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. 2005 Sep;25(17):7900-16.
doi: 10.1128/MCB.25.17.7900-7916.2005.

Regulation of RelB Expression During the Initiation of Dendritic Cell Differentiation

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Free PMC article

Regulation of RelB Expression During the Initiation of Dendritic Cell Differentiation

Pedro J Cejas et al. Mol Cell Biol. .
Free PMC article

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 CbetaII (PKCbetaII) expression in KG1a cells allows them to differentiate into DCs. We show that PKCbetaII also downregulated constitutive expression of NF-kappaB 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 PKCbetaII expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKCbetaII-mediated regulation of transcriptional initiation and elongation.

Figures

FIG. 1.
FIG. 1.
RelB is upregulated early in the differentiation of DC progenitor KG1 cells. (A) RelB mRNA content is lower in KG1 cells than in KG1a cells, but it is soon upregulated after PMA treatment. KG1 and KG1a cells were stimulated with PMA (10 ng/ml), and RelB mRNA expression levels were analyzed in a Northern blotting assay. The membrane was subsequently hybridized to a GAPDH cDNA probe to confirm equal loading. Hybridization signals were quantified by phosphorimaging before normalizing the RelB mRNA content in each lane to the respective GAPDH signal (right side). Values at the top of the bars indicate n-fold expression over untreated KG1 cells. Shown is one representative result from at least three independent experiments. (B) RelB protein expression follows the regulation observed at the mRNA level. Western blot analyses were performed on lysates of KG1 and KG1a cells stimulated with PMA (10 ng/ml) using an anti-RelB polyclonal antibody. Shown is one representative result from at least three independent experiments. n.s. = nonspecific band. (C) Expression levels of RelB flanking genes are not affected during PMA stimulation. The relB gene expands over ∼37 kb of DNA, and it is flanked by the CLPTM1 and SFRS16 genes (CLPTM1 and SFRS16 are not represented in their entirety). Total RNA was isolated from KG1 and KG1a cells left untreated or stimulated with PMA for 2 h for a global expression analysis using the Affymetrix HG-U133 Plus 2.0 array. All probe set signal values for the CLPTM1, relB, and SFRS16 genes were called present by the GeneChip algorithm employed in the analysis. n-Fold induction in each cell line was determined by dividing the signal values of each gene at 2 h by the respective values at 0 h.
FIG. 2.
FIG. 2.
Changes in RelB promoter activity do not correlate fully with the regulation of RelB in KG1 cells. (A) Cloning of the promoter-containing RelB 5′ genomic region into the pGL2-basic vector. A 4.6-kb DNA fragment containing the RelB promoter-enhancer region was cloned into the pGL2-basic vector to drive the expression of luciferase (pGR4647). The pGR599 and pGR171 deletion constructs were generated by restriction digestion of the original clone. The pGR4647R construct containing the 4.6-kb DNA fragment in reverse orientation was cloned as a negative control. NF-κBI and -II indicate the two NF-κB-binding regulatory sites previously identified in the RelB promoter-enhancer region. Pol, polymerase. (B) PMA treatment induces only twofold upregulation of the RelB promoter activity in KG1 cells. KG1 and KG1a cells were transiently transfected with the reporter constructs containing the promoter-enhancer region of RelB. Renilla luciferase-encoding plasmid pRL-CMV was cotransfected to allow normalization of transfection efficiency. After overnight incubation, cells were treated with PMA for 5 h before assaying for luciferase activity. Relative light units (RLU) were determined by dividing the firefly luciferase activity by the respective Renilla luciferase activity values. Values at the top of the bars indicate n-fold induction over untreated samples. The data represent the mean ± the standard deviation of triplicate measurements from a representative experiment of three. (C) RelB promoter activity correlates with thesteady-state mRNA levels in untreated KG1 and KG1a cells. Standardized light units were determined by normalizing the relative light units from the reporter constructs in untreated KG1 and KG1a cells to the relative cytomegalovirus promoter activity in both cell lines. The relative cytomegalovirus promoter activities were determined by adjusting the Renilla luciferase activity values from the pRL-CMV plasmid to the transfection efficiency as determined by flow cytometry after cotransfection with the pDsRed2-C1 vector. Data represent the mean ± the standard deviation of triplicate measurements from a representative experiment of three.
FIG. 3.
FIG. 3.
