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B of Human 25-Hydroxyvitamin D3 1
-Hydroxylase Promoter
Orthopedic Department (R.E., M.J., M.U., D.S., J.M.-W., F.J.), University of Wuerzburg, Wuerzburg, Germany; and Institute of Experimental Genetics (J.A.), GSF-National Research Center for Environment and Health, Neuherberg, Germany
Address all correspondence and requests for reprints to: Franz Jakob, M.D., Experimental and Clinical Osteology, Orthopedic Department, University of Wuerzburg, Brettreichstrasse 11, D-97074 Wuerzburg, Germany. E-mail: f-jakob. klh{at}mail.uni-wuerzburg.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)
B sites, 10 of which were found to bind NFB in EMSA experiments. Cotransfection of NFB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NFB-responsive element almost abolished NFB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFB inhibitor, resulted in enhanced 1-hydroxylase mRNA production. Down-regulation of 1-hydroxylase promoter through NFB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-HYDROXYLASE, converts liver-derived 25-hydroxyvitamin D3 to its active hormonal metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2 vitamin D3]. The p450 enzyme is expressed mainly in the proximal tubule cells of the kidney and plays a key role in systemic vitamin D metabolism and in calcium homeostasis. The product suppresses enzyme activity in a negative feedback regulation (12). Although suppression of promoter activity by 1,25-(OH)2 vitamin D3 has been reported, no vitamin D3 responsive element could be localized within the promoter (9). In systemic calcium homeostasis, PTH stimulates production of 1,25-(OH)2 vitamin D3. This was verified in vitro in transient transfection studies, where stimulation of cells with PTH resulted in an increase in 1-hydroxylase promoter activity (9). In contrast, the phosphaturic compound fibroblast growth factor 23 (FGF-23) inhibits 1-hydroxylase mRNA synthesis (18, 20). Single cells such as keratinocytes, monocytes, and macrophages are also able to secrete active 1,25-(OH)2 vitamin D3 hormone (4, 13, 23). In human endothelial cells, forskolin, lipopolysaccharide, and TNF enhanced 1-hydroxylase enzyme activity (22). In patients with granulomatous diseases such as sarcoidosis, high serum concentrations of 1,25-(OH)2 vitamin D3 can be measured due to an increased expression of 1-hydroxylase in this tissue (7). In patients with rheumatoid arthritis, where levels of 1,25-(OH)2 vitamin D3 are diminished, high concentrations of TNF can be detected (15). In patients with transplantation-associated osteoporosis a down-regulation of 1- hydroxylase activity is also observed (6).
Little is known about the promoter regulation of human 1-hydroxylase in this context. In signal transduction cascades, TNF acts on gene expression via the activation of the transcription factor NFB (nuclear factor B). NFB exists mainly as a heterodimer of p50 and p65 subunits. Both proteins are located in the cytoplasm, where the inhibitory protein IB binds to the nuclear localization sequence of the p50 subunit. Signal-dependent phosphorylation of IB leads to its dissociation, and the p50/p65 heterodimer forms and is transported to the nucleus where it binds to NFB-responsive elements in promoters. The consensus sequence of NFB motifs is 5'-GGGRNNYYCC-3', but other putative elements are known (8). Here we report on the down-regulation of human 1-hydroxylase promoter by the transcription factor NFB in human embryonal kidney (HEK)-293 cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Hydroxylase Promoter-hydroxylase and the promoter region of genomic DNA isolated from five healthy donors was sequenced and compared with the GenBank entry (GenBank accession no. AF072470). We found four base changes and three deletions (see Table 1
) in all sequenced samples. The observed base replacements are of main importance as the base exchange C-171A has consequences for basal promoter activity. The promoter construct pGL2–1413 C-171 was twice as active as pGL2–1413 A-171 in luciferase assays in HEK-293 cells (data not shown).
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B response elements within the cloned 1413-bp fragment, which did not differ from a canonical NFB element in more than three bases (Fig. 1A and Table 2). Five of these elements were described previously (NFB1–5) but not further characterized (9, 14).
