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B Responsiveness of the Human dio2 Gene
Laboratory of Endocrine Neurobiology (A.Z., J.M., Z.L., C.F., B.G.), Institute of Experimental Medicine, Hungarian Academy of Sciences, and Department of Neuroscience (Z.L.), Faculty of Information Technology, Péter Pázmány Catholic University, Budapest H-1083, Hungary; Department of Physiology and Biochemistry (M.D., I.K.), Faculty of Veterinary Science, Szent István University, H-1400 Budapest, Hungary; Division Medical Biochemistry (M.C.H.), Biocenter, Innsbruck Medical University, A-6020 Innsbruck, Austria; Thyroid Section (L.P.C., W.S.d.S., A.C.B.), Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Tupper Research Institute and Department of Medicine (C.F.), Division of Endocrinology, Diabetes, and Metabolism, Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: Dr. Balázs Gereben, Institute of Experimental Medicine, Laboratory of Endocrine Neurobiology, Szigony u. 43, Budapest H-1083 Hungary. E-mail: gereben{at}koki.hu.
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Abstract |
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B (NF-B) inhibitor sulfasalazine. Next, the human dio2 5'-flanking region promoter assay was used in HC11 cells and the p65/NF-B responsiveness mapped to the 3' approximately 600-bp region of hdio2 5'-flanking region, with an approximately 15-fold induction. Semiquantitative EMSA identified the strongest NF-B binding sites at the positions –683 bp (called no. 2) and –198 bp (no. 5) 5' to the transcriptional starting site. Despite the very similar NF-B binding affinity of these two sites, site-directed mutagenesis and promoter assay indicated that only site no. 5 possessed transactivation potency in the presence of the p65 subunit of NF-B. Other cytokine mediators such as signal transducer and activator of transcription-3 (STAT3) or signal transducer and activator of transcription-5 (STAT5) did not induce transcription of the dio2 gene. Our results indicate that inflammatory signals regulate D2 expression predominantly via the NF-B pathway in a direct transcriptional manner and could contribute to the changes in thyroid economy observed in nonthyroidal illness syndrome during infection. |
Introduction |
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The negative regulation of the dio2 gene by T3 forms the basis of D2-mediated homeostatic regulation of tissue T3 levels (2). D2 expression in astrocytes of the cerebral cortex is regulated predominantly in a homeostatic manner, which prevents changes in cortical T3 levels despite changes of peripheral T4 levels (8, 9, 10). However, although D2 expression in tanycytes in the mediobasal hypothalamus is relatively insensitive to changes in thyroid hormone levels, it responds robustly to stimuli during infection (11). Consequently, D2 induction in tanycytes and the resulting increase in T3 production could affect the hypothalamic-pituitary-thyroid axis via changing the T3 levels in the region of TRH-secreting neurons in the paraventricular nucleus of the hypothalamus. This negative feedback mechanism could play a role in the generation of nonthyroidal illness syndrome.
Using an infection model, we have previously shown that bacterial lipopolysaccharide (LPS) treatment increases D2 mRNA levels in rat tanycytes (12) independent of the LPS-induced fall in serum thyroid hormone levels (13). The LPS-induced increase of D2 mRNA in the hypothalamus was also observed in mice, immediately followed by other changes in thyroid economy, e.g. decreased expression of thyroid receptor ß2, TSHß in the pituitary, and decreased D1 mRNA in the pituitary and liver (14).
LPS is recognized by Toll-like receptor (TLR) 4, which contains an extracellular leucine-rich domain and an intracellular Toll/IL-1 receptor signaling domain (15). TLR4 signals through the adapter protein MyD88, similarly to other TLRs (with the possible exception of TLR3) (16). TLR activation leads to an early translocation of the nuclear factor-B (NF-B) with consequent up-regulation of proinflammatory cytokines, costimulatory molecules, and chemokines including IL-1ß, TNF-
, interferon (IFN)-
, and IL-6 (17, 18). As a consequence, NF-B is a crucial effector of LPS or LPS-induced cytokines (e.g. TNF-) and plays an important role in the signaling of other cytokine receptors as well (19, 20, 21). Therefore, NF-B is an eminent candidate for transcription factors potentially involved in LPS-induced D2 up-regulation, along with signal transducer and activator of transcription (STAT) proteins, which are involved in the signaling of some cytokine molecules (IL-6, IFN) and play a crucial role in host defense in infection (22). Although we have previously shown that D2 expression is induced by NF-B, the molecular mechanism of this response has not been established (12).