RelB expression is not regulated by changes in mRNA stability. (A) Determination of RelB mRNA half-life in KG1, KG1a, and PMA-treated KG1 cells. KG1 and KG1a cells left unstimulated or stimulated with PMA for 2 h were treated with actinomycin D to arrest transcription before isolation of total RNA at the indicated time points. RelB and GAPDH transcripts were detected in Northern blotting assays as previously described. (B) PMA treatment does not increase the RelB mRNA half-life in KG1 cells. Hybridization signals for the RelB and GAPDH mRNAs were quantified by phosphorimaging in KG1 cells left untreated or treated with PMA for 2 h. Each lane was normalized for equal loading by dividing the RelB content by the respective GAPDH signal and expressed relative to RelB content at 0 h for each condition. The mRNA half-life was calculated by linear regression analysis to be 3.0 h and 2.5 h for untreated and PMA-treated KG1 cells, respectively. The dashed line parallel to the x axis represents 50% mRNA decay. The data represent the mean ± the standard error from two independent experiments. (C) RelB mRNA half-life is the same in KG1 and KG1a untreated cells. A RelB mRNA half-life of 3.0 h was determined for untreated KG1a cells as described for panel B. The data represent the mean ± the standard error from two independent experiments.
FIG. 4.
FIG. 4.
RelB upregulation in KG1 cells results from an increase in transcription initiation and elongation. (A) Cloning of RelB genomic probes for nuclear transcriptional runoff assays. The relB gene contains 12 exons extended over 37 kb of DNA sequence. Exons 4 and 5 are separated in the relB gene by 9.8 kb of intronic sequence. Hybridization probes A, B, C, and D were obtained by restriction digestion of bacterial artificial chromosome clone RP11-91A12 and expand over the 5′ end, exon 4, exon 5, and the 3′ end of the relB gene, respectively. (B) Nuclear transcriptional runoff assays. Nuclei were isolated from KG1 and KG1a cells left untreated or treated with PMA for 4 h. Nascent transcripts were labeled in vitro with [α-32P]UTP and hybridized to the indicated sense (s) and antisense (as) ssDNA probes. (C) RelB transcriptional elongation is enhanced exclusively in PMA-treated KG1 cells. Hybridization signals for each probe were quantified by phosphorimaging and corrected for thymidine content. The resulting values were normalized against the respective actin intensities and expressed relative to the 3′-end probe value in untreated KG1 cells. The graph shows representative results from one of three independent experiments. (D) PMA stimulation induces an 11-fold average upregulation in full RelB transcripts. Changes in n-fold expression were determined by dividing the signal intensity values of each probe at 4 h by the corresponding values in untreated samples. The graph shows representative results from one of three independent experiments.
FIG. 5.
FIG. 5.
Inhibition of PKCβII expression in primary human monocytes inhibits RelB upregulation in response to cytokine-induced DC differentiation. (A) Monocytes were enriched from PBMC as described in Materials and Methods. CD14-positive cells are shown as an open histogram and are overlaid with the isotype control (filled histogram). Percent CD14-positive cells is indicated. PE, phycoerythrin. (B) Enriched monocytes were left untransfected or transfected with pEGFP-siEmpty or pEGFP-siPKCβ constructs and analyzed for EGFP expression 2 days after transfection by fluorescence-activated cell sorter. The data are illustrated as transfected monocytes (open histogram) overlaid with untransfected monocyte controls (filled histogram). The percentage of cells expressing EGFP is indicated. FITC, fluorescein isothiocyanate. (C) Total cell lysates were made from enriched monocytes prior to transfection, untransfected or transfected monocytes incubated for 2 days in medium alone, and untransfected or transfected monocytes stimulated with GM-CSF plus IL-4 for 2 h, 2 days posttransfection. Proteins were separated by SDS-polyacrylamide gel electrophoresis and analyzed for expression of PKCβII, RelB, and actin. The data shown are representative results from one of two independent experiments.
FIG. 6.
FIG. 6.
Generation of single cell clones of PKCβII-transfected KG1a cells expressing different levels of kinase expression. (A) Cell clones βII-E11, -E9, -C10, and -D8 express different levels of PKCβII protein. Flow cytometry analysis of KG1a cell clones expressing different levels of PKCβII-EGFP fusion protein. FITC, fluorescein isothiocyanate. (B) Untreated PKCβII-expressing KG1a cells have significant levels of PKC enzyme activity. Total PKC activities were determined in KG1, KG1a, KG1a-neo, βII-E9, and βII-E11 untreated cell lysates by phosphorylation of a PKC-specific peptide substrate. PKC values are normalized to total protein content and are expressed relative to the PKC activity in KG1a-neo cells. The data represent the mean ± the standard deviation from three independent experiments. (C) The PKCβII-EGFP fusion protein translocates to the cell membrane after PMA stimulation. KG1a cells and βII-E9 cells were left untreated or treated with PMA (10 ng/ml) for 30 min. KG1a cells were stained for PKCβII using a fluorescein isothiocyanate-conjugated secondary antibody before visualization by confocal microscopy. βII-E9 cells were visualized directly for the EGFP tag. (D) PKC enzyme activity at the cell membrane increases after PMA stimulation. KG1, KG1a-neo, and βII-E9 cells were left unstimulated (open bars) or stimulated (closed bars) with PMA (10 ng/ml) for 45 min. Cell membrane lysates were generated for all samples before determining PKC activity levels as explained for panel B.
