USF-1 and USF-2 trans-repress IL-1beta -induced iNOS transcription in mesangial cells

Ashish K. Gupta and Bruce C. Kone

Department of Internal Medicine and Department of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682-1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region -904/-883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the -893/-888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the -893/-888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1beta (IL-1beta ) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the -893/-888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1beta -simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the -904/-883 region augmented IL-1beta stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at -893/-888 serves to trans-repress basal expression and IL-1beta induction of the iNOS promoter.

transcription; glomerulus; inducible nitric oxide synthase; promoter; inflammatory cytokines; transcription factors; upstream stimulatory factor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is an important molecular mediator of numerous physiological processes in virtually every organ. NO is synthesized from L-arginine by the NO synthase (NOS) isozymes. Neuronal and endothelial NOS isozymes have restricted tissue distributions and are regulated in part by intracellular Ca2+ transients. Inducible NOS (iNOS) is expressed in a number of cell types in mammals after induction by cytokines and/or lipopolysaccharide (LPS) and, once expressed, is active at resting levels of intracellular Ca2+ (12). Induction of iNOS principally involves transcriptional activation, so considerable effort has been dedicated to identify the cis element and trans-acting factors that control its expression.

In the kidney, NO plays prominent roles in the homeostatic regulation of glomerular, vascular, and tubular function, as well as a variety of fundamental cellular functions, including DNA replication, transcription, energy metabolism, and apoptosis (12, 13, 17, 44). Although NO serves beneficial roles as a messenger and host defense molecule, excessive NO production can be cytotoxic, the result of NO's reaction with reactive oxygen species, leading to peroxynitrite anion, nitroxyl radical, and hydroxyl radical production and protein tyrosine nitration (2, 16). Recent studies provide clear evidence for participation of iNOS-generated NO in the induction, progression, or protection of several types of experimental and human glomerulonephritis. In human glomerulonephritis, iNOS gene expression has been described in glomerular mesangial cells, as well as in local and infiltrating macrophages (6, 41). Mesangial cells contribute prominently to the pathogenesis of glomerulonephritis, in part by producing a variety of cytokines and NO via iNOS. Consequently, the mesangial cell has been a center of investigational focus in this disease.

Structure-function analyses of the murine iNOS promoter/enhancer region have identified several response elements that are functionally active. LPS inducibility of the iNOS promoter activity in the mouse macrophage cell line RAW 264.7 is largely dependent on a nuclear factor-kappa B (NF-kappa B)-binding element (-85/-76) (46) and an LPS response element (45) in its proximal region. The synergistic effect of interferon (IFN)-gamma to activate iNOS promoter activity requires inclusion of distal promoter elements, including an IFN regulatory factor-1-binding element (-923/-913) (20), two sequential IFN-stimulated response elements, and an IFN-gamma -activated site (7). The finding that LPS induces footprinting of other regions of the murine iNOS promoter suggested that other cis elements and trans-acting factors contribute to iNOS induction. In particular, we were intrigued by the observation from in vivo footprinting of RAW 264.7 macrophage cells (8) that LPS induces protection of guanines in the region -904/-883, which contains a sequence -893 CATGTG 888 that conforms to an E-box element (CANNTG), the suggested target site for DNA binding of basic helix-loop-helix (bHLH) transcription factors. The identical sequence is similarly positioned in the rat iNOS promoter.

The bHLH superfamily of transcription factors regulates growth and differentiation in a variety of tissues by forming transcriptionally active heterodimers that bind to E-box elements in the promoters of target genes. bHLH factors are important in developmental and tissue-specific expression of numerous genes but have not been classically attributed to regulation of proinflammatory genes (24). This class of transcription factors includes, among others, c-Myc (9), Max (9), E2A (11), sterol regulatory element-binding protein (34), and upstream stimulatory factors (USFs) (10). USF was originally identified as a factor that activates the adenovirus major late promoter (32). USF activity involves two polypeptides with apparent molecular masses of 43 and 44 kDa, which are referred to as USF-1 and USF-2, respectively (31, 33). The USF proteins form hetero- and homodimers (37) and bind to the E-box motif (37). USF proteins are thought to be involved in cell cycle regulation, including an antagonistic action against the function of c-Myc (3). In addition, USFs have been implicated in the control of several genes, including the genes for C/EBP (38), liver-type pyruvate kinase (39), type 1 collagen (27), and fatty acid synthase (40).