We assessed the potency of NF-B to increase human D2 activity in a nonheterologous expression system, examining the effect of LPS-induced NF-B activation on endogenous D2 expression of the human mesothelioma (MSTO-211H) cell line. Furthermore, we characterized the NF-B binding sites of the human dio2 (hdio2) 5'-flanking region (5'FR) to obtain evidence for the direct transcriptional regulation of the hdio2 gene by NF-B. Finally, we studied the responsiveness of the dio2 gene to STAT3 and STAT5 to identify additional effectors that could increase D2 expression during infection.
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Materials and Methods |
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The hdio2-Luc constructs were generated by Vent polymerase, and the hdio2 5'FR cassette was inserted between SacI and NheI into a pGL3-basic vector (hdio2-short-Luc) containing the 95-bp hdio2 short promoter (hdio2-short-Luc) between NheI and HindIII. Names of the constructs contain the positions of the fragments. Constructs generated via this strategy are as follows: hdio2–5'-Luc, –3880 to –6860; hdio2-PstI-Luc, –2079 to –3880 PstI fragment; hdio2-PstI/PacI-Luc, PstI-PacI fragment –2079 to –901; and the constructs containing mutagenesis of no. 1, 2, and 5 hdio2 binding sites for the p65 subunit of NF-B. Oligonucleotides used for hdio2–1,2,5wt-Luc and its derivatives were: hdio2–2,5wt-Luc, Bp383–385; hdio2–2,5Mut-Luc, Bp384–386; hdio2–2wt-5Mut-Luc, Bp38-Bp386; hdio2–2Mut-5wt-Luc, Bp384–385; hdio2–2wt-5RatMut-Luc, Bp383–402; hdio2–1,2,5wt-Luc, Bp381–385; hdio2–1Mut-2wt-5Mut-Luc, Bp382–386; hdio2–1Mut-2wt-5wt-Luc, Bp382–385 (see Table 2
). Position of the mutated fragments in the hdio2 promoter (used for studies shown; see Fig. 7) was depicted (see Fig. 3).
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) and its reverse complementer].
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DNA transfection and luciferase promoter assay
NF-B, STAT3
HC11 [a D2-expressing mouse mammary cell line (28) that has been successfully used to analyze the NF-B pathway (29)], HEK-293, and U87 cells were cultured as described and transfected by the polyethylenimine (HC11, HEK-293) or calcium phosphate method (U87) when they reached approximately 70% confluency in 6-well plates (12, 23, 30). Neither HEK-293 [a human kidney cell line with established potential to study STAT-mediated pathways (28)] nor U87 (a human glioblastoma) express D2 endogenously (28). In one well, 800 ng of the pGL3Basic constructs were cotransfected with 200 ng of cytomegalovirus promoter (CMV)-driven p65 subunit NF-B (27) or the same amount of the empty pCI-neo vector (Promega) and 1800 ng of pGEM-T vector (Promega) as carrier plasmid DNA. To monitor transfection efficiency, 4 ng hu-ß-actin promoter-driven Renilla luciferase construct (pRL-actin) was cotransfected. The same cotransfection was performed for STAT3 experiments, with the exception that the p65 vector was replaced by 200 ng of a vector expressing STAT3c, a constitutively active form of STAT3 (25).
For transfection of U87 cells with calcium phosphate, 2400 ng pGL3Basic construct, 600 ng p65 expression vector or empty pCI-neo vector, and 20 ng pRL-actin were transfected. Luciferase activity was assessed using the dual-luciferase reporter assay system (Promega) and a Luminoskan Ascent luminometer (Thermo Electron Corp. Labsystems, Vantaa, Finland). Each construct was transfected at least three times.
STAT5
To produce an active phosphorylated STAT5, prolactin was added to the cells along with cotransfection of the prolactin receptor, as described (31). We used this pathway to activate STAT5 in COS-7 and HEK-293 cells that were grown in 6-well-plates and transfected with TransFastTH transfection reagent (Promega). COS-7, a monkey kidney cell line without endogenous D2 expression is an established system for studies on the D2 promoter (28) and STAT mediated pathways (28). A total of 2200 ng DNA comprising 500 ng prolactin receptor PRL-R, 500 ng STAT5a (31), 1000 ng of pGL3-based constructs /hdio2–6.9-Luc, hdio2-only linker, rdio2–3.6-Luc (12), and pGL3(–344/–1) containing the rat ß-casein promoter from –344 to –1 (31)/ and 200 ng of SV40 Renilla expression constructs was used for each 6-well plate. Thirty-six hours after transfection, cells were treated with 5 µg/ml ovine prolactin (Sigma, St. Louis, MO) or vehicle alone. Twelve hours after hormone stimulation, cells were washed with ice-cold PBS and luciferase activity was measured.
