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Department of Pharmacology and Toxicology, Magdeburg University, Magdeburg, Germany
Received for publication October 25, 2007.
Accepted for publication December 21, 2007.
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Abstract |
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). The majority of effects is mediated by CB1 and CB2, which belong to the class of G-protein-coupled receptors. Earlier, CB1 was termed "central" cannabinoid receptor, because its expression is most abundant in neuronal cells. In contrast, the CB2 receptor was termed "peripheral" because of its expression in peripheral tissue, including immune tissue (Howlett et al., 2002). Today, this classification needs to be redefined, because CB1 receptors are also expressed in many peripheral tissues, particularly in immune effector cells (Klein et al., 2004; Börner et al., 2007). Moreover, recent reports also suggest the expression of CB2 receptors in neuronal cells (Ashton et al., 2006; Gong et al., 2006). According to the expression profile of their receptors, cannabinoids most prominently modulate neuronal and immune functions. Moreover, the cannabinoid system provides a biochemical basis for multiple neuroimmune interactions. In addition to neuronal functions, it was shown that CB1 receptors are involved in several anti-inflammatory effects of cannabinoids, such as the up-regulation of the interleukin (IL)-1 receptor antagonist and the inhibition of proinflammatory cytokines like IL-6 and IL-8 (Molina-Holgado et al., 2003; Nakajima et al., 2006). Furthermore, it was demonstrated that CB1 receptors are involved in cannabinoid-induced T-helper cell biasing, promoting the differentiation of T-helper cells type 2 (Klein et al., 2004). Despite considerable effects mediated by CB1 receptors in T lymphocytes, their expression level in resting T lymphocytes is very low (Daaka et al., 1995, 1996). Similar results were found by our group; however, we recently reported that expression of the CB1 gene in T lymphocytes may be markedly up-regulated by stimuli, including cannabinoids themselves (Börner et al., 2007). This feedback induction involves several steps and requires, as one of the first steps, cannabinoid-induced expression of IL-4. Once released from the cells, IL-4 then induces transcription of the CB1 gene. IL-4 is the typical cytokine released from T-helper cells type 2 and has anti-inflammatory properties (Curfs et al., 1997). Effects of IL-4 are normally mediated by the transcription factors GATA3 and STAT6. They bind to cis-active, regulatory elements, which are normally located on the target genes' promoters. A typical binding sequence for GATA3 is 5'-AGATAA-3'. All seven members of the STAT transcription factor family bind to similar sequences, the majority of which contain the six base pair palindrome 5'-TTCN2-4GAA-3'. In an attempt to define sequences, which were specific for the individual members of this family, DNA elements with defined, extended eight base pair palindromes (e.g., 5'-TTCCN2GGAA-3') were reported to specifically bind STAT6 (Kraus et al., 2003b). Thus far, little is known about regulatory promoter elements of the human CB1 gene, because its genomic organization, intron/exon structure, and promoter sequences were determined only recently (Zhang et al., 2004). The CB1 gene is located on chromosome 6, where it covers more than 20 kb and consists of 4 exons. The region that codes for two protein isoforms of the gene is entirely located on exon 4. A first analysis revealed a marked promoter activity of sequences flanking the 5' end of exon 1 in neuronal cell lines (Zhang et al., 2004). Basing on these studies, one aim was to characterize the molecular basis for the transcriptional induction of the gene in T cells in response to the cytokine IL-4, which includes the identification of IL-4-responsive promoter elements and the characterization of the transcription factors binding to these sequences. Another aim was to identify regions on the promoter of the human CB1 gene, which may be involved in controlling the basal transcription of the gene in Jurkat cells as a model for T lymphocytes. |
Materials and Methods |
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Reporter Gene Constructs. All reporter gene constructs are based on the pBLCAT2/pBLCAT3 system and contain the CAT reporter gene. Plasmid pBLCAT2 additionally contains the tk minimal promoter (Luckow and Schütz, 1987). The series of phCB1-CAT plasmids contain sequences of the human CB1 promoter instead of the tk promoter in front of CAT. Their construction was as follows: the CB1 gene is contained on human genomic clone RZPDB737B0120D (RZPD, Berlin, Germany). A SacI fragment of this clone spanning from nt -3086 to +142 (referring to the first nucleotide of exon 1; Zhang et al., 2004) of the CB1 gene was excised and cloned into the polylinker of the promoterless pBLCAT3 vector yielding phCB1-CAT-3086. Five prime deletion constructs of this plasmid were constructed either by site