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B Responsiveness of 1-Hydroxylase Promoter-hydroxylase promoter activity, the full-length fragment (pGL3–1413 construct) was transfected into HEK-293 cells. The luciferase activity of the promoter construct was 500 times higher than background activity, defined as luciferase activity of the promoterless pGL3basic plasmid. To identify regulatory sequences within the 5'-flanking region of the 1-hydroxylase gene, progressive deletion constructs were transfected into HEK-293 cells. Luciferase activities of the deletion constructs are shown in Fig. 1B
. The pGL3–937 construct revealed a 1.45-fold stimulation in luciferase activity compared with the full-length construct in HEK-293 cells. Elimination of another 230 bp (pGL3–748 construct) resulted in a luciferase activity of 50% compared with that of pGL3–982 construct. Further deletions showed slightly weaker or unchanged luciferase activity with a descending trend. By deleting the region between –240 and –155, basal promoter activity was lost. The pGL3–240 construct displayed 30% luciferase activity compared with the full-length construct, and a minimal promoter of 155 bases showed very low basal activity.
To determine responsiveness of 1-hydroxylase promoter to the transcription factor NFB, p50 and p65 subunits (pcDNA3.1-p50 and pcDNA3.1-p65 plasmids) were cotransfected with the promoter full-length and deletion constructs in HEK-293 cells. Promoter activity was reduced dramatically by overexpression of NFB after 48 h. The minimal promoter pGL3–155 comprising the NFB15 response element depicted very low basal activity; significant modulation by p50/p65 overexpression could not be shown (Fig. 1B).
As a control, the NFB-responsive vector pNFB-LUC (obtained from Stratagene, La Jolla, CA), comprising five NFB consensus sites located 3' of the firefly luciferase gene, was cotransfected with pcDNA3.1-p50 and pcDNA3.1-p65 in HEK-293 cells. Measurement of luciferase activity showed a 11.5-fold activation (Fig. 1C). Because we wanted to confirm that transfection of NFB subunits resulted in enhanced expression of protein, the time course of NFB overexpression after transfection was analyzed by Western blot. After 24 and 48 h of overexpression of NFB subunits, the amount of detected p50 and p65 protein was increased compared with control samples (data not shown).
However, the responsiveness of firefly luciferase reporter vectors and transfection efficiency control vectors turned out to be a serious problem. From various firefly luciferase reporter vectors tested we have chosen pGL2basic and pGL3basic, which were inversely regulated by NFB overexpression. All other vectors used were down-regulated and thus not suitable in our experimental setting. Transfection efficiency control vectors showed marked responsiveness to NFB overexpression. pSV-ß-galactosidase control vector activity was up-regulated 7-fold by NFB overexpression, and the activity of the Renilla luciferase control vectors phRG-TK and pRL-TK were down-regulated more than 50-fold and more than 6-fold, respectively (data not shown). Thus we decided to normalize on protein content, the most reliable system in this context.
Characterization of Putative NFB-Response Elements
HEK-293 cells were transfected with expression plasmids for the NFB subunits p50 and p65 or with expression plasmid for p50 alone before nuclear extracts were prepared and analyzed by EMSAs using 17 oligonucleotides comprising putative NFB elements described in Table 2. The oligonucleotides used for the EMSAs also contained the flanking bases from the 1-hydroxylase promoter. All 17 oligonucleotides displayed retarded bands and were tested in supershift experiments using an antibody against the p50 subunit. Ten of 17 oligonucleotides showed a supershift band and were used for further characterization (Fig. 2B). The remaining seven elements were not further analyzed. The binding of the 10 putative elements could be competed by incubation with a 100-fold excess of unlabeled oligonucleotide and a NFB consensus element. The results are summarized in Table 2 and Fig. 2B. No binding of nuclear extracts to mutated forms of oligonucleotides NFB 1–14 could be detected. The elements were mutated in such a way that at least four bases differed from the consensus element.