We have examined the functional importance of the -893/-888 E-box element in the transcriptional competency of the murine iNOS promoter in cultured mesangial cells. We provide evidence that this E-box is required and USF-1 and USF-2 are in vivo components of iNOS regulation by correlating functional assays and USF-binding activities to the E-box. When the E-box was mutated, it was no longer a target for USF binding, and the activity of the iNOS promoter was enhanced. Cotransfection of USF-1 and USF-2 expression vectors with the iNOS promoter suppressed iNOS promoter activity, and dominant-negative USFs lacking the DNA binding activity augmented interleukin-1beta (IL-1beta ) induction of the iNOS promoter activity. Our results suggest that the USF proteins play important roles in constraining iNOS induction in mesangial cells and, thus, may serve to limit untoward effects of excessive NO in the mesangium and glomerulus.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents. Poly(dI-dC)-poly(dI-dC) was purchased from Pharmacia-LKB Biotech. Oligonucleotides were custom synthesized by Genosys (The Woodlands, TX). The Dual-Luciferase Reporter Assay System and the luciferase vectors pGL3-basic and pRL-TK were obtained from Promega, the bicinchoninic acid protein estimation kit from Pierce Chemical, RNAzol reagent from TEL-TEST (Friendswood, TX), the endotoxin-free plasmid Maxi-prep kit from Qiagen (Santa Clarita, CA), and mouse recombinant IL-1beta from Genzyme (Cambridge, MA). Rabbit polyclonal IgG antibodies raised against bHLH proteins USF-1 (C-20) (21, 23), USF-2 (C-20) (21), c-Myc (sc-764) (42), E2A (sc-416) (15), Id-1 (sc-427) (43), and BETA3 (sc-6045) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); these antibodies have been shown to recognize the corresponding mouse proteins (see manufacturer's catalog and Refs. 28-32).

Cell culture. Mouse mesangial cells (American Type Culture Collection) were cultured at 37°C in complete medium (Ham's F-12 + DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% FBS). Vehicle or IL-1beta (10 ng/ml) was added to the cells as indicated.

Plasmids and site-directed mutagenesis. The iNOS promoter-luciferase construct piNOS-luc, which contains a DNA fragment comprising nucleotides -1486 to +145 of the murine iNOS promoter/enhancer in pGL3-basic, was used as the wild-type iNOS promoter-luciferase construct. Deletion and site-directed mutation of the -904/-883 segment in piNOS-luc was accomplished by PCR splicing using overlap extension, with the wild-type iNOS promoter/enhancer cDNA as template. For deletion of the -904/-883 element, forward primer P1 was used with mutagenic reverse primer P3 (5'-TCCAATAAAGCATTCAGCATGGAATTTTC-3') in the upstream reaction, and mutagenic forward primer P4 (5'-AATGGAAAATTCCATGCTGAATGCTTTAT-3') and reverse primer P2 were used in the downstream PCR. For mutation of the -893/-888 E-box (CATGTG replaced with ACTGCT), the upstream reaction contained forward primer P1 and mutagenic reverse primer P5 (5'-TCCAATAAAGCATTCAAGCAGTGCATGGAATTT-3'), whereas mutagenic forward primer P6 (5'-AATGGAAAATTCCATGCACTGCTTGAATGCTTTA-3') and reverse primer P2 were used in the downstream reaction. The full-length site-deleted or -mutated iNOS promoter/enhancer was then constructed in a PCR containing 50-fold dilutions of the upstream and downstream PCR products from the initial PCR together with primers P1 and P2. The mutated P1-P2 promoter fragment PCR products were first cloned into pCR2.1, sequenced to verify the presence of the desired mutations and the absence of spurious mutations, and then subcloned into pGL3-basic to create the recombinant molecules piNOS-delE-box-luc (deleted E-box) and piNOS-Delta E-box-luc (mutated E-box). Expression plasmids psv-USF1 and psv-USF2, as well as a dominant-negative psv-USF2Delta B, were provided by Dr. Michele Sawadogo (University of Texas M. D. Anderson Cancer Center). psv-USF2Delta B encodes a USF-2 protein that lacks the DNA-binding domain but is able to dimerize with USF-1 or USF-2 and, thereby, inhibits them in a dominant-negative fashion (22).

