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.


Introduction
The transmembrane lectin CD69 is best characterized and widely used as an early T-lymphocyte activation marker that is expressed upon inflammatory stimuli [1]. A major function of CD69 is to shut down lymphocyte egress from lymphoid organs via inhibition of sphingosine 1-phosphate signalling [2]. However, CD69 expression has not only been found on lymphocytes, but on all bone marrow-derived cells except erythrocytes (reviewed in [1]). Regarding its expression on monocytic cells, one report exists that describes constitutive expression on CD14 positive monocytes [3], but in a subsequent study only 10% of total monocytes were found to be positive for CD69. In that study the basal level of CD69 was enhanced by stimulation with leptin, lipopolysaccharide or phorbol 12-myristate 13-acetate (PMA) [4].
With respect to its role in monocytes, CD69 has been functionally linked to 5-lipoxygenase (5-LO), the key enzyme in the conversion of arachidonic acid to leukotrienes [3]. Leukotrienes are potent lipid mediators involved in inflammatory disorders, including asthma, arthritis as well as allergic reactions, and have been implicated in the pathogenesis of atherosclerosis and different neoplasms [5]. Cross-linking of CD69 on monocytes coincided with Ca 2+ influx, arachidonic acid release and leukotriene B 4 production [3]. Moreover, induction of apoptosis by anti-CD69 antibodies in LPS-stimulated human monocytes or monocytic THP-1 could be blocked by 5-LO inhibitors [6]. 5lipoxygenase is a known TGF-b/1a,25(OH) 2 D 3 target gene in monocytes [7,8], and several other genes are established to be regulated by this combination of chemically unrelated mediators [9]. The signalling pathways of TGF-b and 1a,25(OH) 2 D 3 alone are well understood, respectively. The lipophilic hormone 1a,25(OH) 2 D 3 acts on mRNA expression via its nuclear receptor, the vitamin D receptor (VDR). Together with its heterodimeric binding partner, the retinoid X receptor (RXR), VDR binds to vitamin D responsive elements (VDREs) in regulatory DNA regions. Upon ligand binding, a complex of coactivator proteins is recruited, which subsequently acts on the basal transcription machinery [10]. On the other hand, the cell-impermeant peptide TGF-b signals through a specific cell surface receptor, the TGF-b receptor complex. Activation regulates mRNA biosynthesis either via the canonical Smad transcription factor pathway [11], or via non-Smad signalling pathways in which TGF-b activated kinase 1 (TAK1) is a central component and other mitogen activated protein kinases (MAPKs) like p38, Jnk and Erk are major players [11,12,13]. Smad proteins bind to their cognate binding elements on the DNA in cooperation with other transcription factors, where the complexes interact with the basal transcription machinery, whereas the signals of the non-Smad pathways are channelled through specific transcription factors that receive the signals of the different kinases. TGF-b and 1a,25(OH) 2 D 3 share common effects on cell growth and differentiation, but the exact mechanisms of their interaction still remain to be elucidated.
Regarding mechanisms of CD69 gene regulation in monocytes, no systematic studies are available to date. We report here that CD69 is a novel TGF-b/1a,25(OH) 2 D 3 target gene that is cell type specifically regulated in monocytic cells, but exhibits differences in the profile and the underlying mechanisms of gene regulation as compared to 5-LO. The main distinctions are that CD69 gene regulation by TGFb/1a,25(OH) 2 D 3 , in contrast to 5-LO, depends on Smad3 and seems to be regulated by a signalling axis involving TAK1-mediated p38 activation.