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shows a representative EMSA experiment where oligonucleotide NFB5 was used for binding to nuclear extracts prepared from HEK-293 cells. In lane 1 a band appeared that could be suppressed by coincubation with a 100-fold excess of unlabeled oligonucleotide or NFB consensus oligonucleotide (lanes 2 and 3). After overexpression of the NFB subunits p50 and p65, the retarded band was more intensive (lane 4). Incubation with antibodies against the p50 and p65 subunits, respectively, led to the formation of supershift complexes (lanes 5 and 6). After overexpression of the p50 subunit alone, the binding of nuclear extracts to the oligonucleotide NFB5 was confirmed by supershift assay using an antibody against the p50 subunit (lanes 7 and 8). Using a mutated form of the putative NFB5 element, no band appeared (lanes 9 and 10).
Localization and Characterization of the Most Important 3'-Response Element
EMSA Experiments.
According to the deletion construct experiments (Fig. 1B) no putative NFB-responsive element upstream –240 seemed to contribute to the NFB responsiveness of the full-length promoter to a relevant extent. In the case of the element NFB15 (base –92 to –83) the first EMSA experiments were done using an oligonucleotide derived from the published promoter sequence (GenBank no. AF072470). The element was identified as a functional NFB element by supershift assay using nuclear extracts from p50 overexpressing HEK-293 cells (Fig. 2B). After sequence analysis of our 1413-bp promoter fragment and comparison of both promoter sequences, a single base exchange (A-95G) was observed, located three bases 5' to the described element. The EMSA experiments were repeated using an oligonucleotide based on the new promoter sequence. Nuclear extracts still bound to the new oligonucleotide, and the intensity of the band was reduced by incubation with a p50 antibody, but no supershift band could be detected. The putative sites NFB14.1 and NFB14.2 located within the pGL3–240 construct were characterized as functional response elements (Fig. 2B).
Site Directed Mutagenesis.
The first effort to functionally elucidate the role of the three putative very 3'-located NFB sites was the mutation of these elements by site-directed mutagenesis analogous to the oligonucleotide mutations described in the EMSA experiments. NFB15 (sequence 5'-GGGGCGTCAT-3') was mutated to 5'-CGTGCGTCAT-3' in the pGL2–1413 promoter as well as in the –155 bp construct and luciferase activities were compared. The promoter of the pGL2–1413NFB15mut plasmid showed a 4.8-fold increased luciferase activity compared with the wild-type construct (Fig. 3B). This NFB site was also mutated in the minimal promoter-containing plasmid pGL3–155. The luciferase activity of the pGL3–155NFB15mut was increased 26-fold compared with the non-mutated deletion construct (data not shown). We tested both mutated constructs for responsiveness on promoter activity and the mutated oligonucleotide for NFB binding in EMSA analysis using nuclear extracts prepared from p50 overexpressing cells. A retarded band was enhanced after mutation of the oligonucleotide; this band could not be displaced by an unlabeled NFB consensus element and was not supershifted by a p50 antibody. The mutated full-length and the pGL3–155 construct still retained NFB responsiveness (data not shown). To characterize the contribution of the NFB14.1 and NFB14.2 elements to the NFB inhibition of the promoter activity, both elements were mutated in the pGL3–1413 construct. The sequence of the NFB14.1 element was changed from 5'-GGGAGTTGTG-3' to 5'-CGTAGTTGTG-3', and the sequence of the NFB14.2 element was changed from 5'-GTCAGCCCCA-3' to 5'-GTCAGCCGCA-3' by site-directed mutagenesis. Basal activity of the pGL3–1413mut14.2 and the pGL3–1413mut14.1 constructs were unchanged or reduced to 25% compared with the wild-type construct, respectively. Both mutated elements did not bind NFB protein in EMSA analyses, but both promoter constructs were still NFB responsive in overexpression experiments (data not shown).