EMSA, supershift assay, and semiquantitative EMSA
HeLa (a cell line without endogenous D2 expression) and HC11 cells were treated with 20 ng/ml TNF- (Sigma) for 1 h followed by the preparation of nuclear extracts with CelLytic nuclear extraction kit (Sigma). Sense and antisense oligonucleotides containing hdio2 putative NF-B binding sites were annealed to produce double-stranded DNA (Table 1
). Labeled probes were generated with T4 polynucleotide kinase using 1 µl -[32P]ATP (
5 µCi) followed by purification through a Sephadex mini Quick Spin column (Roche, Indianapolis, IN). Labeled double-stranded DNA was incubated with nuclear extract to achieve protein-DNA binding. The binding buffer contained 4 mM HEPES (pH 7.9), 20 mM KCl, 0.4 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.5 mg/ml BSA, 50 µg/ml poly(dI-dC) (Sigma), 0.1% IGEPAL (Sigma), and 4% glycerol. Alternatively, reaction mixture contained unlabeled oligonucleotides for competition assay or antibody to the p65 subunit of NF-B (H-286, Santa Cruz Biotechnology, Santa Cruz, CA) for supershift assay. For comparison of signal strength, 400,000 cpm of each probe were added to the mixture. Probes were run on a nondenatured [4% (wt/vol)] acrylamide gel in 0.5x Tris-borate EDTA at 18 C. Semiquantitative EMSA was performed by adding excess of unlabeled probe to the binding reaction mixture (see Fig. 6). After electrophoresis, gels were dried and exposed for several hours. For analysis of the semiquantitative EMSA results, the signal intensities of the specific bands were determined by Quantiscan (Biosoft, Cambridge, UK). Signal strength of each lane was calculated as percentage of the value in the control lane (probe without competitor) and plotted against the amount of the added unlabeled competitor DNA. Each gel shift experiment was performed at least twice. Semiquantitative EMSAs were run in triplicate.
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) and subjected to D2 activity measurement as previously described (32). In short, cells were harvested, washed, and sonicated in buffer of 0.1 M potassium phosphate, 1 mM EDTA (pH 6.9) containing 10 mM DTT and 0.25 M sucrose. Fifteen micrograms cell homogenate were assayed for deiodination of freshly purified 2 nM 125I-labeled T4 in the presence of 20 mM DTT and 1 mM propylthiouracil for 3 h at 37 C. D2 activity was reported as femtomoles per hour per milligram protein. Sulfasalazine (Sigma) was used as a NF-B inhibitor (33). Sulfasalazine was dissolved in dimethylsulfoxide (DMSO) and added at concentrations of 0.1, 0.3, and 0.5 mM (Fig. 1B). DMSO was added at concentrations of 0.02, 0.06, and 0.1%, respectively, as a control. Cells were incubated for 30 min with sulfasalazine or DMSO. After their removal, cells were incubated with 1 µg/ml LPS or PBS. Cells were harvested 4 h after treatment and samples assayed for D2 activity as described above.
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Results |
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B increases D2 activity in MSTO cellsB in human cells, we used the MSTO-211H mesothelioma cell line, which endogenously expresses both D2 (32) and NF-B (34). Cells were exposed to 1 µg/ml LPS for 4 or 10 h to induce NF-B. D2 activity was significantly increased after 4 h (Fig. 1A). Furthermore, 30 min treatment with the NF-B inhibitor sulfasalazine partially blocked the LPS- (4 h treatment) induced increase in D2 activity (Fig. 1B). These data indicate that the LPS-NF-B signaling pathway is capable of stimulating D2 expression in human cells endogenously expressing D2.
