets and Desensitizes Cells to TLR2-Dependent Activation

“…1F). Because NF-kB and TRAF6 were shown to regulate myelopoiesis and DC subset differentiation (41,47,48) we analyzed whether ectopic miR146a might influence myelopoiesis. Thus, we generated a retroviral expression construct for miR-146a by inserting the genomic sequence of miR-146a 6 250 bp flanking regions into the retroviral vector backbone murine stem cell virus-GFP (Fig.…”