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B15B elements, we hypothesized that the mutated NFB sites could still be low-affinity response elements under conditions of overexpression. Therefore we decided to delete these sites in the full-length promoter and the pGL3–155 construct starting with NFB15 (Fig. 3A). The basal activity of the pGL3–1413KO15 construct was 30% compared with the wild-type promoter (Fig. 3B). Cotransfection of successive 0.1 and 0.05 µg p50 and p65 subunits inhibited luciferase activity of the pGL3–1413 wild-type construct as usual, whereas the promoter activity of the pGL3–1413KO15 construct was almost abolished, indicating that NFB15 was the absolute predominant element in mediating NFB responsiveness (Fig. 3C). The pGL3–155KO15 construct could not be analyzed in cotransfection experiments due to very low basal promoter activity (30 relative light units vs. 500,000 relative light units of the full-length promoter).
Regulation of Endogenous 1-Hydroxylase mRNA Expression by Sulfasalazine
After 24, 48, and 72 h of incubation with H2O2, the amount of endogenous NFB protein detected by polyclonal antibodies against the p50 and the p65 subunits, respectively, was equally high throughout the experiment and could not be further stimulated (Fig. 4A), indicating very high basal expression and activation of NFB in HEK-293 cells. The same results were achieved using nuclear extracts from HEK-293 cells (data not shown). After treatment of untransfected HEK-293 cells with 2 mM sulfasalazine, a potent inhibitor of NFB, for 2–4 h the expression of 1-hydroxylase mRNA was determined by real-time PCR. 1-Hydroxylase mRNA levels were enhanced more than 9-fold compared with mRNA levels in untreated cells by 2 h (Fig. 4B). The data presented in Fig. 4B were obtained from two independent experiments and were normalized to the expression levels of ß-actin as a housekeeping gene. In another independent experiment we measured a 3.4-fold stimulation of 1- hydroxylase mRNA levels after 4 h of incubation (data not shown). According to the Relative Expression Software Tool (REST), P = 0.001 (17).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase promoter fragment to the GenBank entry AF072470, we found four base exchanges and three deletions. We confirmed our data by genomic sequencing of five fragments isolated from peripheral blood. Our published sequence was verified by other groups (Hewison, M., Birmingham, UK; personal communication). The regulation of human 1-hydroxylase activity in calcium homeostasis is well established. The main source of 1,25-(OH)2 vitamin D3 hormone is the kidney, where the activating enzyme is expressed in the proximal tubuli. Here, PTH enhances the synthesis of 1,25-(OH)2 vitamin D3 via stimulating promoter activity as previously shown by Murayama et al. (14). In a negative feedback loop 1,25-(OH)2 vitamin D3 suppresses further vitamin D3 hormone synthesis as shown in enzyme kinetics, although no vitamin D3 response element could be localized within the published promoter sequence. However, it was shown, that the PTH promoter comprises a negative 1,25-(OH)2 vitamin D3 response element that is responsible for the negative feedback regulation of PTH secretion in calcium homeostasis (2). 1-Hydroxylase transcription is also regulated in the context of phosphate homeostasis. Recent data show stimulation of the 1-hydroxylase promoter in the renal proximal tubule by a low-phosphate diet (10, 20). Low-phosphate intake stimulates the sodium-phosphate cotransporter (NPT2a) as well as 1-hydroxylase expression in the kidney, to enhance duodenal phosphate uptake via 1,25-(OH)2 vitamin D3 and renal tubular reabsorption via NPT2a. The phosphaturic compound FGF-23, on the contrary, inhibits 1-hydroxylase mRNA synthesis. Thus, FGF-23 hypersecretion by mesenchyme derived tumors with consecutive renal tubular wasting of inorganic phosphate is accompanied by low serum levels of 1,25-(OH)2 vitamin D3 causing oncogenic osteomalacia as we and others have shown previously (18, 20).
