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Department of Pharmacology and Toxicology, University of Magdeburg, Magdeburg, Germany
Received June 10, 2004; accepted September 21, 2004
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Abstract |
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-opioid receptor mRNA levels are not influenced. The mRNA increase in these cells is followed by an increase in µ-opioid receptor-specific binding. Using transcription factor decoy oligonucleotides, direct evidence was provided that the up-regulation of µ-opioid receptor mRNA in intact cells is dependent on the transcription factors signal transducers and activators of transcription 1 (STAT1) and STAT3, whereas other transcription factors, such as activator protein-1, nuclear factor (NF)-
B, or NF-interleukin-6 are not involved. STAT1 and STAT3 bound to a site located at nucleotide -1583 on the promoter of the human µ-opioid receptor gene, as shown by transient transfection experiments, electrophoretic mobility shift assays, and transcription factor decoy oligonucleotides. A mutation analysis of the 5'-TTCATGGAA-3' STAT1/3 element (palindrome underlined) was performed to determine nucleotide residues that are necessary for the binding of STAT1 and STAT3. It suggested that only the palindromic half sides and the two adjacent central nucleotides are required. Neither mutation of the nucleotides outside the palindrome nor mutation of the central nucleotide affected STAT1/3 binding.
; Leon, 2002). In addition, it induces a variety of opposing effects in the nervous system that are not well understood. On the one hand, interleukin-6 is among the candidates of inflammatory mediators involved in the pathology of Alzheimer's disease. For example, it increases amyloid precursor protein expression in neurons (Del Bo et al., 1995) and synergistically contributes to neuronal damage caused by 
-amyloid peptide and N-methyl-D-aspartate (Qiu and Gruol, 2003). On the other hand, several reports demonstrated beneficial effects of interleukin-6 in terms of neuronal repair and neuroprotection. Nerve injury in the peripheral and central nervous system results in a rapid elevation in interleukin-6 levels (Gadient and Otten, 1997; Flatters et al., 2003), which accelerates post-traumatic neuronal regeneration (Hirota et al., 1996; Swartz et al., 2001). In addition, ischemic brain damage could be reduced by intracerebroventricular injection of interleukin-6 (Loddick et al., 1998). Furthermore, this cytokine is a neuroprotectant in cerebral ischemia in transgenic animal models with central nervous system-targeted interleukin-6 expression (Penkowa et al., 2003). There is increasing evidence that interleukin-6 also elicits beneficial effects in the regulation of neuropathic and inflammatory pain. Thus, spinal administration to rats with nerve injury results in antinociceptive effects, indicating that this cytokine is a modulator of neuropathic pain (Flatters et al., 2003). In addition, interleukin-6 was shown to induce analgesic effects in an animal model of inflammation contributing to the activation of the endogenous opioid system, which is induced in response to peripheral inflammation (Czlonkowski et al., 1993). This inflammation-induced analgesia involves the release of endogenous opioid peptides from immune cells that activate their cognate receptors, the µ-, -, and -opioid receptors (Pol and Puig, 2004). Furthermore, there is an up-regulation of opioid receptors in inflammation as well, which may additionally contribute to the antinociceptive effects of endogenous opioids (Pol et al., 2001, 2003; Cahill et al., 2003; Stein et al., 2003). The µ-opioid receptor is an important mediator of the analgesic effects of opioids and is the main subtype for analgesic drugs of the morphine type. A study with interleukin-6-deficient mice supports the idea that this cytokine is important for the regulation of opioid receptors and pain (Bianchi et al., 1999). These mice showed reduced analgesic response to morphine compared with wild-type animals. In addition, it was reported that such mice have a lower µ-opioid receptor density in the gray matter of the midbrain, which is known to be critical for the analgesic actions of opioids. The aim of this study was to investigate the influence of interleukin-6 on the expression of the µ- and -opioid receptor genes and the molecular mechanisms underlying the interleukin-6-induced up-regulation of the µ-opioid receptor gene in the human neuroblastoma cell line SH SY5Y, which serves as a model for neuronal cells. |
Materials and Methods |
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; Kraus et al., 2003a).
µ-Opioid Receptor Binding Studies. [3H]DAMGO binding was performed on membranes from SH SY5Y cells essentially as described earlier (Ammer and Schulz, 1997; Koch et al., 1998).