Transient transfection and reporter gene assays. Mesangial cells were seeded in six-well plates and grown to ~70% confluence in DMEM + 10% FBS without antibiotics. On the following day, the cells were cotransfected with 4.5 µg/well of pGL3-basic, piNOS-luc, piNOS-delE-box-luc, or piNOS-Delta E-box-luc, together with 0.5 µg/well of the Renilla luciferase expression plasmid pRL-TK, using the LipoFectamine PLUS reagent according to the manufacturer's protocol. At 24 h after transfection, the medium was replaced with complete medium and vehicle or IL-1beta . After 16 h, cell lysates for measurement of firefly and Renilla luciferase activities were prepared using Passive Lysis Buffer (Promega) according to the manufacturer's directions, and firefly and Renilla luciferase activities were measured as described in our previous report (10a). For trans-repression assays, pGL3-basic or piNOS-luc was transfected with psv-USF1, psv-USF2, or psv-USF2Delta B, together with 0.5 µg/well of pRL-TK.

Electrophoretic mobility shift and supershift assays. Nuclear extracts were prepared from mesangial cells, and electrophoretic mobility shift assay (EMSA) was performed as previously described (14). Double-stranded oligomers for use as probes or competitors (Table 1) were generated by annealing complementary single-stranded oligonucleotides and were 5'-end-labeled with [gamma -32P]ATP (3,000 Ci/mmol) using T4 polynucleotide kinase. Binding reactions (20 µl) containing 5-10 µg of nuclear extract protein, 1.75 pmol of duplex DNA probe (~2 × 105 cpm), and reaction buffer [25 mM HEPES, pH 8.0, 50 mM KCl, 0.1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 10% glycerol, and 50 µg/ml poly(dI-dC)-poly(dI-dC)] were conducted for 30 min at room temperature. To demonstrate sequence specificity of the protein-DNA interactions, binding reactions were conducted in the presence of a 50-fold molar excess of nonradiolabeled specific heterologous oligomers. For supershift assays, the probe-nuclear protein complexes were allowed to form for 15 min at 25°C, and then antibodies (2 µg) specific for USF-1, USF-2, c-Myc, E2A, Id-1, or BETA3 transcription factors or a comparable amount of nonimmune IgG were added to the binding reaction and incubated at room temperature for another 30 min. Aliquots of all reactions were electrophoresed through 5% native polyacrylamide gels, and the dried gels were subjected to autoradiography for detection of the shifted bands.

                              
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Table 1.   Oligonucleotides

Ultraviolet cross-linking analysis. Scaled-up EMSAs (100 µl reaction volume) were performed as described above, but using 50 µg of nuclear extract and 106 cpm of -904 to -883 probe, which had been modified to contain bromodeoxyuridine and bromodeoxycytosine in place of dT and dC (Genosys) on both strands to increase ultraviolet (UV) cross-linking efficiency. After electrophoresis of the binding reaction, autoradiograms of the wet gel were prepared to localize the shifted complexes. The complexes were individually excised, and the gel slices were UV irradiated (254 nm) in a Stratalinker (Stratagene) at 4°C for 1 h, boiled in Laemmli's sample buffer for 2 min, and electrophoresed on SDS-10% polyacrylamide gels. The gels were dried and autoradiographed to detect the constituent protein bands.