Real-time PCR (RT-qPCR)
Total RNA was extracted from the cells using TRIzol reagentH (Life Technologies) following the manufacturer's instructions, with some modifications: Cell pellets were lysed with TRIzol reagent using a 1 ml syringe with a 20 G Sterican hypodermic needle (Braun, Germany) and incubated at room temperature for 10 minutes. Then, 200 ml chloroform were added, samples were vortexed, incubated at room temperature for 15 minutes and centrifuged at 15000 g, 45 min, at 4uC. Clear supernatant (450 ml) was transferred into a new tube and 5 ml 3 M sodium acetate pH 6.5 and 500 ml isopropanol were added. After vortexing, RNA was allowed to precipitate for 3 h at -20uC. Samples were centrifuged 1 h at 15000 g, 4uC. RNA pellets were washed with 1 ml 75% ethanol and centrifuged 1 h at 15000 g, 4uC. Supernatants were removed and pellets were allowed to dry for a few minutes and dissolved in 50 ml DEPC-treated water (55uC, 10 min). RNA was quantified spectrophotometrically at 260 nm using a NanoPhotometerH (Implen, Munich, Germany). RNA integrity was verified by agarose gel electrophoresis. Subsequently, RNA was treated with DNaseI (Fermentas) to eliminate genomic DNA contamination. For cDNA synthesis, a constant amount of total RNA (2 mg) was reversely transcribed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems) following the manufacturer's instructions. Absolute quantities (expressed as mRNA copies of the target gene per 10 6 mRNA copies of the reference gene) were calculated from standard curves constructed from serial dilutions of linearized plasmids, which contained 100 ng/ml yeast tRNA as carrier. Data for standards and corresponding samples were always generated in the same PCR run. Standard plasmids were: pcDNA-CD69 (encoding full-length human CD69 cDNA), pcDNA Exon 1-Intron 1 and pcDNA Intron 1 (containing parts of the CD69 gene) pcDNA-B2M (containing parts of the human B2M coding sequence), pcDNA3.1-5LO (encoding full-length human 5-lipoxygenase), pCGN-Smad3 (encoding full-length human Smad3). B2M was chosen as reference gene after it had been determined as the most stable reference gene out of 5 candidates (b-actin, B2M, UBC, GAPDH, HPRT1) using geNorm software on datasets generated from THP1 and Mono Mac 6 cells that were treated with or without TGFb/1a,25(OH) 2 D 3 . B2M mRNA levels remained unchanged upon TGFb/1a,25(OH) 2 D 3 or PMA treatment (data not shown). Primer pairs used for qPCR are listed in Table 2.
Mono Mac 6 and THP-1 were transfected by electroporation using BioRadH Gene Pulser II.
Electroporation of Mono Mac 6 was performed according to Klan et al. [20] with some modifications. Cells were grown for 1 d, washed with PBS pH 7.4 and resuspended in RPMI without additives at a density of 4610 6 per ml. 300 ml of cell suspension were transferred into a 0.4 cm electroporation cuvette (Invitrogen) and 50 ml of plasmid solution, containing 20 mg reporter plasmid and 1 mg pRL-SV40 (Promega) as internal control, were added. Electroporation was conducted at 975 mF and 200 V. Cuvettes were immediately transferred on ice for 20 minutes. Cells were transferred into 50 ml prewarmed growth medium without phenol red and were treated. Eight hours after treatment cells were harvested, washed with PBS and luciferase assay was performed as described for HeLa cells.
Minor modifications for transfection of THP-1 were applied: Electroporation was conducted in BTXpress Electroporation Solution at 250 V and 950 mF.
Cells were transferred into growth medium immediately after electroporation and luciferase assay was performed 12 h after treatment.

Lentiviral gene silencing
293T cells were seeded in 24-well plates at a density of 6610 4 cells per well. After 24 h, cells were transfected using the calcium phosphate method. Transfection mixes contained 10 mg of MISSIONH shRNA TRCN0000330056 plasmid with pLKO.1puro backbone (Sigma Aldrich) plus 6.5 mg and 3.5 mg of the packaging plasmids pCMVdR8.91 [21] and pMD2.G (www. tronolab.epfl.ch), respectively. Both packaging plasmids were kindly provided by Dr. Manuel Grez, Georg-Speyer-Haus, Frankfurt, Germany. Four hours after transfection, medium was replaced with 1 ml THP-1 growth medium. Virus production was allowed for 72 h. Then, the supernatant of transfected cells was collected and filtered trough a 0.22 mm filter (Roth, Karlsruhe). Four hours prior to transduction, THP-1 cells were seeded in 24well plates in growth medium containing protaminsulfate (4 mg/ ml, Calbiochem). Each well contained 3 6 10 5 cells in 0.5 ml medium. Six wells were treated with 50 ml of 293T supernatant, respectively. Supernatant of untransfected 293T was used as control for selection. In order to obtain a negative control cell line, 5 ml Mission non Target shRNA control transduction particles (Sigma Aldrich) were used per well. Plates were centrifuged for 90 minutes at 10006 g, 32uC. Subsequently, medium was added to a final volume of 1 ml in each well and cells were incubated at 37uC, 5% CO 2 for 3 days.
