Cyp26b1 Regulates Retinoic Acid-Dependent Signals in T Cells and Its Expression Is Inhibited by Transforming Growth Factor-β

The vitamin A metabolite, retinoic acid (RA), plays important roles in the regulation of lymphocyte properties. Dendritic cells in gut-related lymphoid organs can produce RA, thereby imprinting gut-homing specificity on T cells and enhancing transforming growth factor (TGF)-β-dependent induction of Foxp3+ regulatory T cells upon antigen presentation. In general, RA concentrations in cells and tissues are regulated by its degradation as well. However, it remained unclear if T cells could actively catabolize RA.We assessed the expression of known RA-catabolizing enzymes in T cells from mouse lymphoid tissues. Antigen-experienced CD44+ T cells in gut-related lymphoid organs selectively expressed Cyp26b1, a member of the cytochrome P450 family 26. However, T cells in the spleen or skin-draining lymph nodes did not significantly express Cyp26b1. Accordingly, physiological levels of RA (1-10 nM) could induce Cyp26b1 expression in naïve T cells upon activation in vitro, but could not do so in the presence of TGF-β. Overexpression of Cyp26b1 significantly suppressed the RA effect to induce expression of the gut-homing receptor CCR9 on T cells. On the other hand, knocking down Cyp26b1 gene expression with small interfering RNA or inhibiting CYP26 enzymatic activity led to enhancement of the RA-induced CCR9 expression.Our data demonstrate a role for CYP26B1 in regulating RA-dependent signals in activated T cells but not during TGF-β-dependent differentiation to Foxp3+ regulatory T cells. Aberrant expression of CYP26B1 may disturb T cell trafficking and differentiation in the gut and its related lymphoid organs

GM-CSF and IL-4 synergistically trigger dendritic cells to acquire retinoic acid-producing capacity

Retinoic acid (RA) produced by intestinal dendritic cells (DCs) imprints gut-homing specificity on lymphocytes and enhances Foxp3+ regulatory T-cell differentiation. The expression of aldehyde dehydrogenase (ALDH) 1A in these DCs is essential for the RA production. However, it remains unclear how the steady-state ALDH1A expression is induced under specific pathogen-free (SPF) conditions. Here, we found that bone marrow-derived dendritic cells (BM-DCs) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) expressed Aldh1a2, an isoform of Aldh1a, but that fms-related tyrosine kinase 3 ligand-generated BM-DCs did not. DCs from mesenteric lymph nodes (MLN) and Peyer's patches (PP) of normal SPF mice expressed ALDH1A2, but not the other known RA-producing enzymes. Employing a flow cytometric method, we detected ALDH activities in 10–30% of PP-DCs and MLN-DCs. They were CD11chighCD4−/lowCD8αintermediateCD11b−/low F4/80low/intermediateCD45RBlowCD86highMHC class IIhighB220−CD103+. Equivalent levels of aldehyde dehydrogenase activity (ALDHact) and ALDH1A2 expression were induced synergistically by GM-CSF and IL-4 in splenic DCs in vitro. In BM-DCs, however, additional signals via Toll-like receptors or RA receptors were required for inducing the equivalent levels. The generated ALDH1A2+ DCs triggered T cells to express gut-homing receptors or Foxp3. GM-CSF receptor-deficient or vitamin A-deficient mice exhibited marked reductions in the ALDHact in intestinal DCs and the T cell number in the intestinal lamina propria, whereas IL-4 receptor-mediated signals were dispensable. GM-CSF+CD11c−F4/80+ cells existed constitutively in the intestinal tissues. The results suggest that GM-CSF and RA itself are pivotal among multiple microenvironment factors that enable intestinal DCs to produce RA

Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

<div><p>Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal dehydrogenase 2 (RALDH2) encoded by <i>Aldh1a2</i> is a key enzyme for generating RA in DCs. Granulocyte–macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to <i>Aldh1a2</i> expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced <i>Aldh1a2</i> expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and <i>Aldh1a2</i> expression. Sp1 and the RARα/RXRα complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse <i>Aldh1a2</i> promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RARα/RXRα and Sp1 synergistically enhanced <i>Aldh1a2</i> promoter-reporter activity. GM-CSF did not significantly induce <i>Aldh1a2</i> expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the <i>Aldh1a2</i> promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the <i>Aldh1a2</i> promoter, and can be regulated by a DNA methylation-independent mechanism.</p></div

Methylation of the CpG island in the <i>Aldh1a2</i> promoter prohibits Sp1 to activate the promoter, whereas the <i>Aldh1a2</i> promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs.

<p>(<b>A</b>) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. (<b>B</b>) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. (<b>C</b>) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. (<b>D</b>) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of <i>Aldh1a2</i> mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the <i>Aldh1a2</i> mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's <i>t</i> test (***<i>p</i><0.001). Data in (A, B, and D) are representative of at least three independent experiments. (<b>E</b>) Genomic DNA was isolated from BM-pDCs and BM-cDCs, denatured, modified with sodium bisulfite, and used in nested PCR (−424 to −30) for bisulfite sequencing. Eleven and thirteen independent clones of pDCs and cDCs, respectively, were analyzed. The methylation patterns of 5 representative clones of each cell type are shown. Closed circles indicate methylated CpG and open circles indicate unmethylated CpG.</p

Sp1 participates in the <i>Aldh1a2</i> expression.