specific restriction enzyme deletion (-2490, AfeI; -1950, SpeI; -1653, EagI; and -648, SphI) or by a deletion strategy using the sequence-unspecific enzyme Bal31 (-1099, -955, -875, -559, and -223). Plasmid pS1-tk-CAT contains the sequence 5'-GATCTTAGGTTTCTGCTTGGAATTTCATAGAATTCAGTGAAGAAACCCAACGTCAATCAGAAG-3' cloned into the multiple cloning site 5' to the tk promoter. Plasmid pS2-tk-CAT has the insertion 5'-GATCTGGCTGAGTTGAGATACAAGATTTCCATGGAATACCTTTCACAGGTTGATGCTGACG-3' and pmuS2-tk-CAT has the insertion 5'-GATCTGGCTGAGTTGAGATACAAGATGTCGATGGAGTACCTTTCACAGGTTGATGCTGACG-3' in front of the tk promoter. The inserted DNAs were synthesized by Metabion (Martinsried, Germany). The correct insertion of sequences and the deletions of all plasmids were verified by sequencing.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total RNA from T cells was extracted using the Nucleospin RNA II kit from Macherey-Nagel (Düren, Germany). One microgram of total RNA was used for cDNA synthesis with Moloney murine leukemia virus reverse transcriptase, RNase H minus (Promega, Mannheim, Germany), and diluted to 50 µl. Two microliters of cDNA was used for RT-PCR reactions. Quantitative real-time RT-PCR was performed in a total volume of 20 µl on a LightCycler instrument using the LightCycler-Fast Start DNA Master SYBR Green I kit (both from Roche). Conditions were as follows: β-actin, 5'-GGTCCACACCCGCCACCAG-3' and 5'-CAGGTCCAGACGCAGGATGG-3' primers; preincubation, 8 min at 95°C; 50 cycles:5sat 95°C,5sat 60°C, and 22 s at 72°C. CB1, 5'-CACCTTCCGCACCATCACCAC-3' and 5'-GTCTCCCGCAGTCATCTTCTCTTG-3' primers; preincubation: 8 min at 95°C; 50 cycles:5sat 95°C,5sat 68°C, and 10 s at 72°C. The primers were synthesized by Metabion.
Decoy Oligonucleotide Approach. The transcription factor decoy oligonucleotide approach, its efficiency, and specificity were described in detail in previous publications from our group (Kraus et al., 2003a,b; Börner et al., 2004a,2004b). In general, in the decoy oligonucleotide approach, short double-stranded oligonucleotides with specific binding sequences for transcription factors are introduced into living cells. In the cells, transcription factors interact with the excess of decoy oligonucleotides rather than bind to the natural regulatory motifs of genes. Thus, the decoys selectively disrupt the function of a desired transcription factor. Because the decoys act within living cells, they are highly specific. The sequences of the decoy oligonucleotides used for the experiments described in Fig. 3 were as follows: STAT6, 5'-CTAGTTCTTCTCAGAAGCATATGT-3'; nSTAT6, 5'-CTAGTTGATCTCAGATCCATATGT-3'; GATA3, 5'-CTAGAGGAAGTCTTCAGATAAAAAAGATAACAA-3'; and nGATA3, 5'-CTAGAGGAAGTCTTCACTTAAAAAACTTAACAA-3'. The sequences of the decoy oligonucleotides used for the experiments described in Fig. 5 (S1, S2, muS2) are given in the figure (only the top strand is given). As demonstrated earlier, Jurkat cells take up the decoy oligonucleotides passively (Kraus et al., 2003a). Cells were incubated with decoy oligonucleotides (160 nM) for 16 h before stimulation. Decoy oligonucleotides were synthesized by Metabion (Martinsried, Germany) as complementary single strands.
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). Here, we investigated the direct effect of IL-4 on the transcription of the human CB1 gene. IL-4 induced a significant increase in the rate of transcription of the gene in primary T cells from peripheral human blood (Fig. 2, A and B) and in Jurkat T cells (Fig. 2C). Experiments in Jurkat cells additionally revealed that the transcriptional induction is almost complete after 3 h of IL-4 stimulation.
Transcriptional Induction of CB1 in Response to IL-4 Is Mediated by the Transcription Factor STAT6 but Not GATA3. The next question to address was which transcription factor mediated the induction of the gene by IL-4. In T cells, most transcriptional effects of IL-4 are mediated by the transcription factors STAT6 and GATA3. Therefore, transcription factor decoy oligonucleotides against these factors were introduced into Jurkat cells before the stimulation with IL-4. In the cells, the stimulus-induced transcription factors then bind to the excess of decoy oligonucleotides rather than to the gene's regulatory elements, which results in attenuation of the induction of the gene. The specificity of the decoy oligonucleotides for the desired transcription factors and that of the respective mutated negative control oligonucleotides had been shown in previous reports from our group (Börner et al., 2006, 2007). Decoy oligonucleotides directed against STAT6 completely abolished IL-4-mediated induction of CB1 (Fig. 3), indicating that this factor plays a pivotal role in the induction of CB1 by this cytokine. Control decoy oligonucleotides, in which the STAT6 binding site was destroyed (nSTAT6), had no effect on the induction. In contrast to STAT6, decoy oligonucleotides directed against GATA3 had no effect on the induction of the gene, indicating that GATA3 is not involved in up-regulation of the gene by IL-4.