The responsiveness of the hdio2 promoter to the p65 subunit of NF-B is restricted to the 3' region
To characterize the molecular basis of the NF-B responsiveness of the human dio2 gene, we assessed the effect of the p65 subunit of NF-B on different regions of the hdio2 5'FR. Computer-assisted inspection, Transcription Element Search System (TESS) (35) identified 12 putative NF-B binding sites at –5520, –5344, –4018, –3684, –2487, –2334, –878, –683, –546, –254, –198, and –111 in the 6.9-kb 5'FR (numbers indicate the position of the starting nucleotide of the element 5' in relation to the TSS). Thus, we performed promoter assays with luciferase expression constructs containing the hdio2 5'FR fragments, with Renilla luciferase as internal control. Coexpression of the human p65 subunit of NF-B with the 6.9-kb hdio2 5'FR fragment (hdio2–6,9-Luc, Fig. 2) in HC11 cells, a D2-expressing mouse mammary cell line (28) that has been successfully used to analyze the NF-B pathway (29), resulted in a 140-fold increase in transcriptional activity, confirming previous results (12). This construct was also tested in the U87 glioblastoma and astrocytoma derived human cell line without endogenous D2 expression (23), in which it was induced approximately 55-fold by p65 coexpression.
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), and used for promoter assay after p65 coexpression in HC11 cells. Whereas hdio2 5'FR regions between –6860 and –901 showed no p65 responsiveness (Fig. 2), fragments containing portions of the approximately 1 kb proximal to the hdio2 TSS were clearly induced except for the 95-bp short hdio2 promoter (hdio2-short-Luc) and the 117-bp 3' region of the hdio2 5'FR (hdio2–117-Luc) (Fig. 2). These findings indicate that the proximal portion of the hdio2 5'FR contains the functional p65 binding sites. Six NF-B binding sites (no.1–6) were predicted within this region (Fig. 3). The 901- and 584-bp Luc constructs (hdio2–901-Luc and hdio2–584-Luc) showed no significant difference in p65 response (Fig. 2), indicating that the most potent binding sites responsible for p65-mediated transcriptional activation were located in the approximately 600-bp proximal region. To test the NF-B binding affinity, we subjected all six putative binding sites to EMSA including site no. 6, although it is located in the unresponsive 3' 117-bp region.
No. 2 and no. 5 are the most potent NF-B binding sites in hdio2 5'FR EMSA
We used EMSA and supershift assays to determine the NF-B binding affinity of the six putative NF-B binding sites in this proximal approximately 0.9-kb hdio2 region (Fig. 3). Nuclear extract of TNF--treated HeLa or HC11 cells was incubated with double-stranded oligonucleotides labeled with 32P (Table 1). As positive control we used the oligonucleotide containing a high-affinity NF-B binding site (36), which showed two shifted bands in accordance with the findings of the gel shift assay system (Promega) (Fig. 4, lane 2) and no band in probes prepared without nuclear extract (Fig. 4, lane 1). The shifted bands were abolished by an excess of unlabeled control DNA (Fig. 4, lane 4), but only the lower band could be supershifted with an anti-p65 NF-B antibody (Fig. 4, lane 3, open arrow), confirming the p65-DNA binding. Based on these findings, we considered the lower shifted band of the control probe to represent the authentic p65-DNA complex (Fig. 4, solid arrow). All six putative NF-B binding sites in the 0.9-kb 5'FR of the hdio2 promoter region were tested for NF-B binding by EMSA. Equal counts per minute activity of each probe was loaded to make signal strengths comparable. In the presence of the nuclear extract, the hdio2 site no. 1 showed a faint band, whereas no. 2 and no. 5 showed an intense shifted band migrating similarly to the lower band of the positive control (Fig. 4, lanes 5, 7, and 13, respectively, solid arrow). The shifted band could be abolished by adding unlabeled control DNA (Fig. 4, lanes 6, 8, and 14). The specific bands of all three sites disappeared on incubation with an NF-B antibody, whereas the supershifted band (open arrow) was visible only in the presence of the no. 2 and no. 5 probes (Fig. 5A, lane 5, and B, lanes 6 and 8, for the sites no. 2 and no. 5, respectively, not shown for no. 1).
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, lane 5), we repeated lanes 5 and 6 of Fig. 4 with longer run (see inset in Fig. 4). This indicated that the fast migrating band is a doublet (left lane of the inset), and the upper band of this could be clearly suppressed with consensus competitor oligo (right lane of the inset).
In the presence of extract, the site no. 3 probe produced an intense band that migrated more slowly than the NF-B complex, the intensity of which could not be decreased by unlabeled control DNA (Fig. 4, lanes 9 and 10) or nuclear extract of TNF- untreated HeLa cells (not shown), whereas no. 4 did not show any band corresponding the size of the positive control (Fig. 4, lanes 11 and 12). The site no. 6 produced a band of slightly bigger size than the upper band of the control, and it could be eliminated with unlabeled control DNA (Fig. 4, lanes 15 and 16) but could not be supershifted (not shown).