Northern analysis. Total cellular RNA was extracted from cell monolayers using RNAzol II. The samples were quantitated by spectrophotometry at 260 nm. For Northern analysis, cDNA probes specific for the murine iNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14) were labeled with 32P by the random primer method according to the manufacturer's instructions (Prime-a-Gene, Promega, Madison, WI). Fifteen micrograms of total RNA per lane were separated by size on 1% agarose-2% formaldehyde gels and blotted to nylon membranes (Hybond N, Amersham). After UV cross-linking, the blots were visualized under UV light and prehybridized for 2 h at 68°C in QuickHyb solution (Stratagene). The blots were sequentially hybridized with the murine iNOS and GAPDH DNA probes, with the blots being stripped before the next analysis, and washed to a final stringency of 0.1× saline-sodium citrate-0.1% (wt/vol) SDS at 60°C. Autoradiographs of the blots were prepared at -70°C.

Cis-element decoy assays. Double-stranded phosphorothioate oligonucleotides containing a four-repeat palindrome of the wild-type -904/-883 sequence (sense strand -904 5'-AATGGAAAATTCCATGCCATGTGTGAATGCTTTATT-3' -883) or the -904/-883 sequence bearing a mutated E-box (5'-AATGGAAAATTCCATGCACTGCTTGAATGCTTTA-3') were generated by annealing complementary oligonucleotides in 1× saline-sodium citrate at 95°C. Double-stranded oligonucleotides (150 nM) were transfected into mesangial cells as described above for plasmid preparations. At 16 h after transfection, RNA was harvested from the cells for Northern analysis, or, in separate plates, nuclear extracts were prepared for EMSA.

Data analysis. The intensities of bands on the Northern blot autoradiograms were measured by whole band densitometry software running on a SPARC Station IPC (Sun Microsystems, Mountain View, CA) equipped with an image analysis system (Bio Image, Ann Arbor, MI). Quantitative data are presented as means ± SE and were analyzed for significance by ANOVA. Significance was assigned at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of nuclear proteins to the -904/-883 region of the iNOS promoter. To determine whether the -893/-888 E-box could bind nuclear proteins from mesangial cells, EMSAs were performed using wild-type and E-box-mutated oligomers derived from the murine iNOS promoter as probes or competitors. Probe A (Table 1) contains the E-box element as well as the neighboring guanine residues that were protected in previous in vivo footprinting studies (8). EMSA using nuclear extracts from vehicle-treated cells with probe A resulted in formation of a DNA-protein complex (Fig. 1A). IL-1beta treatment resulted in greater amounts of the complex. Sequence specificity of the binding activity was verified in competition experiments with excess unlabeled probe A or heterologous DNA (Fig. 1A). Excess unlabeled probe A exhibited comparable sequence-specific competition for the DNA-protein complexes from the vehicle and the IL-1beta -treated cells (Fig. 1A). To determine the sequence boundaries important for formation of the DNA-protein complexes, competition experiments were performed in EMSAs with oligomer A as probe and 50-fold molar excesses of unlabeled oligomer B, C, or D as competitors. These competitor oligomers represent partially overlapping segments of oligomer A. The DNA-protein complex was partially competed by excess oligomer B or C (Fig. 1B), both of which overlap the E-box. Oligomer D, which does not overlap the E-box, did not competitively suppress binding. Furthermore, no DNA-protein complex was formed in EMSAs using the mutated E-box probe AEboxm (Fig. 1C). Mutation of the E-box in the probe abolished formation of the gel shift complex in nuclear extracts prepared from vehicle- and IL-1beta -treated cells (Fig. 1C). Thus, although it is possible that other trans-acting factors contribute to the DNA-protein complex of the entire -904/-883 region, they do not contribute to the gel shift complex specific for the E-box.