Characterization of a STAT6 Element on the CB1 Gene Promoter. To characterize IL-4-responsive sequences on the promoter of the human CB1 gene, reporter gene assays in transiently transfected Jurkat cells were performed. A significant inducibility with IL-4 was revealed for construct phCB1-CAT-3086, which comprises 3086 bp of sequences flanking 5' to exon 1 of the CB1 gene (Fig. 4). The loss of inducibility after deletion of 5' sequences up to nt -2490 demonstrated that the IL-4-responsive sequences are located between nts -3086 and -2490 and indicated that these sequences contain one or more STAT6 binding sites. Sequence comparisons within this region suggested two possible STAT6 elements. First, a tandem repeat of the 5'-TTC... GAA-3' motif, which is common to the binding sequences for all members of the STAT family (S1 sequence; Fig. 5A); second, an eight base pair motif, similar to a STAT6 binding sequence defined previously (S2 sequence; Fig. 5A) (Kraus et al., 2003b). Therefore, we next tested whether sequences comprising these motifs were sufficient to confer inducibility with IL-4 on the heterologous tk promoter of pBLCAT2. As shown in Fig. 4, only S2 conferred inducibility with IL-4. A mutated form of this element (muS2 sequence; Fig. 5A) used as a control, in which the binding site for STAT6 was destroyed, and S1 were not responsive to IL-4. Next, it was tested whether S2, when used as a decoy oligonucleotide, would inhibit the IL-4-mediated up-regulation of the gene in Jurkat cells (Fig. 5A). Indeed, S2 decoy oligonucleotides completely abolished the IL-4-inducible transcription of the gene, whereas S1 and muS2 did not, confirming that S2 is the STAT6 site of the CB1 gene. In addition, we tested wether S2 decoy oligonucleotides had any effect on basal transcriptional activity (Fig. 5B). Therefore, construct -3086 was transiently expressed in Jurkat cells in the presence of decoy oligonucleotides. However, neither S2 nor muS2 decoy oligonucleotides had any effect on basal activity of this construct, indicating that STAT6 is not involved in the regulation of basal CB1 transcription.
Inhibition of cAMP Accumulation. The above data show the induction of CB1 at the level of RNA. Next, we addressed the question of whether there was also an increase in the amount of functional CB1 receptor protein after stimulation of Jurkat cells with IL-4. It is known that short-term binding of cannabinoids to their receptors causes inhibition of forskolin-induced cAMP accumulation via coupling to Gi proteins (Felder et al., 1995). First, the kinetics of induction of functional receptors was studied (Fig. 6A). In Jurkat cells prestimulated with IL-4 for 1 and 2 days, R(+)-methanandamide, which preferentially binds to CB1 receptors, caused no inhibition of forskolin-induced cAMP accumulation. In contrast, prestimulation of cells with IL-4 for 3 and 4 days caused a significant inhibition of forskolin-induced cAMP accumulation by R(+)-methanandamide. This effect was blocked by the CB1-selective antagonists AM251 and AM281, indicating that it is mediated by CB1. Furthermore, the effect in cells stimulated for 4 days was dose-dependent (Fig. 6B). In contrast, in naive, non-IL-4-prestimulated cells, R(+)-methanandamide only caused an inhibition of forskolin-induced cAMP formation at the highest concentration tested (Fig. 6B). Although R(+)-methanandamide preferentially binds to CB1 receptors at 500 nM, a possible activation of endogenous CB2 receptors by R(+)-methanandamide was excluded by the addition of the CB2-specific antagonist AM630 (500 nM) to all samples of the experiments shown in Fig. 6. Taken together, these data indicate that the IL-4-prestimulated cells contain significantly more functionally expressed CB1 receptor protein than unstimulated cells.