1,25-(OH) vitamin D3 also has immunomodulatory function. 1,25-(OH)2 vitamin D3 is immunosuppressive via diminishing Th1 cell activity and preventing the differentiation of immature dendritic cells from monocytes. In mature dendritic cells, 1,25-(OH)2 vitamin D3 induces apoptosis and reduces their T cell stimulatory potential (11, 16). There is evidence that the key enzyme of 1,25-(OH)2 vitamin D3 activation is itself regulated by cytokines. In human umbilical vein endothelial cells, proinflammatory cytokines such as TNF increased 1-hydroxylase enzyme activity (22). In contrast, in inflammatory diseases such as rheumatoid arthritis, clinical observations show high serum levels of TNF and IL-1ß coincident with diminished serum concentrations of 1,25-(OH)2 vitamin D3 (15) indicating the down-regulation of 1-hydroxylase activity in the kidney.
Within the 1.4-kb cloned promoter fragment, we characterized 17 putative binding sites for the transcription factor NFB. We chose NFB elements that differed from the NFB consensus site 5'-GGGRNNYYCC-3' in no more than three positions. In EMSA experiments, 10 of 17 oligonucleotides indicated functionality by showing a supershift band after incubation with nuclear extracts and an antibody against the p50 subunit of NFB complexes. Although we analyzed 17 putative NFB elements, we cannot exclude the existence of even more NFB sites within the 1.4-kb promoter fragment. We cloned progressive 5'-deletion constructs of 1-hydroxylase promoter and determined luciferase activity and NFB responsiveness. Overall promoter activity of deletion constructs varied probably due to deletion of enhancer/silencer elements. NFB almost abolished promoter activity in cotransfection studies.
When analyzing 1-hydroxylase promoter activity and regulation by NFB, we came upon technical and methodical problems. HEK-293 cells show a very high basal expression and activation of NFB, which could not be further enhanced by stimulation with H2O2 or TNF. Therefore, we cotransfected expression plasmids for NFB subunits to investigate NFB effects on 1-hydroxylase promoter activity. In our overexpression studies we observed stimulatory effects on pGL3basic and pGL2basic luciferase reporter vectors as well as stimulatory and inhibitory effects on the internal control vectors for ß-galactosidase and for Renilla luciferase, respectively, suggesting the existence of intrinsic response elements within the vector sequences. Thus we had to normalize the obtained luciferase values to protein content.
All promoter deletion constructs investigated were markedly NFB responsive. Upon analysis of the minimal promoter fragment –155 we found a slight but significant enhancement of promoter activity on a very low activity level. However, as discussed earlier, this might also be due to the stimulatory effect on the empty pGL3basic reporter vector.
When the putative NFB15 response element of this minimal promoter fragment was mutated by site-directed mutagenesis, basal promoter activity was enhanced. Especially with respect to the fact that the activity of the pGL3–155 construct is 10-fold below the activity of the empty reporter vector pGL3basic, one can conclude already at this point that the putative NFB15 response element in fact comprises a negative regulatory element. However, under conditions of overexpression of NFB components, the inhibitory effect was still present. Thus we decided that we had to delete the element to demonstrate its functional relevance. After deletion of the NFB15 element in the pGL3–1413 construct, the responsiveness of the promoter to NFB overexpression was almost abolished (Fig. 3C), indicating that this element mediates the down-regulation of 1-hydroxylase promoter activity by NFB. The basal activity of the pGL3–1413KO15 promoter construct was 33% compared with the pGL3–1413 construct, which might be due to the existence of more putative binding sites for stimulatory transcription factors in this region. In fact, when we analyzed the minimal promoter –155 in silico by using the most recent version of AliBaba2.1 we found a putative cAMP response element-binding protein binding site (base –91 to –80), an AP1 site (base –89 to –80), and a SP1 site (base –93 to –86) which was previously described by Kong et al. (9) overlapping with the putative NFB15 element (base –92 to –85) (Fig. 3A). This may indicate that a very intensive competition of various transcription factors occurs in this critical region. Thus, even if the NFB15 response element does not show the strongest binding compared with other response elements, we would assign it as a low-affinity, high-efficiency binding site in a DNA stretch where multiple signal pathways are integrated.