Decoy Oligonucleotide Approach. The use of transcription-factor decoy oligonucleotides to study interactions with transcription factors in living cells has been described in detail in earlier publications from our laboratory (Kraus et al., 2003a,b). Decoy oligonucleotides were used in a final concentration of 120 nM.
Oligonucleotides. All oligonucleotides were synthesized by Metabion (Martinsried, Germany). To obtain double-stranded decoy oligonucleotides, equimolar amounts of both strands were heated to 90°C in a water bath which then was allowed to slowly cool down to room temperature. Then the ends of the oligonucleotides were filled in with Klenow enzyme, and the annealed oligonucleotides were stored at -20°C. Oligonucleotide sequences are shown in Table 1. Oligonucleotides M1 mu2 to M1 mu18 are mutations of M1(-1583), as depicted in Fig. 5. For all oligonucleotides, only the sequences of the sense strands are given.
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Western Blot Analysis. In brief, 1400 cells/mm2 were seeded. After 1 day, the medium was changed to 1% fetal calf serum. After another 24 h, cells were treated for 15 and 30 min with interleukin-6 or vehicle. Then medium was removed, and cells were lysed with 80°C hot sample buffer. Cell lysis, blotting, and antibody incubations were performed as suggested in the Western immunoblotting protocol (New England Biolabs, Frankfurt, Germany). Aliquots (20 µl) were separated on an 8% polyacrylamide gel. Primary antibodies [Phospho-STAT1(Tyr701) and Phospho-STAT3(Tyr705) were from New England Biolabs, and STAT1(E23) and STAT3(C20) were from Santa Cruz Biotechnology, (Heidelberg, Germany)] were used in a 1:1000 dilution.
Real-Time RT-PCR. RNA isolation, cDNA synthesis, and real-time RT-PCR procedures have been described previously (Kraus et al., 2003a). The amounts of µ- and -opioid receptor transcripts were calculated relative to those of the housekeeping gene GAPDH. Amplification of GAPDH and µ-opioid receptor transcripts have been described in detail earlier (Kraus et al., 2003a). -Opioid receptor transcripts were amplified with the primers 5'-ACGTGCTTGTCATGTTCGGCATCGT-3' and 5'-ATGGTGAGCGTGAAGATGCTGGTGA-3' (located on different exons to avoid amplification of genomic DNA) as follows: preincubation for 8 min at 95°C, and then 50 cycles for 5 s at 95°C, for 5s at 63°C, and for 13 s at 72°C.
Reporter Gene Plasmids. All reporter plasmids are derived from the pBLCAT2/pBLCAT3 vector system (Luckow and Schütz, 1987). The construction of the human µ-opioid receptor promoter reporter constructs -2624, -2229, -1702, -1372, -779, -2229/-1854.tk, -1854/-1227.tk has been described in previous publications (Kraus et al., 2001, 2003a; Börner et al., 2002). Plasmid -2624/-2291.tk was generated by digestion of the µ-opioid receptor promoter with Sau3A and shotgun cloning of the fragments into pBLCAT2's BamHI site. The plasmid -2229
-1580/-1080 was created by opening construct -2229 with BglII (-1372) and cutting with enzyme Bal31. Insertion of oligonucleotides into pBLCAT2 was performed according to a method described previously (Kang and Inouye, 1993). All plasmids were sequenced from both sides to ensure correct orientations and sequences of the inserts.
Extraction of Nuclear Proteins and EMSA. Both procedures have been described earlier (Kraus et al., 2001). For immunoshift experiments, STAT1(E23)X and STAT3(C20)X antibodies (both from Santa Cruz Biotechnology) were used.
Statistical Analysis. For statistical evaluation of the experiments, Student's t tests were performed. Asterisks indicate significantly different values (
, p < 0.05; , p < 0.01; , p < 0.001).