After incubation, cells from each 6 wells were pooled, counted, washed with PBS pH 7.4 and seeded in fresh medium with 1.5 mg/ml puromycin at a density of 3610 5 cells/ml. After two weeks of puromycin selection, cells were used for experiments.

Statistics
Data are presented as mean + standard deviation (SD), derived from a minimum of three independent experiments. Statistical analysis was conducted using the software Graph Pad Prism, version 5.0 (GraphPad Software, San Diego, California, USA). Significance of differences between two groups was analyzed using two tailed, unpaired t-tests. Data expressed as fold changes relative to a reference group were analyzed using one sample t-tests. Analysis of variance (ANOVA) was performed for significance analysis of more than two groups, combined with multi comparison post tests (Dunnett's or Bonferroni's, as indicated in the respective figure legends).
Induction of CD69 mRNA expression by TGF-b/ 1a,25(OH) 2 D 3 occurs rapidly and in a concentration dependent manner To examine the concentration dependence of CD69 mRNA induction, THP-1 cells were either treated with TGF-b 1 ng/ml in combination with 1a,25(OH) 2 D 3 in concentrations between 50 pM and 50 nM, or conversely with 50 nM 1a,25(OH) 2 D 3 and TGF-b in varying concentrations from 1 pg/ml to 1 ng/ml. This was compared with single TGF-b (1 ng/ml) or 1a,25(OH) 2 D 3 (50 nM) treatments, or with untreated controls ( Fig. 2A). Neither TGF-b nor 1a,25(OH) 2 D 3 alone induced CD69 mRNA expression. For the combination of TGF-b and 1a,25(OH) 2 D 3 , a TGF-b concentration of 0.1 ng/ml or higher was required to induce CD69 mRNA expression together with 1a,25(OH) 2 D 3 . When different concentrations of 1a,25(OH) 2 D 3 were combined with TGF-b 1 ng/ml, 1a,25(OH) 2 D 3 acted concentration dependently. In contrast, the presence of TGF-b was not a prerequisite for 5lipoxygenase mRNA induction by 1a,25(OH) 2 D 3 , as 1a,25(OH) 2 D 3 alone led to a 7.5-fold increase in mRNA expression (Fig. 2B). As observed for CD69, 1a,25(OH) 2 D 3 also acted concentration dependently on 5-LO mRNA expression in presence of TGF-b in a concentration of 1 ng/ml.
In order to analyze the time course of CD69 mRNA induction, THP-1 cells were treated with TGF-b 1 ng/ml in combination with 1a,25(OH) 2 D 3 50 nM for 2, 8, 24 or 48 h. CD69 mRNA abundance was analyzed by RT-qPCR using two primer pairs. Alongside the primer pair specific to the proximal part (exon 1/2-2), which was used for the initial results ( Fig. 1-Fig. 2B), an additional primer pair binding to the distal (exon 4/5-5/39UTR) region of the CD69 gene was employed (Fig. 3A). In comparison to CD69 mRNA levels, 5-LO mRNA expression was monitored. CD69 mRNA expression was induced rapidly, rising from undetectable levels to 250 mRNA copies per 10 6 B2M mRNAs after 2 h (Fig. 3B). The increase in expression continued almost linearly up to 24 h and reached a plateau afterwards. In contrast, 5-lipoxygenase gene induction was not detectable before 2 h after treatment (Fig. 3D). For transcripts comprising the distal part of the CD69 gene, a basal level of 270 mRNA copies per 10 6 B2M was detected in untreated cells, which was raised exactly in parallel with the transcripts including the proximal part. In addition, primary CD69 transcripts were quantified using a primer pair specific for the sequence around the junction of exon 1 and intron 1 and one encompassing the first intron of the CD69 gene. The kinetics of the increase in nascent pre-mRNA copy numbers (Fig. 3C) correlated with the increase in mature RNA species (Fig. 3B) with the ratio between the two species averaging at 1:5. Taken together, the rapid onset of CD69 mRNA induction by TGF-b/1a,25(OH) 2 D 3 , where primary and mature transcripts rise in parallel, indicates that CD69 is a primary TGF-b/ 1a,25(OH) 2 D 3 target gene in monocytic cells that is regulated on the level of transcriptional activation by the combination of these compounds.

