CD69 is a TGF-β/1α,25-dihydroxyvitamin D3 target gene in monocytes

CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene

Post-Transcriptional Regulation of 5-Lipoxygenase mRNA Expression via Alternative Splicing and Nonsense-Mediated mRNA Decay

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression

Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics

5‐Lipoxygenase (5‐LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5‐LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter‐interacting proteins, obtained by DNA pulldown and label‐free quantitative mass spectrometry. Protein preparations from myeloid and B‐lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel‐like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G‐quadruplex structures. As G‐quadruplexes are implicated in gene regulation, spectroscopic and antibody‐based methods were used to confirm their presence within the GC‐rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G‐quadruplex structures, which may play a functional role in ALOX5 gene regulation

3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes:evidence for activation by cytochrome P450 1A1 and P450 1A2

3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2 3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA

Oligonucleotides used for cloning by PCR.

a)<p>F =  forward, R =  reverse; non-genomic sequences (overhangs, restriction sites, Kozak sequence) are underline.</p

Effects of sequential treatment with TGF-β and 1α,25(OH)₂D₃.

<p>(A and B) THP-1 were treated with (TV) or without (w/o) 1 ng/ml TGF-β and 50 nM 1α,25(OH)₂D₃ for 24 h or pretreated for 12 h with either 1 ng/ml TGF-β (T12h V24h) or 50 nM 1α,25(OH)₂D₃ (V12h T24h), washed with PBS and subsequently treated with the respective other compound for another 24 h. CD69 and 5-LO mRNA levels were determined by qPCR. Statistical analysis was conducted with one way ANOVA and Bonferroni's post test (*P<0.05).</p

Reporter gene analysis of the proximal CD69 promoter.

<p>(A) HeLa cells were transfected with the indicated reporter plasmids and treated with or without 1 ng/ml TGF-β and 50 nM 1α,25(OH)₂D₃ (TV) or 15 ng/ml phorbol 12-myristate 13-acetate (PMA). Luciferase activity was measured 24 h after treatment as described under “Materials and Methods”. The values are expressed as fold induction over untreated cells. Statistical analysis was performed with one sample t-test (*P<0.05; # = 0.0538). (B) HeLa cells were transfected with the indicated reporter plasmids and expression vectors encoding human vitamin D receptor (VDR), retinoid X receptor (RXR), Smad3 and Smad4 or the corresponding empty vectors. Cells were either treated with 1 ng/ml TGF-β and 50 nM 1α,25(OH)₂D₃ (TV) or left untreated (w/o). Luciferase activity was measured 24 h after treatment. The values are expressed as fold inductions over untreated cells cotransfected with empty vectors. Statistical analysis was conducted with one sample t-test (*P<0.05; ***P<0.001). (C) THP-1 or Mono Mac 6 cells were transfected with the indicated reporter plasmids and were treated with or without 1 ng/ml TGF-β and 50 nM 1α,25(OH)₂D₃ (TV). After 12 h (THP-1) and 8 h (Mono Mac 6), luciferase assay was performed. The values are expressed as fold induction over untreated cells.</p

Time dependence of CD69 and 5-LO mRNA induction by TGF-β/1α,25(OH)₂D₃.

<p>(A) Schematic overview of primer positions on CD69 mRNA (exon and intron sizes are not to scale). (B, C and D) Cells were treated for 2, 8, 24 and 48 h with (TV) or without (w/o) TGF-β/1α,25(OH)₂D₃ (1 ng/ml and 50 nM, respectively) and mRNA levels were determined by qPCR. Statistical analysis was done by two way ANOVA and Bonferroni's post test (*P<0.05; **P<0.01; ***P<0.001). The two parts of CD69 gene were analyzed independently. In (B and C), significances as determined by the Bonferroni post test on the data for each part of the gene, compared with the corresponding untreated control, are displayed.</p