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Results |
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-Opioid Receptor mRNA Levels and µ-Opioid Receptor-Specific Binding. The human neuroblastoma cell line SH SY5Y serves as a common model for neuronal cells and is known to express µ- and -opioid receptors constitutively. In addition, SH SY5Y cells were shown to express interleukin-6 receptors and contain functional signaling pathways for this cytokine (e.g., activated STAT1 and STAT3, and p42/44 mitogen-activated protein kinase) (Schumann et al., 1999). Therefore, this cell line was used to study the effect of interleukin-6 on the amounts of µ- and -opioid receptor mRNA by quantitative real time RT-PCR (Fig. 1, A and B). It was shown that µ-opioid receptor gene transcription is strongly induced by interleukin-6, initiating after 6 to 8 h. The protein synthesis inhibitor cycloheximide did not influence the effect of interleukin-6 on µ-opioid receptor gene transcription, indicating that it is a direct transcriptional effect. Contrasting with this, -opioid receptor gene transcription was not regulated by interleukin-6. Next, we investigated whether the µ-opioid receptor mRNA increase was followed by an increase in µ-opioid receptor-specific binding (Fig. 1C). Binding studies on membranes prepared from SH SY5Y cells with [3H]DAMGO, which is highly selective for the µ-opioid receptor subtype, demonstrated a modest increase in the receptor number after stimulation, which was significant at the 48-h time point.
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Identification of the Transcription Factors Mediating Interleukin-6 Induction of µ-Opioid Receptor mRNA. To address the question of which transcription factor(s) mediate(s) the interleukin-6-induced up-regulation of µ-opioid receptor transcription, we used an approach that combines the use of transcription factor decoy oligonucleotides and quantitative real-time RT-PCR. Generally, in the decoy approach, double-stranded oligonucleotides with specific binding sequences for transcription factors are used, which are brought into living cells to selectively disrupt the function of these factors. In the cells, transcription factors then interact with an excess of decoy oligonucleotides instead of binding to the natural regulatory motifs of genes. In our experiments, we disrupted the function of STAT1 and STAT3, AP-1, NF-B, and NF-interleukin-6 in SH SY5Y cells using decoy oligonucleotides corresponding to sites of the mouse metallothionein-I gene (STAT1/3) (Lee et al., 1999), human µ-opioid receptor gene (AP-1 and NF-B) (Börner et al., 2002; Kraus et al., 2003a), and the commercially available NF-interleukin-6 binding oligonucleotide (sequence from Santa Cruz Biotechnology). These factors are known to directly or indirectly mediate effects of interleukin-6. On the promoter of the human µ-opioid receptor gene two AP-1 (-2388 and -1434) and three NF-B (-2174, -557, and -207) binding sites were already identified (Börner et al., 2002; Kraus et al., 2003a). In addition, there are two putative NF-interleukin-6 binding sites located at -525 and -331 in the promoter region. Furthermore, AP-2 decoy oligonucleotides (corresponding to a site in the mouse -opioid receptor gene) (Wöltje et al., 2000) were selected as a negative control, because AP-2 is unrelated to interleukin-6 signaling. Efficiency and specificity of the decoy oligonucleotides were confirmed earlier in transfection experiments in SH SY5Y cells (data not shown) and identification of NF-B regulatory elements on the µ-opioid receptor gene (Kraus et al., 2003a). Our experiments (Fig. 2, A and B) revealed that interleukin-6 induction of µ-opioid receptor transcription was not significantly affected by AP-1, NF-B, NF-interleukin-6, and AP-2 decoy oligonucleotides, suggesting that these transcription factors most probably are not involved in mediating the effect. In contrast, loss of STAT1/3 function attenuated transcriptional induction of the µ-opioid receptor gene in the presence of interleukin-6, demonstrating that this regulation is dependent on STAT1 and/or STAT3. In line with this, increasing amounts of phosphorylated STAT1 and STAT3 were observed in Western blot experiments after 15 and 30 min of incubation of SH SY5Y cells with interleukin-6 (Fig. 2C).