Simultaneous presence of TGF-b and 1a,25(OH) 2 D 3 is required for CD69 and 5-LO mRNA induction
In order to investigate whether induction of CD69 and 5-LO mRNA by TGF-b and 1a,25(OH) 2 D 3 requires the simultaneous presence of both agents, or if one of the agents alone was capable of priming the cells for the action of the other, we performed sequential treatments. For this, we treated THP-1 with either agent alone for 12 h, exchanged medium and incubated with the respective other compound for another 24 h. As controls, we used cells treated with TGF-b/1a,25(OH) 2 D 3 for 24 h and untreated cells. We found that pre-treatment with neither of the compounds induced mRNA levels to the same extent as simultaneous incubation (Fig. 4). For the CD69 gene, only pre-treatment with 1a,25(OH) 2 D 3 followed by TGF-b treatment led to modestly enhanced mRNA levels compared to untreated control (Fig. 4A). In contrast, levels of 5-LO mRNA were increased moderately in both ways of pre-treatment as compared to untreated cells (Fig. 4B), however the extent of induction was only slightly higher than that obtained by incubation with the single compounds as observed in the concentration series (Fig. 2B). Thus, combined treatment is necessary for strong induction of both mRNA species and pre-treatment more or less reflects the setting of single treatment.  by TGF-b/1a,25(OH) 2 D 3 , respectively, but not by PMA. The positive control construct p3TP-Lux, which contains adjacent SBEs and TPA/PMA responsive elements (TREs), was activated 3-fold by TGF-b/1a,25(OH) 2 D 3 and 7-fold by PMA (Fig. 5A).
Next, the reporter constructs were either cotransfected with expression vectors encoding the transcription factors vitamin D receptor (VDR), retinoid X receptor (RXR), Smad3 and Smad4, or with empty vectors. Luciferase activity was increased for all  constructs by coexpression of the transcription factors, and the inducibility of the control constructs for (1a,25(OH) 2 D 3 ) and TGFb response increased dramatically, but induction of CD69 promoter constructs by TGF-b/1a,25(OH) 2 D 3 was still not detectable (Fig. 5B). Similarly, CD69 promoter constructs did not respond when THP-1 and Mono Mac 6 as monocytic cells were transfected (Fig. 5C).
From this, we concluded that (1) regulatory elements located outside the CD69 proximal promoter regions might be responsible or needed additionally in order to activate CD69 mRNA transcription (2) complex chromatin remodelling events, that can not be properly reproduced in transient reporter assays might be involved in TGF-b/1a,25(OH) 2 D 3 action, or (3) that TGF-b/ 1a,25(OH) 2 D 3 treatment acts on CD69 mRNA stability.

CD69 mRNA stability is not influenced by TGF-b/ 1a,25(OH) 2 D 3
In order to assess whether the combination of TGF-b and 1a,25(OH) 2 D 3 acts on CD69 mRNA stability, qRT-PCR analysis was carried out after incubation with the transcription inhibitor actinomycin D. As THP-1 cells were not suitable for this approach due to their lack of basal CD69 mRNA expression, Mono Mac 6 cells were used. Mono Mac 6 cells were preincubated with TGFb/1a,25(OH) 2 D 3 or left untreated for 24 h. Then 1 mg/ml actinomycin D was added and CD69 mRNA level was analyzed at the indicated time points. CD69 mRNA half life was comparable (less than 2 h) between treated and untreated samples (Fig. 6A).