<p>(<b>A</b>) The genomic organization of the mouse <i>Aldh1a2</i> gene and its 5′-flanking region is shown. A fragment containing exon 1 and its 5′-flanking region from −2,600 to +182 was inserted into reporter vectors. DNA binding sites (STAT-binding sites, NF-κB binding sites, a SREBP binding site, and putative RARE half-sites (RARE-h)), the TATA box, and the GC-rich region in the fragment are indicated. (<b>B</b>) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 1 µM mithramycin A. After the culture, <i>Aldh1a2</i> mRNA expression was assessed by real-time PCR (<i>Left panel</i>), and protein expression of RALDH2 (ALDH1A2) and α-tubulin was analyzed by Western blotting (<i>Right panel</i>). Relative mRNA expression levels were calculated by defining the <i>Aldh1a2</i> mRNA expression level in the cells incubated with medium alone for 16 h was set to 1 (<i>Left panel</i>). Data are representative of three (<i>Left panel</i>) or two (<i>Right panel</i>) independent experiments. (<b>C</b>) Serial-deletion fragments derived from the 5′-flanking region of the mouse <i>Aldh1a2</i> gene were inserted in the reporter vector, pGL3-basic. COS-7 cells were transfected in triplicate with one of the deletion constructs (1.25 µg) or the pGL3-RALDH2 (−2,600) reporter vector (1.25 µg) in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after the transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL3-basic alone as 1. Data are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's <i>t</i> test (**<i>p</i><0.01, ***<i>p</i><0.001). Data are representative of three independent experiments.</p

The short DNA regions containing the GC-rich region, the RARE half-site, and the TATA box in the 5′-flanking region of the <i>Aldh1a2</i> genes are well conserved among various species.

<p>The sequence of the 5′-flanking region of the mouse (<i>Mus musculus</i>) <i>Aldh1a2</i> gene was compared with that of the human (<i>Homo sapiens</i>) <i>ALDH1A2</i> gene, that of rat (<i>Rattus norvegicus</i>) <i>Aldh1a2</i> gene, that of cattle (<i>Bos taurus</i>) <i>ALDH1A2</i> gene, that of chicken (<i>Gallus gallus</i>) <i>ALDH1A2</i> gene, and that of zebrafish (<i>Danio rerio</i>) <i>aldh1a2</i> gene. The sequence data were obtained using the NCBI MapViewer. The shaded regions indicate homology with the mouse sequence. The locations of the conserved RARE half-sites (RARE-h) and TATA boxes are indicated by boxes.</p

The MEK1-ERK-signaling pathway and the p38 MAPK-signaling pathway are required for the GM-CSF-induced <i>Aldh1a2</i> expression and nuclear translocation of Sp1 in BM-DCs.

<p>(<b>A</b>) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF for 16 h in the presence or absence of 50 µM PD98059 (PD) or 25 µM SB203580 (SB). After the culture, <i>Aldh1a2</i> gene expression was assessed by real-time PCR. The <i>Aldh1a2</i> mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. The results are shown as the mean + SD of triplicate cultures. Statistical significance was determined by the Student's <i>t</i> test (***<i>p</i><0.001). (<b>B</b>) BM-DCs were cultured with or without 10 ng/ml GM-CSF in the presence or absence of 50 µM PD98059 or 25 µM SB203580 for 16 h. After the culture, cytosolic and nuclear proteins were analyzed for the presence of Sp1, α-tubulin, and lamin B1 by Western blotting. (<b>C</b>) Structures of the deletion constructs used in this figure are shown. Δ1, Δ2, and Δ3 constructs were prepared by deleting the region spanning from the −56 to −41, −99 to −78, and −139 to −123, respectively, from the pGL3-RALDH2 (−373) reporter vector. COS-7 cells were transfected in triplicate with the 1.25 µg of pGL3-RALDH2 (−373) reporter vector or the deletion mutants in combination with 0.5 µg of the pCMV-Myc-Sp1 expression vector or control empty vector. One day after the transfection, cells were stimulated with or without 5 ng/ml PMA, and luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL3-basic alone as 1. Data are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's <i>t</i> test (**<i>p</i><0.01, ***<i>p</i><0.001; NS, not significant). Data are representative of three independent experiments.</p

Sp1 binds to the <i>Aldh1a2</i> promoter region.

<p>(<b>A</b>) The locations and nucleotide sequences corresponding to Probe A, Probe B, and Probe C in the 5′-flanking region of the mouse <i>Aldh1a2</i> gene are shown. COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1 or control empty vector. One day after transfection, cell lysates were analyzed for DNA-binding activity by DNAP assay using the indicated biotinylated DNA probes and anti-Myc Ab. <b>(B)</b> Flt3L-generated BM-DCs were cultured in the presence or absence of 10 ng/ml GM-CSF. After 16 h, nuclear extracts were analyzed for the presence of lamin B1 and Sp1 by Western blotting using anti-lamin B1 and anti-Sp1 Abs (<i>left panel</i>), or assessed for DNA binding activity by DNAP assay using biotinylated DNA Probe C and anti-Sp1 Ab (<i>right panel</i>). Data are representative of at least three independent experiments.</p

Botulinum ADP-ribosyltransferase activity as affected by detergents and phospholipids

AbstractGTP-binding proteins with Mr values of 22 000 and 25 000 in bovine brain cytosol were ADP-ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3-catalyzed ADP-ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3-catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP-ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADP-ribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other