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). Thus, at least the distal promoter sequences located upstream of approximately -2 kb had contrary effects on basal transcription of the CB1 gene in the immune and the neuronal cells, indicating different control mechanisms in cell types derived from different tissue. In general, the basal expression of CB1 is much weaker in immune cells compared with neuronal cells. This situation is well reflected by the CB1 promoter activity. Thus, basal transcriptional activity of the CB1 promoter in T lymphocytes was lower than that of plasmid pBLCAT2, which lacks additional enhancer elements, whereas the activities in the neuronal cells were reported to be similar to a highly active, SV40 enhancer-driven reporter gene (Zhang et al., 2004). Although the two different sets of experiments may not directly be comparable because of the use of different control-reporter genes, they reflect the much weaker expression rate of the gene in T cells compared with distinct neuronal cells. The silencing effects of the distal promoter regions defined in the experiments presented here may additionally contribute to the weak expression of the gene in T cells. Several transcriptional start sites for the CB1 gene have been reported, and other promoter regions upstream of other exons have been discussed (Zhang et al., 2004). Nevertheless, the marked promoter activity of the sequences flanking exon 1, in both neuronal and T cells, supports the hypothesis that these sequences constitute the main promoter of the gene. The hypothesis is further supported by our findings that these sequences contain a typical promoter element, which is the binding site for STAT6, conferring responsiveness of the gene to IL-4. It is interesting that the IL-4-responsive STAT6 site of the gene is located within a region that silences basal transcription of the gene. However, as indicated in the experiments, in which basal activity of this region was tested in the presence of STAT6 decoy oligonucleotides (see Fig. 5B), STAT6 seems not to influence basal transcription. In general, promoters seem to consist of a marked number of cis-active modules, to which specialized trans-acting factors bind. Some factors mediate stimulus-induced transcription, whereas others regulate basal transcription. Unless a cell is not subjected to a constant level of a certain stimulus, inducible factors contribute little to basal transcription of a gene. STAT factors are specialized factors that respond to cytokines and growth factors. Most transcriptional effects of IL-4 are mediated by the transcription factors STAT6 and GATA3 (Hou et al., 1994; Kraus et al., 2003b; Börner et al., 2006). Experiments using decoy oligonucleotides excluded the involvement of GATA3 in the IL-4-mediated up-regulation of CB1 but clearly established the role of STAT6 (Fig. 3). Once activated by Jak kinases, STAT6 dimers bind to defined regulatory sequences and enhance transcription of the target gene (Hou et al., 1994). Because of the close similarity of the binding sequences for all STAT members, thus far, the decoy oligonucleotide approach, with the oligonucleotides acting in living, intact cells, has proven to be the most selective method for studying DNA-STAT interactions. In contrast, classic in vitro binding assays such as electrophoretic mobility shift assays often gave unspecific results (Kraus et al., 2003b). The relevant regulatory sequence of the CB1 gene promoter, termed S2 in this study, is located at nt -2769. It consists of an extended, eight base pair palindrome and thus sticks to the rule defined previously for STAT6 binding sequences (Fig. 5A; Kraus et al., 2003b). In addition to transcriptional induction of the CB1 gene by IL-4, an increase in functional receptors in response to IL-4 stimulation of Jurkat cells was demonstrated. It was astonishing to find that although IL-4 caused an increase in CB1 mRNA after 3 h, an increase in functional CB1 protein was only observed after 3 days. This is probably due to an overall small amount of CB1 receptor protein in the T cells or to post-translational effects needed for full receptor activity. From a physiological standpoint, CB1 receptors in T cells may participate in the regulation of the T-helper cell balance. It is known that cannabinoids alter this balance in favor of the type 2 phenotype. Among other mechanisms, such as the inhibition of the expression of the T helper cell type 1 cytokine interferon-
, cannabinoids lead to the induction of IL-4, which is a key factor for the T-helper cell type 2 differentiation and its maintenance (Curfs et al., 1997; O'Garra and Arai, 2000). This cannabinoid-induced induction of IL-4 is mediated by CB2 receptors, which are abundantly expressed in resting T lymphocytes (Börner et al., 2006). IL-4 then in turn induces genes, which further promote T-helper cell type 2 development and, as reported now, the CB1 receptor gene. CB1 additionally participates in T-helper cell biasing. It was shown that cannabinoids, via CB1 receptors, inhibit the expression of IL-12Rβ2, which is the binding and signal-transducing component of the IL-12 receptor complex, which is necessary for T-helper cell type 1 development (Klein et al., 2000, 2004). There is an increasing awareness that cannabinoids, via CB1 receptors, not only regulate neuronal functions but also potently modulate immune functions. Furthermore, being expressed on cells of the nervous and the immune system, CB1 receptors enable communication between the two systems and allow neuroimmune interactions. The extent of the CB1-mediated cross-talk may be regulated at the level of gene expression (e.g., by IL-4).
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Footnotes |
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ABBREVIATIONS: IL, interleukin; AM630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-methoxyphenyl)methanone; CAT, chloramphenicol acetyl transferase; tk, herpes simplex thymidine kinase; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; STAT, signal transducer and activator of transcription; AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-mo rpholinyl-1H-pyrazole-3-carboxamide; nt, nucleotide(s); kb, kilobase(s); ELISA, enzyme-linked immunosorbent assay; bp, base pair.
Address correspondence to: Dr. Jürgen Kraus, Department of Pharmacology and Toxicology, Magdeburg University, 44 Leipzigerstrasse, 39120 Magdeburg, Germany. E-mail: juergen.kraus{at}med.ovgu.de
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