The 39UTR region of the CD69 gene is known to contain destabilizing AU-rich elements [24], and stabilization of mRNAs containing AU-rich elements by TGF-b has been shown [25]. Therefore, we inserted the CD69 39UTR into a SV40-promoter driven luciferase reporter vector and analyzed changes in relative luciferase activity as compared to the empty vector with or without TGF-b/1a,25(OH) 2 D 3 treatment. As expected, insertion of the CD69 39UTR decreased luciferase activity 5.8-fold, but this effect could not be reverted by TGF-b/1a,25(OH) 2 D 3 (Fig. 6B).

Induction of CD69 mRNA expression by TGF-b/ 1a,25(OH) 2 D 3 depends on Smad3
Next, we analyzed whether TGF-b contributes to mRNA upregulation via the classical Smad pathway. For this purpose, we employed lentiviral shRNA for functional knockdown of Smad3 expression in THP-1 cells. At first, five independent shRNA constructs were analyzed for Smad3 mRNA reduction by transient transfection of 293T cells, and the most effective construct was subsequently used to generate a stable cell line (data not shown). A negative control cell line was established using non target shRNA. The Smad3 shRNA construct led to a 37% and 73% reduction in mRNA and protein level, respectively, compared to untransduced cells ( Fig. 7A and B).
The cells were incubated with TGF-b/1a,25(OH) 2 D 3 for 24 h, and CD69 and 5-LO mRNA levels were analyzed by RT-qPCR. Smad knockdown resulted in a 71% decrease of CD69 mRNA induction by TGF-b/1a,25(OH) 2 D 3 . In contrast, induction of 5-LO mRNA was not altered significantly (Fig. 7C). Therefore, we clearly demonstrate that CD69 mRNA induction by TGF-b/ 1a,25(OH) 2 D 3 depends on Smad3, whereas 5-LO upregulation seems to be independent of Smad3.  are major routes of non-Smad pathways of TGF-b signalling [11,12], and PKC inhibitors were employed due to the fact that the proximal CD69 promoter is known to contain AP1 binding sites [26] and is responsive to phorbol ester stimulation [23]. Moreover, AP1 is a known interaction partner of activated Smad3/4 as TGF-b effector proteins [27].
With the PKC inhibitors calphostin C and staurosporine, we found that neither calphostin C at 0.1 or 0.2 mM nor staurosporine 10 nM potently blocked CD69 mRNA induction, only at 100 nM staurosporine a 60 and 78% reduction was detectable for proximal and distal parts of CD69 mRNA, respectively. 5-LO mRNA abundance was not affected by both inhibitors (Fig. 8D).
Taken together, TGF-b/1a,25(OH) 2 D 3 act differently on CD69 and 5-LO mRNA regulation regarding the involvement of the MAPK pathway, with inhibition being prominently detected for the CD69 gene. It is unlikely that PKC plays a major role in the regulation of CD69 gene expression by TGF-b/1a,25(OH) 2 D 3 in monocytic cells.

TGF-b/1a,25(OH) 2 D 3 induce p38 phosphorylation that is blocked by TAK1 inhibition
Our inhibitor studies revealed that inhibitors of TAK1 and p38 MAPK reduced CD69 mRNA induction to exactly the same extent ( Fig. 8B-C). This is in line with findings that activation of p38 by TGF-b is mediated by TAK1 in HEK293 cells [28]. Therefore, we first analyzed whether TGF-b/1a,25(OH) 2 D 3 activate p38 MAPK by phosphorylation in THP-1 cells. Interestingly, we found that only the combination of TGF-b and 1a,25(OH) 2 D 3 , but not the single compounds, led to prominent p38 phosphorylation (Fig. 9A). Subsequently, we tested whether TAK1 inhibition abrogates p38 phosphorylation using the TAK1 inhibitor oxozeaenol and the control compound zeaenol in TGFb/1a,25(OH) 2 D 3 treated THP-1 cells. Oxozeaenol almost completely inhibited p38 phosphorylation, whereas zeaenol had no effect on the phosphorylation status (Fig. 9B).