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Identification of the Interleukin-6-Inducible Promoter Element of the Human µ-Opioid Receptor Gene. To characterize the molecular mechanisms underlying the induction of the µ-opioid receptor gene by interleukin-6, transfection experiments in SH SY5Y cells were performed. Using reporter gene constructs containing various lengths of the 5'-flanking region of the gene, we localized the promoter region required for interleukin-6 inducibility (Fig. 3A). Plasmid pBLCAT2, which was used as a vector containing the herpes simplex virus thymidine kinase promoter instead of the µ-opioid receptor promoter in front of the CAT reporter gene, was not responsive to interleukin-6 (lane 10). The basal activity of the -2624 reporter gene construct containing the µ-opioid receptor promoter had approximately 55% of the activity of the herpes simplex virus thymidine kinase promoter in the SH SY5Y cells. Construct -2624, and consecutive 5'-deletions of it up to -1702 were inducible by interleukin-6 (lanes 1-3). Two constructs with further 5'-deletions showed no induction by this cytokine (lanes 4 and 5). The constructs -2229-1580/-1080 (lane 6), -2624/-2291.tk (lane 7), and -2229/-1854.tk (lane 8) were not interleukin-6-inducible either. In contrast, construct -1854/-1227.tk (lane 9) was inducible. These results suggested that the STAT1/3 binding site(s) is located in the region between -1580 and -1372 (highlighted in gray).
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STAT1 and STAT3 transcription factors predominantly bind to sequences with the motifs 5'-TTCNNNGAA-3' and 5'-TTNNNNNAA-3'. Apart from this rule, several STAT1 and STAT3 binding sites with one mismatch in the 5'-TTCNNNGAA-3' motif were identified in studies with other genes (Ehret et al., 2001). These criteria were chosen to designate six sequence motifs within the interleukin-6-responsive region of the µ-opioid receptor promoter as putative STAT1/3 binding sites (M1-M6; Fig. 3B). To identify sequences that really bind STAT1/3 in vivo, we performed experiments in which construct -1702 was cotransfected with decoy oligonucleotides containing the putative binding sites (Fig. 3C). Cotransfection of decoy oligonucleotides that do not bind STAT1/3 will retain interleukin-6 inducibility of construct -1702, whereas cotransfection of decoys that bind STAT1/3 will abolish inducibility of the reporter gene construct. The mouse metallothionein-I STAT1/3 decoy oligonucleotides served as positive controls, and decoy oligonucleotides containing the unrelated STAT6 binding site of the human µ-opioid receptor gene promoter (hSTAT6) (Kraus et al., 2001) were used as negative controls. The experiments revealed that M1(-1583) is a STAT1/3 binding site, whereas M2(-1546) to M6(-1422) do not bind STAT1/3.
Next, the oligonucleotides used in the decoy experiments shown in Fig. 3C were cloned in front of the thymidine kinase promoter in pBLCAT2, and we tested whether they would mediate interleukin-6 responsiveness (Fig. 3D). Indeed, the M1(-1583) motif mediated interleukin-6 induction independently of its orientation. In contrast, no induction was observed for the constructs containing motifs M2 to M6, and a mutated M1 motif (M1mu1-tk-CAT), confirming the results with the decoy oligonucleotides.
Binding of STAT1/3 to the Interleukin-6 Response Element on the Promoter of the Human µ-Opioid Receptor Gene In Vitro. EMSAs were performed to further characterize the binding of STAT1 and STAT3 to the M1(-1583) element (Fig. 4). First, M1 (-1583) probe was incubated with nuclear extract of unstimulated and interleukin-6-stimulated SH SY5Y cells (Fig. 4A). The intensities of the specific bands were strongly enhanced in extracts of stimulated cells (lanes 1 and 2). Experiments with various competitor DNAs showed that both M1(-1583) and mouse metallothionein-I STAT1/3 oligonucleotides can compete with both M1(-1583) and mouse metallothionein-I STAT1/3 probes for STAT1/3 binding, whereas M1 mu1 competitor DNA (with an exchange of two nucleotides compared with M1) does not bind the transcription factors (Fig. 4: A, lanes 3-9, and B, lanes 1-7). In immunoshift experiments, a specific antibody can either further retard the electrophoretic mobility of the DNA-protein complex or prevent formation of a specific DNA-protein complex. The latter case was observed in our experiments. Using specific antibodies against STAT1 and STAT3, we showed that both factors can bind to M1(-1583) (Fig. 4A, lanes 10-12). Efficiency of these antibodies was demonstrated in immunoshift experiments with metallothionein-I STAT1/3 probe (Fig. 4B, lanes 8-10).