The shortcomings of transfection studies are the artificial setting for analyzing promoter regulations and the possibility of squelching effects of different transcription factors. Thus, we treated untransfected HEK-293 cells, expressing high levels of endogenous NFB under basal conditions, with the NFB inhibitor sulfasalazine and evaluated the mRNA levels of 1-hydroxylase by real-time PCR. Sulfasalazine acts by repressing IB phosphorylation, leading to the inhibition of IB degradation and thus blocking NFB translocation to the nucleus (21). Endogenous 1-hydroxylase mRNA levels were enhanced 9-fold by sulfasalazine, indicating a suppressive effect of NFB under basal conditions. These results are compatible with those obtained in the cotransfection system, indicating that, even in the cellular context, and the whole promoter context of untransfected cells, there is evidence for down-regulation of 1-hydroxylase mRNA by NFB. The results are also in line with the clinical situation in which sulfasalazine is used as a therapeutic agent in chronic inflammatory diseases such as Crohn’s disease and rheumatoid arthritis.
The promoter of 1-hydroxylase is suppressed after activation of NFB although NFB is otherwise mainly involved in activating gene transcription in inflammatory diseases. NFB binding sites are found in more than 50 promoters and enhancers of genes that are known to be activated upon inflammation (1). The fact that in different cell systems, such as endothelial cells, proinflammatory signals stimulate 1-hydroxylase activity may be due to a different cellular context and requires further investigations. However, the fact that a positive NFB-LUC control reporter gene vector is definitely stimulated within the same cellular context makes us confident in our observed data concerning suppressive 1-hydroxylase responsiveness.
In summary, we show here the down-regulation of the 1-hydroxylase promoter by NFB and stimulation of endogenous 1-hydroxylase mRNA expression by the NFB inhibitor, sulfasalazine, in HEK-293 cells. This would be in line with clinical findings in inflammatory diseases, in which low 1,25-(OH)2 vitamin D3 levels are coincident with high levels of proinflammatory cytokines involving the NFB signaling pathway. Systemic down-regulation of 1-hydroxylase may contribute to the pathogenesis of inflammation and transplantation-associated osteoporosis.
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MATERIALS AND METHODS |
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Cloning of the 1-Hydroxylase Promoter Fragment
A 1.4-kb fragment of the promoter of human 25(OH) vitamin D3 1-hydroxylase was isolated from a PAC-clone (Resource center of the German human genome project, clone ID LLNLP70PI0129Q2). A genomic PAC library (female RPCI6 709) with 4-fold coverage and an average insert length of 130 kb was obtained from the Resource Center, Primary Database of the German Human Genome Project at the Max-Planck-Institute for Molecular Genetics (Berlin, Germany). The library was screened with a randomly primed [32P]dCTP-labeled probe corresponding to the full-length of the 1.5-kb cDNA of human 1-hydroxylase amplified from human kidney cDNA by PCR. To amplify a 1413-bp promoter fragment from the PAC DNA, primer 122 and primer 1NCO (Carl Roth GmbH, Karlsruhe, Germany, see Table 3) and Herculase Enhanced Polymerase (Stratagene, Amsterdam, The Netherlands) were used according to the manufacturer’s instructions. The PCR product was cloned into pCRII vector (Invitrogen, Karlsruhe, Germany), excised with NcoI and KpnI (New England Biolabs GmbH, Frankfurt, Germany), and cloned into luciferase reporter vector pGL3basic (Promega GmbH, Mannheim, Germany; GenBank no. U47295). For cloning the 1-hydroxylase promoter 1413 bp into pGL2basic (GenBank no. X65323), the promoter fragment was amplified with 122 and 123, cloned into pCRII, excised with SacI and KpnI restriction enzymes, and subcloned into pGL2basic vector. The cloned promoter fragment was confirmed by sequencing.
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NCO as the reverse primer and 1K155, K240, K437, K748, and 1K927 as forward primer (see Table 3 for primer sequences). As template, 100 ng of pGL3–1413 were used. For amplification the Herculase Enhanced Polymerase system was also used as described previously. All constructs were confirmed by sequencing.