Combined effects of TGF-b/1a,25(OH) 2 D 3 and PMA on CD69 mRNA induction revert over time
In lymphocytes, CD69 expression has been studied extensively, and it is well known that the almost undetectable CD69 mRNA levels in these cells are induced strongly upon PMA treatment [23,29,30,31]. We determined absolute CD69 mRNA levels in PMA treated Jurkat, MOLT-4 and Rec-1 cells and observed several hundred-fold higher mRNA levels compared to those in TGF-b/1a,25(OH) 2 D 3 treated monocytic cells ( Fig. 10A; Fig. 1A). Analogously, PMA treatment of monocytic cells led to significantly lower CD69 mRNA levels than in equally stimulated lymphocytes (Fig. 10B). In Mono Mac 6 cells, CD69 mRNA levels were slightly higher after PMA treatment compared to incubation with TGF-b and 1a,25(OH) 2 D 3 , whereas in THP-1 cells TGF-b/ 1a,25(OH) 2 D 3 treatment led to 4.8 fold higher CD69 mRNA levels than PMA treatment ( Fig. 10B; Fig. 1A).
We investigated the possibility that TGF-b/1a,25(OH) 2 D 3 act on the CD69 gene locus of monocytic cells by converting the chromatin architecture into a permissive state and therefore prime cells for PMA response. To elucidate this, we treated THP-1 cells with TGF-b/1a,25(OH) 2 D 3 or PMA alone for 48 h, or cells were sequentially incubated with or without TGF-b/1a,25(OH) 2 D 3 for 24 h, and PMA was added for further 24 h.
For further analysis, we performed a time course experiment with combined treatment of TGF-b/1a,25(OH) 2 D 3 and PMA, as well as either treatment alone, or no treatment (Fig. 10D). Remarkably, we observed a strong synergism of TGF-b/ 1a,25(OH) 2 D 3 and PMA after 2 and 4 h, whereas PMA abolished the effect of TGF-b/1a,25(OH) 2 D 3 after 12 and 24 h of treatment. Discussion CD69 has been functionally linked to 5-lipoxygenase, the key enzyme in leukotriene biosynthesis, in a study with CD14-positive monocytes [3]. Both CD14 and 5-lipoxygenase are known target genes for TGF-b and 1a,25(OH) 2 D 3 in this cell type [7,8,32,33]. In contrast, at present no data are available on the mechanism of CD69 gene regulation in monocytic cells. Here, we demonstrate that CD69 is a novel TGF-b/ 1a,25(OH) 2 D 3 target gene and is therefore coexpressed with 5lipoxygenase, but that individual mechanisms account for TGF-b/ 1a,25(OH) 2 D 3 action regarding the two genes.
We could find that CD69 mRNA upregulation by TGF-b/ 1a,25(OH) 2 D 3 occurs cell-specifically in monocytes, but not in Tor B-cells. This expression pattern conforms to the 5-LO gene, where TGF-b/1a,25(OH) 2 D 3 do not induce expression in T-and B-cells either (Dieter Steinhilber, unpublished data). It must however be noted as a difference between the two genes that Tcells do express CD69 upon cellular activation by different signalling molecules [29], but are 5-LO negative cells [34]. The similarities in the expression pattern could indicate that the proteins have a monocyte-specific functional relationship.
The two monocytic cell lines THP-1 and Mono Mac 6 differed in the occurrence of basal CD69 mRNA expression; no CD69 mRNA could be detected in untreated THP-1 cells whereas Mono Mac 6 cells exhibited a basal level of CD69 mRNA. It has been shown that these two cell lines diverge in their differentiation status, with Mono Mac 6 being more differentiated towards mature monocytes than THP-1 [35]. Therefore, a basal level of CD69 expression is possibly acquired during monocyte maturation.