To test the efficiency of TNF- treatment on HeLa, the no. 2 containing probe was incubated with nuclear extracts from untreated HeLa cells, resulting in no specific binding (Fig. 5A, lane 1). To further analyze NF-B binding affinity of the hdio2 promoter sequence at site no. 2, we generated mutations of this NF-B binding site (Table 1, no. 2-MutA and no. 2-MutB) and tested them by EMSA. Neither no. 2-MutA nor no. 2-MutB showed NF-B binding affinity, as evidenced by the absence of the band (Fig. 5A, lanes 6 and 7) migrating similarly to the NF-B-DNA complex (Fig. 5, solid arrow). Moreover, incubation of binding reaction complex with unlabeled version of no. 2-MutB oligonucleotide did not compete with labeled wild-type site no. 2 for NF-B binding, as indicated by the presence of labeled NF-B-DNA complex (Fig. 5A, lane 4).
The signals of shifted no. 2 and no. 5 probes were stronger than those of no. 1 (Fig. 4), suggesting a higher NF-B binding affinity of the former two sites. To establish the affinity for NF-B binding of these three sites, we used semiquantitative EMSA. Different amounts of no. 1, 2, and 5 unlabeled DNA were used to decrease binding of the positive control probe to the nuclear extract of HeLa cells treated with TNF- (Fig. 6A). Quantitative analysis of this experiment is depicted in Fig. 6B. In line with the previous experiments (Figs. 4 and 5), sites no. 2 and 5 interfered with binding of the positive control to NF-B more strongly than did no. 1. Although no. 2 and 5 showed a very similar inhibitory potency, their binding capacities were still weaker than that of the positive control.
The no. 2 and 5 NF-B binding sites of hdio2 show strikingly different transactivation potency
The data presented above identified one weak (no. 1) and two strong (no. 2 and 5) NF-B binding sites at the most 3' approximately 900 bp of the hdio2 5'FR. To test their transactivation potency in response to p65, sites no. 1, 2, and 5 were subjected to site-directed mutagenesis. Oligonucleotides used for generation of fragments for luciferase constructs are listed in Table 2, and the positions of the generated 5'FR fragments in relation to the binding sites are depicted in Fig. 3. The mutant hdio2 fragments were inserted 5' to a D2 minimal promoter governing the luciferase reporter gene and tested in promoter assay in HC11 cells as described above.
The wild-type constructs hdio2–2,5wt-Luc and hdio2–1,2,5wt-Luc showed a 5- and 4-fold response to p65, respectively (Fig. 7). However, the construct harboring both no. 2 and 5 mutations (hdio2–2,5Mut-Luc) remained unresponsive to p65. The construct containing a mutation in binding site no. 1, but not in sites no. 2 and 5 (hdio2–1Mut-2wt-5wt-Luc), was induced by p65 similarly to the wild-type, indicating that site no. 1 is not critical in NF-B-induced transcription, in good accordance with the relatively weak NF-B binding of this site, as shown with EMSA. Interestingly, the hdio2–2Mut-5wt-Luc construct containing a mutant site no. 2 and wild-type no. 5 performed similarly to the hdio2–2,5wt-Luc. However, none of the constructs containing a mutated no. 5 and wild-type no. 2 (hdio2–1Mut-2wt-5Mut-Luc; hdio2–2wt-5Mut-Luc) responded to p65. These data indicate that despite the similar NF-B binding affinity of no. 2 and 5 hdio2 sites, no. 5 is the primary NF-B binding site of the hdio2 gene. Whereas the rat dio2 (rdio2) promoter completely lacks the no. 2 binding site of the hdio2, the no. 5 site in the hdio2 is highly similar in both species, except that a T was replaced by C in the rdio2 5'FR at –193 5' to the TSS. The rat-like site responded to p65 similarly to the hdio2–2,5wt-Luc as assessed by promoter assay in HC11 cells (not shown).