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Mutational Analysis of the Interleukin-6 Response Element M1(-1583). Next, the question was addressed which nucleotides of the M1(-1583) sequence may be necessary for STAT1/3 binding. The interleukin-6-responsive reporter gene construct -1702 construct was cotransfected with various decoy oligonucleotides containing one or several base pair sequence exchanges compared with M1(-1583) (Fig. 5). It was shown that decoy oligonucleotides containing the wild-type motif M1(-1583) bind STAT1/3, whereas M1 mu1, with an exchange of two nucleotides within the core palindrome, did not bind to STAT1/3, confirming the EMSA and transfection results. Then in one series of experiments, the effects of nucleotide exchanges directly outside of the core palindrome were tested. M1 mu2 and M1 mu3 with four altered nucleotides on each side of the core palindrome both bound STAT1/3, indicating that these nucleotides are not required. In another series of experiments, motifs each having one base pair exchange in the 5'-TTC... GAA-3' sequence were tested. These experiments (M1 mu4 to M1mu9) demonstrated that only M1 mu4 bound STAT1/3, whereas M1 mu5 to M1 mu9 showed no STAT1/3 binding. In addition, effects of mutations of the nucleotides inside the palindromic half sides were investigated. The experiments with M1 mu10 to M1 mu12 indicated that the central one of these three nucleotides has no influence on STAT1/3 binding. At nucleotide 5' of the central position (M1 mu16 to M1 mu18), either an A (wild-type) or C (M1 mu16) residue was required, whereas at 3' of the central position (M1 mu13 to M1 mu15), a G (wild-type) residue was needed for STAT1/3 binding.
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Discussion |
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, 1995). The analgesic effects of peripherally applied opioids are augmented under conditions of tissue injury and inflammation (Stein et al., 2003). For example, injection of a low dose of morphine into inflamed submucous tissue of patients undergoing dental surgery reduced supplementally required doses of analgesic agents and induced prolonged postoperative analgesia, whereas administration into noninflamed tissue or perineural was not effective (Likar et al., 2001). One underlying mechanism of such effects is an up-regulation of peripheral opioid receptors under inflammatory conditions (Pol et al., 2001, 2003; Stein et al., 2003; Zöllner et al., 2003). Cytokines may play an important role in this mechanism by up-regulating µ-opioid receptor expression in neuronal cells, as it was shown for interleukin-4 (Kraus et al., 2001) and interleukin-1 (Ruzicka et al., 1996). Another study indicated a regulation of µ-opioid receptors by interleukin-6, demonstrating that interleukin-6-deficient mice show decreased µ-opioid receptor levels in the gray matter of the midbrain compared with wild-type animals, whereas no differences in -opioid receptor densities were found (Bianchi et al., 1999). Our study provides evidence that µ-opioid receptor mRNA and protein is induced by interleukin-6 in SH SY5Y cells, which serves as a model for neuronal cells. In contrast, -opioid receptor mRNA levels are not influenced by this cytokine, which is in good accordance with the results described above (Bianchi et al., 1999). An earlier study demonstrated that interleukin-6 has antinociceptive effects in an animal model of peripheral inflammation (Czlonkowski et al., 1993). A specific µ-opioid receptor antagonist abolished these effects. In contrast, a specific -opioid receptor antagonist did not influence interleukin-6-induced analgesia. This indicates that µ-opioid receptors are involved in these effects, whereas -opioid receptors are not. The results obtained from our cell model support the idea that interleukin-6 contributes to antinociceptive effects by enhancing µ-opioid receptor expression. However, this model cannot predict whether µ-opioid receptors may be up-regulated only at sites of injury or inflammation, or throughout the body. In the whole organism, the analgesic effect of interleukin-6 may involve different or additional mechanisms. It is interesting that morphine treatment can increase interleukin-6 levels in plasma (Houghtling et al., 2000) and in spinal cord (Raghavendra et al., 2002). In this context, our results indicate that this cytokine is probably involved in mechanisms underlying an up-regulation of µ-opioid receptors by morphine.
Interleukin-6 binds to its specific receptors, causing an association with two molecules of gp130, thereby activating the intracellular tyrosine kinases of the Janus kinase family (Heinrich et al., 2003). Activated Janus kinases then activate STAT transcription factors by phosphorylation, or a second pathway can be activated involving the G-proteins Ras and Raf, which leads to downstream activation of mitogen-activated protein kinase, resulting in activation of another set of transcription factors. Thus, downstream signaling of interleukin-6 can be mediated by several transcription factors, including STAT1 and STAT3, NF-interleukin-6, NF-B, and AP-1. Using a decoy oligonucleotide approach in living cells, we showed that the interleukin-6-induced µ-opioid receptor mRNA up-regulation in SH SY5Y cells is mediated via the transcription factors STAT1/3, whereas the other transcription factors mentioned above are most probably not involved in this effect.