Site-Directed Mutagenesis
Mutants and deletion constructs of the NFB site 15 were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions. For mutagenesis, primers cNFB15mut and cNFBKO15 (see Table 3) and their complementary oligonucleotides were used. The mutation was confirmed by sequencing.
Transfection Studies
For transfection, HEK-293 cells were seeded in 12-well plates and transfected at 50–70% confluence. Liposome-mediated transfection was performed using LipofectAMINE reagent (Invitrogen) according to the method of Felgner et al. (3). LipofectAMINE (4 µl) and 0.9 µg of the 1-hydroxylase promoter reporter constructs were diluted in 100 µl serum-free medium and incubated for 30 min at ambient temperature. For overexpression of p50 and p65 NFB subunits, 0.1 and 0.05 µg of the expression plasmids pcDNA3.1-p50 and pcDNA3.1-p65 were used. In control experiments, the amount of transfected DNA was adjusted to 0.1 µg by using the empty pcDNA3.1 (Invitrogen) vector. Serum-free medium (400 µl) were added, and the solution was then carefully dripped onto the cells. HEK-293 cells were incubated for 5 h in transfection solution. After removal of the medium, cells were cultivated with serum-containing medium. After 48 h the cells were harvested in 200 µl reporter lysis buffer (Promega GmbH). Cell lysate (20 µl) was used for luciferase measurement. Analysis of luciferase activity was performed using the reporter gene assay provided by Promega in a microplate luminometer (EG&G Berthold, Bad Wildbad, Germany). Protein content of the cell lysate was determined by using Bio-Rad protein assay (Bio-Rad Laboratories GmbH, München, Germany) and measured in a Uvicord III photometer (Amersham Pharmacia Biotech, Freiburg, Germany). For control of transfection efficiency, the obtained luciferase values were divided by the protein concentrations. As a control, the NFB responsive vector pNFB-LUC obtained from Stratagene was cotransfected with pcDNA3.1-p50 and pcDNA-3.1-p65 overexpressing plasmids, and luciferase activity was determined.
For monitoring transfection efficiencies the control vectors pSV-ß-Galactosidase, pRL-TK, a reporter vector for Renilla luciferase and phRG-TK, a synthetic Renilla luciferase reporter vector were cotransfected with promoter luciferase constructs in equal amounts, respectively. Firefly and Renilla luciferase activities were determined using the Dual-Glo luciferase assay system according to the manufacturer’s instructions. ß-Galactosidase activity was measured using the ß-galactosidase enzyme assay system according to the manufacturer’s instructions. All vectors, luciferase, and ß-galactosidase assay systems were obtained from Promega.
Protein-DNA Interactions
The interaction of HEK-293 nuclear proteins with oligonucleotides corresponding to putative NFB elements with the consensus sequence 5'-GGGRNNYYCC-3' (8), and flanking bases found in the promoter of human 1-hydroxylase (see Table 2), were analyzed by EMSAs. HEK-293 cells were seeded in 25-cm2 flasks and transfected with NFB expressing vectors as described above. After 48 h the medium was removed and the cells were washed twice with PBS. Nuclear extracts were prepared according to a modified protocol of Grandison et al. (5). Nuclear cell extracts (5–20 µg of protein) were incubated in a reaction buffer of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.2% Nonidet P-40, 5% glycerol containing 1 µg of poly(dIdC), and approximately 50 fmol of 32P-labeled double-stranded oligonucleotide containing the putative NFB binding sites (15,000–20,000 cpm) in a volume of 15 µl for 30 min at 4 C. The oligonucleotides were annealed and end labeled using T4 polynucleotide kinase (Invitrogen) and [
-32P]ATP (Amersham Biosciences). For all NFB5 elements, additional experiments were done using mutated oligonucleotides.