FIG. 7.
FIG. 7.
Forced expression of PKCβII in KG1a cells results in a mode of RelB regulation similar to that of KG1 cells. (A) PKCβII-transfected KG1a cells regulate RelB expression like KG1 cells. Protein lysates were generated for KG1a-neo, βII-E9, -D8, -C10, and -E11 cells stimulated or not with PMA (10 ng/ml) for 4 h. RelB and actin protein expression was analyzed in Western blot assays as previously described. (B) NF-κB protein expression is downregulated in PKCβII-transfected KG1a cells. Protein lysates were generated for KG1, KG1a-neo, βII-E9, -D8, -C10, and -E11 untreated cells. Expression of the RelA, p105/p50, p100/p52, and actin proteins was analyzed in Western blot assays. (C) RelB mRNA expression has a maximal ninefold upregulation in βII-E9 cells after PMA treatment. KG1a-neo and βII-E9 cells were stimulated with PMA (10 ng/ml) for the indicated times. RelB and GAPDH mRNA levels were determined in Northern blotting assays as previously described. The n-fold increase was calculated after normalizing the RelB mRNA hybridization signals to the respective GAPDH content and dividing by the value obtained at 0 h (right side). Only the maximal n-fold increase (8 h) is shown. The data represent the mean ± the standard deviation from three independent experiments.
FIG. 8.
FIG. 8.
RelB transcription initiation in βII-E9 cells increases after PMA treatment and is not attenuated. (A) PMA treatment induces a sevenfold upregulation of the RelB promoter activity in βII-E9 cells. βII-E9 and KG1a-neo cells were transiently transfected with reporter constructs containing the promoter-enhancer region of RelB. Renilla luciferase-encoding plasmid pRL-CMV was cotransfected to allow normalization of transfection efficiency. After overnight incubation, cells were treated with PMA for 8 h before assaying for luciferase activity. Relative light units (RLU) were determined by dividing the firefly luciferase activity by the respective Renilla luciferase activity values. Values at the top of the bars indicate n-fold induction over untreated samples. The graphs show representative results from one of three independent experiments. (B) PKCβII expression results in downregulation of the RelB promoter activity in untreated βII-E9 cells. Standardized light units were determined by normalizing the relative light units from the reporter constructs in untreated KG1a-neo and βII-E9 cells to the relative activity of the cytomegalovirus promoter in both cell lines. The relative cytomegalovirus promoter activities were determined by adjusting the Renilla luciferase activity values from the pRL-CMV plasmid to the transfection efficiency as determined by flow cytometry after cotransfection with the pDsRed2-C1 vector. (C) Nuclear transcriptional runoff assays. Nuclei were isolated from βII-E9 and KG1a-neo cells left untreated or treated with PMA for 8 h. Nascent transcripts were labeled in vitro with [α-32P]UTP and hybridized to the indicated dsDNA probe inserts. (D) Untreated βII-E9 cells show low transcription initiation rates and no decrease in RelB transcript elongation. Hybridization signals for each probe were quantified by phosphorimaging and corrected for thymidine content. The resulting values were normalized against the respective actin intensities and expressed relative to the average probe value in untreated βII-E9 cells. The graph shows representative results from two independent experiments. (E) PMA stimulation induces a sixfold average upregulation in RelB transcription in βII-E9 cells. Changes (n-fold) in expression for each probe were determined by dividing the signal intensity values at 8 h by the corresponding values in untreated samples. The graph shows representative results from two independent experiments.
FIG. 9.
FIG. 9.
PKCβII controls RelB expression by regulation of transcriptional initiation and elongation. KG1a cells actively initiate transcription from the RelB promoter, but elongation is relatively inefficient. Basal PKCβII activity in untreated KG1 cells downregulates the promoter activity with no increase in elongation efficiency, resulting in lower levels of full-length RelB transcripts. Increased expression (and enzyme activity) of PKCβII in untreated βII-E9 cells enhances transcript elongation, but the stronger downregulation in transcriptional initiation results in even lower levels of RelB mRNA. PMA-mediated activation of PKCβII results in an increase in promoter activity that combines with enhanced elongation to upregulate RelB mRNA levels.

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