STAT1 and STAT3 are members of the STAT transcription factor family (Darnell, 1997). This transcription factor family comprises seven members (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6) that generally are activated in response to cytokines, growth factors, and hormones. After phosphorylation, STATs form homo- or heterodimers and translocate to the nucleus, in which they bind to regulatory motifs in the promoter region and induce gene transcription. STAT1 and STAT3 may form heterodimers and may bind to the same regulatory promoter element. After localizing the STAT1/3 element in the promoter region of the human µ-opioid receptor gene, we performed EMSAs to address the question of whether both transcription factors can bind to the element M1(-1583), which indeed was the case. The question of whether STAT1 or STAT3 homodimers or STAT1-STAT3 heterodimers bind to the characterized M1(-1583) element in vivo remains unsolved, because these two STAT factors are not distinguishable by their size and thus cannot be discriminated by the EMSA technique.
It is interesting that the region containing the STAT1/3 element is conserved also in the rat µ-opioid receptor gene promoter with a similar element at nt -1518, but it remains to be shown whether this putative STAT binding site is a functional element as well.
STAT transcription factors generally bind to sequences containing a 6-base pair 5'-TTC... GAA-3' core palindrome. The different STATs have distinct, nonoverlapping functions, as revealed by studying recombinant mice deficient for the different genes coding for STATs (Darnell, 1997; Akira, 1999). Therefore, it can be assumed that each member of the STAT family binds to different cognate motifs within the regulatory regions of target genes. So far, however, only specific target sequences for regulatory elements for STAT6 were identified (Kraus et al., 2003b). Identification of specific target sequences for the different STAT factors is made even more complex by the fact that STAT1 and STAT3 may form heterodimers, as already mentioned. Addressing the question of which nucleotides in the sequence of this element are required for binding of STAT1/3, we showed that the nucleotides outside of the 6-base pair 5'-TTC..GAA-3' core palindrome do not influence STAT1/3 binding. This is in contrast to STAT6, which requires an enlarged palindrome of 8 base pairs (Kraus et al., 2003b). Nearly all naturally occurring STAT1 and STAT3 binding sites possess three nucleotides in the middle of the palindromic half sides (Ehret et al., 2001). We demonstrated that the central nucleotide of the three does not influence STAT1/3 binding. In addition, our results showed that the first nucleotide in the 6-base pair palindrome of the STAT1/3 site might be an adenine or thymine residue. Our experiments thus indicate that 5'-WTCMNGGAA-3' (W = A or T; M = A or C) may be a consensus sequence for STAT1/3 binding. This is a first step to establish specific target sequences for STAT1 and STAT3, but further studies are necessary.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; STAT, signal transducers and activators of transcription; AP-1, activator protein-1; NF, nuclear factor; CAT, chloramphenicol acetyl transferase; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription polymerase chain reaction; tk, thymidine kinase; nt, nucleotide.
1 Current address: Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, 80539 Munich, Germany. 
Address correspondence to: Dr. Jürgen Kraus, Department of Pharmacology and Toxicology, University of Magdeburg, 44 Leipziger Strasse, 39120 Magdeburg, Germany. E-mail: juergen.kraus{at}medizin.uni-magdeburg.de
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, [
), NF-
), and STAT6 (nt -997; 
) binding sites and the putative NF-interleukin-6 binding sites (nt -525 and nt -331; 
) is shown schematically. Bottom, constructs are depicted relative to the promoter of the gene. CAT activities obtained after transfection of SH SY5Y cells are shown as fold induction (
). C, identification of the interleukin-6-responsive element using decoy oligonucleotides. Top, interleukin-6 inducibility of the reporter gene construct -1702 is shown as fold induction. Bottom, the effect of cotransfected decoy oligonucleotides containing the putative STAT sites on interleukin-6 inducibility of the reporter gene construct is shown. CAT activities are reported as fold induction. Results of at least three independent experiments performed in triplicate plus S.E.M. are shown. 