The transactivation potency of no. 5 site was also assessed in the context of the approximately 6.9-kb-long hdio2 promoter in the presence of the p65 subunit of NF-B. The no. 5 site was mutated in hdio2–6.9-Luc, resulting in hdio2–6.9–5Mut-Luc. This construct contained the same no. 5 mutation as used above in the approximately 600-bp-long hdio2–2wt-5Mut-Luc. The hdio2–6.9–5Mut-Luc responded to p65 14.815 ± 2.24-fold (mean ± SEM, n = 6) in HC11 cells, whereas p65 induced the parallel assayed wild-type hdio2–6.9-Luc 219.3 ± 26.2-fold (mean ± SEM, n = 5) (Fig. 8). Furthermore, the approximately 6.9-kb-long construct containing both mutated no. 2 and 5 sites (hdio2–6.9–2,5Mut-Luc) showed a 11.44 ± 1.75 (mean ± SEM, n = 4)-fold response to p65 (hdio2–6.9–5Mut-Luc vs. hdio2–6.9-Luc P < 0.001; hdio2–6.9–2,5Mut-Luc vs. hdio2–6.9-Luc, P < 0.001; hdio2–6.9–5Mut-Luc vs. hdio2–2,5Mut-6.9-Luc, P > 0.05 by ANOVA followed by Newman-Keuls). These results indicate that the no. 5 site plays a key role in p65 response of the 6.9-kb hdio2 5'FR, whereas mutation of site no. 2 did not have any additional effect.
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Discussion |
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B, unlike STAT3 and STAT5, exerts a direct transcriptional effect on the human dio2 gene. This adds dio2 to the set of key genes regulated by NF-B essential for inflammatory processes and host defense in the central nervous system (20, 38). An important role of this pathway in other tissues should also be considered, as exposure to LPS significantly increased D2 activity in MSTO-211H mesothelioma cells by a mechanism that was partially blocked by the NF-B inhibitor sulfasalazine (Fig. 1). The relatively modest effect of sulfasalazine could be explained by published data indicating that 0.1 mM sulfasalazine blocks NF-B-mediated transcription by only approximately 20% (33).
A key player of NF-B-mediated stimulation is the induction by the heterodimer of p65 and p50 (39), and transient transfection with p65 (RelA) is commonly used to test NF-B sensitivity (40). We studied p65 responsiveness of the human dio2 5'FR to assess the NF-B responsiveness of different regions of this gene. Promoter assays mapped the response of the hdio2 5'FR to p65 to the proximal approximately 600 bp using HC11 (Fig. 2), a D2-expressing mouse mammary cell line (28) that has been successfully used to analyze the NF-B pathway (29). Although upstream 5' regions of the promoter remained unresponsive, the 600-bp region was significantly less responsive to p65 than the approximately 6.9-kb fragment. This discrepancy could be explained by the presence of an enhancer sequence in the more 5' region that could amplify the p65 response of hdio2.
Computer-assisted inspection/TESS (35) identified six putative NF-B binding sites at –878, –683, –546, –254, –198, and –111 in the proximal region of the hdio2 5'FR, indicated in Fig. 3. as sites no. 1, 2, 3, 4, 5, and 6, respectively, showing sequence variability (Table 1). Various consensus sites of NF-B responsive regions have been identified, indicating the existence of a fine-tuning mechanism of the NF-B signaling system (41, 42, 43). One explanation proposed for the well-known phenomenon of variability among NF-B binding sites is the ability of NF-B to modify its conformation (44). In accordance with this suggestion, the sequence differences between sites no. 2 and 5 did not alter NF-B binding affinity as assessed by EMSA (Figs. 4, 6).
EMSA excluded site no. 3 as an NF-B binding site based on its lower migration speed, the independence of its shift from TNF- treatment of the cells used for preparing the nuclear extract and its resistance to suppression by the unlabeled NF-B-positive control DNA in access (Fig. 4).
Site no. 4 was excluded due to the absence of a shifted band migrating similarly to the positive control, whereas no. 6 was not considered as an NF-B binding site due to the lack of supershiftable NF-B-DNA complex paralleled by unresponsiveness of this region in the promoter assay. In contrast, the no. 2 and 5 sites could be supershifted with an NF-B antibody (Fig. 5). The lack of supershift at site no. 1 is probably due to the weak binding, also indicated by the semiquantitative EMSA. Interestingly, whereas semiquantitative EMSA indicated that the NF-B binding of sites no. 2 and 5 were very similar (Fig. 6), their transactivation by p65 was strikingly different, as shown by promoter assay using the hdio2–1,2,5wt-Luc and its derivatives, prepared to assess the effect of point mutations on p65 responsiveness of hdio2. The semiquantitative EMSA also demonstrated that all sites studied have shown a significantly lower affinity to NF-B than the positive control containing a strong consensus NF-B site.