For competition experiments, a 100-fold molar excess of unlabeled oligonucleotide and NFB consensus element, respectively, was added to the reaction mixture before the addition of reaction buffer. For supershift experiments, 2 µg of monoclonal antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) against the p50 and p65 subunits of NFB complexes, respectively, were added to nuclear extracts 30 min before addition of the radioactive labeled oligonucleotide and incubated in reaction buffer at 4 C. Reaction mixtures were analyzed by nondenaturing polyacrylamide gel electrophoresis and autoradiography.
Isolation of DNA from Human Peripheral Blood
DNA from five healthy donors was isolated from peripheral blood using peqGOLD Trifast reagent (Peqlab, Erlangen, Germany) according to the manufacturer’s instructions. Two micrograms were used for PCR to amplify the promoter of 1-hydroxylase as described previously. The DNA was sequenced by cycle sequencing and analyzed on an ABI Prism 310 sequencer (PE Biosystems GmbH, Weiterstadt, Germany).
Western Blot Analyses of NFB Subunits
HEK-293 cells were seeded in 25-cm2 flasks and transfected with expression plasmids for the NFB subunits p50 and p65 as described above. After 48 h, nuclear extracts were prepared as described above or cells were harvested in homogenization buffer (50 mM Tris, 1 mM EDTA, 1 mM Pefabloc, 1 mM dithiothreitol). In an additional approach, cells were stimulated with 300 nM H2O2 for 24, 48, and 72 h and nuclear or whole-cell extracts were prepared. The protein content was determined using the Bio-Rad (München, Germany) protein assay. Protein (20 µg) was boiled for 5 min in SDS-PAGE buffer (100 mM Tris, pH 6.8; 7.5% glycerol; 1% sodium dodecyl sulfate; 0.025% bromphenol blue) and separated by sodium dodecyl sulfate gel electrophoresis. Proteins were transferred to Optitran BA S 85 membranes (Schleicher & Schuell, Dassel, Germany). The membranes were treated with a buffer containing 0.1% Tween 20, 2% horse serum, 2.5% BSA, and 2.5% milk powder in PBS for 2 h to inhibit nonspecific binding. Then, the membranes were incubated in 0.1% Tween 20, 1% horse serum, and 1% milk powder in PBS with polyclonal antibodies against the p50 and the p65 subunits of the NFB complex, respectively (Santa Cruz). Membranes were washed with 10 mM Tris (pH 7.5), 140 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, 1% horse serum, 1% BSA, 1% milk powder, followed by incubation with horseradish peroxidase-conjugated goat antimouse IgG antibody using a solution containing 0.1% Tween 20, 1% horse serum, 1% BSA, and 1% milk powder in PBS. The expression of p50 and p65 was detected by using the ECL system (Amersham Biosciences).
Real-Time PCR of 1-Hydroxylase
For monitoring basal 1-hydroxylase mRNA expression and to confirm its regulation by endogenous NFB, a real-time PCR protocol was established. HEK-293 cells were cultivated with or without 2 mM sulfasalazine (Sigma, Taufkirchen, Germany), a potent inhibitor of NFB activation, for 2–4 h. Total RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. Total RNA (2 µg) was reverse transcribed with M-MLV reverse transcriptase (Promega) in a volume of 20 µl. cDNA (1 µl) was used for 1-hydroxylase and ß-actin amplification as a housekeeping gene, respectively. PCR conditions were as follows: 30 sec, 94 C; 30 sec, 58 C; and 30 sec, 72 C (see Table 3 for primer sequences). Real-time PCR was performed with the DNA Engine Opticon system (MJ Research, Waltham, MA) using SYBR Green as fluorescent dye. For quantification and statistical analyses, 1-hydroxylase mRNA expression was normalized to the expressed housekeeping gene ß-actin using REST (17). Specificity of 1-hydroxylase amplicons was confirmed by melting curve analyses.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: FGF, FiGbroblast growth factor; HEK, human embryonic kidney; N, any base; NFB, nuclear factor B; R, purine; REST, relative expression software tool; Y, pyrimidine.
Received for publication December 30, 2002. Accepted for publication June 22, 2004.
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