We observed marked differences regarding CD69 and 5lipoxygenase mRNA induction by TGF-b/1a,25(OH) 2 D 3 . In both cases, the combination of TGF-b and 1a,25(OH) 2 D 3 was required for the maximum effect, but in case of 5-LO also 1a,25(OH) 2 D 3 alone was sufficient to induce mRNA expression, whereas CD69 expression strictly depends on the presence of TGF-b ( Fig. 2A and B). This is also reflected by the results of sequential treatment (Fig. 4). The results show that cooperation between TGF-b/1a,25(OH) 2 D 3 depends on simultaneous presence of both agents and neither of the agents alone is capable of priming for the full effect. Therefore, for both genes, combined action of TGF-b and 1a,25(OH) 2 D 3 is rather a primary than a secondary effect. Furthermore, we detected kinetic differences between the two genes. CD69 mRNA abundance was induced within 2 h, and thus increased more rapidly than 5-LO mRNA levels, where a significant induction could only be observed later than 2 h. The rapid onset of CD69 mRNA induction also suggests that CD69 is a primary TGF-b/1a,25(OH) 2 D 3 target gene. Methodologically, primary and secondary target genes are often distinguished by the use of protein synthesis inhibitors like cycloheximide. However, due to the fact that the CD69 39UTR contains destabilizing AU-rich elements [24], and such mRNAs are known to be strongly stabilized by cycloheximide [36], this approach was not suitable for the CD69 gene. On the other hand, TGF-b has been shown to increase the half-life of mRNAs which contain AU-rich elements in their 39UTRs [25,37]. Therefore, we investigated whether CD69 mRNA induction by TGF-b/ 1a,25(OH) 2 D 3 is based on mRNA stabilization. However, neither half life studies using the transcription inhibitor actinomycin D nor 39UTR analysis using reporter genes indicated an influence of TGF-b/1a,25(OH) 2 D 3 on CD69 mRNA stability. Analysis of pre-mRNA species revealed that the combination of TGF-b and 1a,25(OH) 2 D 3 directly activates CD69 transcription (Fig. 3C). Regarding the quantification of mature mRNA species over time, we observed that transcripts including the proximal (exons 1-2) and the distal (exon 4-39UTR) part of the CD69 gene increased in parallel upon TGF-b/1a,25(OH) 2 D 3 treatment, however, the number of transcripts comprising the distal part was uniformly higher for all time points, for both treated and untreated cells (Fig. 3B). Several reasons could possibly account for this observation. It is conceivable that a constantly expressed transcript comprising the distal part originates from a second gene that overlaps with the CD69 gene, or from an alternative transcription start site within the distal part of the CD69 gene. Moreover, alternatively spliced transcripts lacking proximal parts, as described for the porcine CD69 gene [38], come into consideration. However, such an overlapping gene, or such transcripts, respectively, have not been described as yet. Furthermore, slow degradation of CD69 mRNA starting from the 59-end may render species that only comprise the distal part.
To further analyze the mechanisms of TGF-b/1a,25(OH) 2 D 3 action, we used (a) lentivirus-delivered stable gene silencing of Smad3 as the central component of Smad-dependent TGF-b signalling, (b) pharmacological inhibitors, (c) reporter gene assays with constructs containing CD69 promoter fragments of different length.
First, we could clearly show by stable silencing of Smad3 expression in THP-1 cells that TGF-b/1a,25(OH) 2 D 3 -mediated CD69 induction depends on Smad3, whereas this is not the case for 5-LO. Regarding 5-LO expression, this is in contrast to findings that overexpression of Smads3/4 induces plasmid-based 5-lipoxygenase expression in reporter gene assays [39]. These differences may be attributed to the fact that the reporter gene assay was conducted in a heterologous cell system and that a Phosphorylated p38 compared to whole p38 protein was analyzed by western blot. Relative protein amount of TGF-b/1a,25(OH) 2 D 3 treated cells was set to 1. Statistical analysis of (A) and (B) was done with one sample t-test (*P,0.05; **P,0.01). doi:10.1371/journal.pone.0064635.g009 CD69 Is a TGFbeta/Vitamin D3 Target Gene reporter construct does not reflect the genomic situation in a 5-LO positive cell as it was used in our approach. Using pharmacological inhibitors, we could clearly show for CD69 that the TGF-b/ 1a,25(OH) 2 D 3 signal completely depends on stimulation of a specific receptor, the TGF-b receptor I. Subsequently, we used inhibitors of TAK1 and the three major MAPK pathways p38 MAPK, Jnk and Erk. Our data reveal that the classical MAPKs and TAK1 play a role in CD69 regulation by TGF-b/ 1a,25(OH) 2 D 3 . Interestingly, TAK1 and p38 MAPK inhibition diminished CD69 induction to the same extent, which pointed to a direct dependence between these two kinases, as demonstrated for TGF-b treated HEK293 cells [28]. This interplay could indeed be confirmed by Western Blot analysis of differences in p38 phosphorylation in the presence or absence of TAK1 inhibitor. Taken together, this suggests that TGF-b/1a,25(OH) 2 D 3 act on CD69 mRNA expression via TAK1-mediated p38 activation.