The response to p65 of the approximately 600- to 800-bp-long hdio2–1,2,5wt-Luc and its derivatives was relatively low, compared with that of hdio2–584-Luc (5- vs. 15-fold), but their basal promoter activity remained unchanged (Fig. 7). The former phenomenon could be explained by an unidentified enhancer in the region between –95 and –180, a region absent from hdio2–1,2,5wt-Luc and its derivatives. Still, this system was suitable to clearly demonstrate the effect of mutations introduced to sites no. 1, 2, or 5 in HC11 cells. Whereas the no. 5 binding site was functional (because its mutation completely abolished transcriptional activation by p65), sites no. 1 and 2 turned out to be silent NF-B sites. The upper band of the fast migrating doublet obtained with the no. 1 site (Fig. 4, inset) might be a p50/p50 homodimer known to be a repressor, as indicated for the ß-casein gene (29). This potential repression along with weak p65 binding to no. 1 might result in silence of this site. The lack of effect of no. 2 mutation on p65 response cannot be explained by retained transactivation potency of the mutagenized sequence because EMSA confirmed the lack of binding of NF-B to the mutant site no. 2 (no. 2-MutB). These findings were further supported by the lack of significant difference between the p65 responsiveness of hdio2–901-Luc and hdio2–584-Luc along with the unresponsiveness of constructs containing a wild-type no. 2 and mutant no. 5 sites.
Because of the relatively low response of the approximately 600- to 800-bp-long hdio2–1,2,5wt-Luc derivatives, we also assessed the transactivation potency of no. 5 and 2 sites in the context of the approximately 6.9-kb-long hdio2 promoter in the presence of the p65 subunit of NF-B. Whereas mutation of the no. 5 site in hdio2–6.9–5Mut-Luc decreased the p65 response of the wild-type 6.9 kb hdio2 promoter by approximately 93%, the additional mutation of no. 2 site (hdio2–6.9–2,5Mut-Luc) did not result in an additional decrease indicating that presence of an intact no. 5 site was inevitable for the robust response of the hdio2 gene to NF-B, whereas the no. 2 site was not (Fig. 8). These data also demonstrate that there is no significant synergism between the no. 2 site and more 5' sites. The retaining approximately 7% response observed in the presence of a mutated no. 5 (and no. 2 site) sites of the 6.9-kb-long 5' hdio2 flanking region could be the result of weak NF-B binding sites potentiating each other. This process then requires the whole 6.9-kb hdio2 5'FR because the putative NF-B binding sites in the –6860 to –2079 region tested as hdio2–5'Luc (–6860 to –3880) and hdio2-PstI Luc (–3880 to –2079) remained unresponsive to p65 in the promoter assay along with the most 3' 117-bp region (hdio2–117-Luc) (Fig. 2). It is also possible that the retaining 7% response could be the indirect result of other factors induced by NF-B. Together, these data indicate that site no. 5 is a major element in the response of hdio2 to p65.
Discrepancies are common between the binding affinity of a site to a specific transcription factor and the transactivation potency of this factor on the same binding site. For example, the C1 and D TTF-1 binding sites of the hdio2 5'FR are similarly involved in TTF-1 response, whereas their binding capacities were very different (23). The contradiction between NF-B binding and transactivation potency at sites no. 2 and 5 (which is more proximal to the TSS) could also arise from a positional effect because the linear arrangement of transcription factors and the three-dimensional organization of enhanceosomes are critical for efficient transcription, as suggested for NF-B binding sites in the promoter of the human IB kinase-related kinase IKKi/IKK
gene (45) and by comparing binding sites HIV-B and IFNß-B (44).
To put the no. 2 and 5 hdio2 binding sites into phylogenetic context, we analyzed the rat dio2 5'FR and found that it completely lacked the no. 2 binding site, whereas site no. 5 of hdio2 was highly similar in both species, showing a C-for-T replacement in rdio2 5'FR at position –193 relative to the rdio2 TSS. The same point mutation is present in the corresponding site of the mouse dio2 5'FR (46). The response to p65 of the C/T mutated site no. 5 was similar to that of the hdio2–2,5wt-Luc, as assessed by promoter assay in HC11 cells, reflecting the evolutionary conservation of the most potent p65 binding site of the dio2 gene.
D2 is normally expressed in astroglial cells in the brain (47), and it is the only deiodinase that generates T3 in the human central nervous system (3). We used the U87 human glioma cell line without endogenous D2 expression (23) to determine whether p65 can induce D2 expression in a glial environment. The approximately 55-fold induction of the hdio2–6.9-Luc in the presence of coexpressed p65 in U87 indicates that this mechanism could be functional in the brain.
LPS-induced D2 activation in the tanycytes of the rat mediobasal hypothalamus indicated a mechanism culminating in increased T3 generation, providing local negative feedback in the hypothalamus and accounting for the impaired response of the hypothalamo-pituitary-thyroid axis observed in nonthyroidal illness syndrome (12). Tanycytes probably serve as a cytoplasmic passage between the cerebrospinal fluid and the blood circulating in the area of the arcuate nucleus and median eminence (48). Thus, changes in D2-catalyzed T3 generation in tanycytes could have important consequences. Recent studies have connected this system with the nonthyroidal illness syndrome because LPS-treated rats showed an enhanced D2 expression in tanycytes (12), increasing local T3 generation, which could suppress the hypothalamo-pituitary-thyroid axis.
Although the upstream signaling components of the NF-B pathway have not yet been resolved for D2-expressing cells in the brain, indirect evidence suggests that NF-B mediated up-regulation of D2 expression could be functional in the mediobasal hypothalamus. It has been shown that the complex LPS-LPS binding protein binds CD14 at the cell surface of myeloid cells followed by the association of the LPS/CD14 receptor complex with TLR4. This is the crucial signal transducer that mediates the effect of LPS on NF-B activation via activation of a complex cascade of cytoplasmic signaling molecules (20, 49). Signaling via TLR4 hence results in an up-regulation of IFN-inducible genes via the adaptor protein TICAM-1 (Toll-IL-1 receptor domain-containing adaptor molecule) or TRIF (TIR domain-containing adaptor-inducing IFN-ß) (50). In addition, TLR4 can signal via another adaptor protein, TRAM (TRIF-related adaptor molecule), and can induce a late NF-B response (51). Importantly, both key components of LPS/TLR4 signaling (i.e. CD14 and TLR4 mRNA) have been detected in the median eminence of the mediobasal hypothalamus (21). It has also been proven that systemic injection of LPS can up-regulate CD14 in the median eminence (52).
Alternatively (or parallel to this mechanism), the role of TNF- should also be taken into account because its expression can be both the consequence and inducing factor that triggers NF-B activation (20, 53). Importantly, the p55 TNF-I receptor is expressed in the median eminence and its level (like that of CD14), is increased by TNF- (54, 55).
Based on these findings, we speculate that LPS acts on D2 expression in tanycytes directly via the CD14/TLR4 complex or indirectly via TNF- released locally, followed in both cases by NF-B activation, which exerts a direct transcriptional effect on the dio2 gene.
The importance of STAT proteins in the brain during inflammation has also been established (53), and members of the STAT family share a relatively similar binding site (56). To look for functional STAT binding sites in the hdio2 promoter, we used STAT5 and STAT3 as members of this protein family that can be effectively activated in the used heterologous expression system. Neither STAT3 nor STAT5 could activate the hdio2 promoter in the cell lines we used. Because species-specific responsiveness of the dio2 gene has been already described (23), we extended this study to the rat dio2 promoter. Again, no response was detected, indicating that these effectors are not relevant for D2 regulation in the systems studied.
In conclusion, our findings show that NF-B exerts a direct transcriptional effect on the human dio2 gene via the no. 5 binding site and that this effect also exists in glial cells. The presented data demonstrate that NF-B could be a major factor in the regulation of D2 activity by infection. This is particularly relevant, given the fact that D2 is the primary source of extrathyroidal T3 generation in the euthyroid human (57). Other factors such as decreased D1 (58) or increased D3 activities (59) have also been reported in patients with nonthyroidal illness syndrome, and tissue-specific changes of D2 expression during infection should be also taken into account, indicating that further studies are needed to clarify the individual contributions of these enzymes to the pathogenesis of this syndrome.
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Acknowledgments |
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Footnotes |
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The results of this work were presented in part at the 13th International Thyroid Congress, Buenos Aires, Argentina, 2005.
The authors have nothing to declare.
First Published Online May 25, 2006
Abbreviations: CMV, Cytomegalovirus promoter; D2, type 2 iodothyronine deiodinase; DMSO, dimethylsulfoxide; DTT, dithiothreitol; 5'FR, 5'-flanking region; IFN, interferon; LPS, lipopolysaccharide; NF-B, nuclear factor-B; STAT, signal transducer and activator of transcription; TESS, Transcription Element Search System; TLR, Toll-like receptor; TSS, transcriptional starting site.
Received December 20, 2005.
Accepted for publication May 15, 2006.
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, nonsignificant difference.