Regarding 5-LO regulation and MAPK involvement, we only found 50% inhibition by the Jnk inhibitor. The 5-LO promoter contains two AP1 response elements, at pos. 22990 to 22984 (TGTCTCA [40]) and at pos. 23246 to 23240 (TGGCTCA [41]) from the translational start site, which could potentially be addressed by activated c-Jun. However, no activation by TGF-b/ 1a,25(OH) 2 D 3 of 5-LO promoter fragments containing these elements could be seen in transient reporter assays [42]. We obtained the same negative result for CD69 promoter fragments (Fig. 5), which contain AP1 sequences near the transcription start site [23]. It is possible that Jnk-mediated protein activation, classically of c-Jun, is not sufficient to address the AP1 elements in the promoters without activation of interacting proteins like c-fos, which is not a Jnk substrate [43]. Jnk might therefore act posttranscriptionally on CD69 and 5-LO mRNA expression. It is also possible that further proteins are needed in the multiprotein complex interacting with the general transcriptional machinery that are not present under the circumstances of transient reporter assays.
The reporter constructs that were used to analyze the effect of TGF-b/1a,25(OH) 2 D 3 on the CD69 promoter included the sequences between positions 20.65 to +0.08 kb and 22.26 to +0.08 kb of the CD69 gene (relative to the transcription start site), respectively. The shorter construct contains two putative vitamin D response elements and two additional potential VDREs are located in the longer construct, as predicted by the in silico promoter analysis program ''NHR Scan'' [44]. Five putative Smad binding elements (SBE) are located in the shorter construct and two additional sites can be found in the 22.26 kb construct, when the sequence is analyzed using the SBE consensus sequence CAGAC, as postulated by Qin et al. [45].
The fact that the constructs comprising proximal promoter regions were not responsive to 1a,25(OH) 2 D 3 is in line with recent findings on other 1a,25(OH) 2 D 3 target genes, for which it was shown that results from assays using proximal promoter constructs were only modest or difficult to interpret. It has been put forward that multiple regulatory sequences, often located many kilobases from the TSS, are needed for expression control [46]. Recent findings on Smad action have established that Smad action depends on cooperation with ''master transcription factors'' [47], which are as yet undefined in monocytes and may be located at other regions of the CD69 gene or its 59 flanking sequence.
Both, the combination of TGF-b/1a,25(OH) 2 D 3 and PMA, are described to induce differentiation events in cells of the monocytic lineage [48,49]. As constitutive expression of CD69 in CD14 positive monocytes is described [3], it is possible that induction of CD69 is a differentiational event. This is in line with the different basal CD69 mRNA levels in Mono Mac 6 and THP-1 together with the fact that THP-1 cells in contrast to Mono Mac 6, are negative for the monocytic marker CD14 [50,51]. However, the mechanisms that lead to CD69 mRNA induction by TGF-b/ 1a,25(OH) 2 D 3 and PMA seem to be diverse and synergism is strongly dependent on the time point that is considered. In conclusion, we show here that CD69 (mRNA) expression is cellspecifically regulated by TGF-b/1a,25(OH) 2 D 3 in monocytic cells. This feature is in common with the gene encoding 5lipoxygenase, the key enzyme in leukotriene formation. The parallelism supports the idea of a functional relation between the two proteins, which needs to be examined in further studies. Moreover, our results demonstrate that the combination of the physiological mediators TGF-b and 1a,25(OH) 2 D 3 act on different genes by individual mechanisms and kinetics.