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Fig. 1.   Interleukin-1beta (IL-1beta ) promotes DNA-binding activity of the -893/-888 E-box in nuclear protein extracts of mesangial cells. A: nuclear proteins extracted from mesangial cells that had been exposed to vehicle (Veh) or IL-1beta (10 ng/ml) for 4 h were subjected to electrophoretic mobility shift assay (EMSA) with 32P-labeled oligomers corresponding to the -904/-883 region of the inducible nitric oxide synthase (iNOS) promoter. To demonstrate binding specificity, reactions were also conducted in the presence of a 50-fold molar excess of unlabeled -904/-883 oligomer or heterologous oligomers. B: EMSAs were performed as described in A, except oligomers consisting of serial truncations of the -904/-883 region were present as competitors in a 50-fold molar excess. C: EMSA was performed as described in A, except probe AEboxm harboring a mutation (M) of the -893/-888 E-box was used in the binding reaction. W, wild-type probe A. Autoradiograms for A, B, and C are each representative of 4 independent experiments performed on separate preparations of nuclear extracts for each assay.

To characterize the factor(s) that is contained within the E-box complex, UV cross-linking reactions were performed using radiolabeled -904/-883 oligonucleotide probe A with nuclear extracts prepared from IL-1beta -treated mesangial cells. The cross-linked products were resolved on an SDS-polyacrylamide gel and detected by autoradiography. Figure 2 shows a clustering of ~45- to 48-kDa bands that were dependent on addition of nuclear extract and exposure to UV light. As a control, a 50-fold molar excess of unlabeled -904/-883 DNA was added as a competitor to determine the specificity of the cross-linking reaction (Fig. 2). Although we do not know the exact contribution of the cross-linked DNA to the clustered proteins, we estimate that the minimal protein size would be ~42 kDa, which is in the size range of the USF proteins.


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Fig. 2.   Ultraviolet (UV) cross-linking of proteins to the -904/-883 region of the iNOS promoter. Binding reactions were performed as for EMSA but contained 50 µg of nuclear extract from IL-1beta -treated mesangial cells and radiolabeled -904/-883 probe, which had been modified to contain bromodeoxyuridine and bromodeoxycytosine in place of dT and dC. In control experiments, a 50-fold molar excess of unlabeled -904/-883 oligomer was used. After electrophoresis of the binding reaction, shifted complexes were individually excised, UV irradiated, and then boiled in Laemmli's sample buffer and analyzed by SDS-PAGE and autoradiography. Results are representative of 4 experiments.

Accordingly, we performed supershift assays with antibodies against the USF proteins and related bHLH proteins. USF-1 and, to a lesser extent, USF-2 produced a supershift band (Fig. 3). In contrast, antibodies against c-Myc, E2A, Id-1, and BETA3 showed no supershifts of the DNA-protein complex (data not shown).


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Fig. 3.   Upstream stimulatory factor (USF)-1 and USF-2 proteins bind to the -904/-883 region of the iNOS promoter in vitro. Polyclonal IgG specific for USF-1 or USF-2 or a nonimmune IgG was used in supershift (SS) experiments with nuclear extracts from IL-1beta -treated (4 h) mesangial cells and the 32P-labeled -904/-888 E-box oligomer. Autoradiograms are representative of 3 independent experiments performed on separate preparations of nuclear extracts.

-893/-888 E-box sequence and USF-1 and USF-2 suppress maximal IL-1beta induction of the iNOS promoter. To determine whether the -893/-888 E-box represents an IL-1beta response element within the iNOS promoter, the promoter activities of a wild-type iNOS promoter-luciferase construct (piNOS-luc) and two derived constructs in which the E-box was deleted (piNOS-delE-box-luc) or mutated (piNOS-Delta E-box-luc) were tested in transient transfection experiments of mesangial cells treated with vehicle or IL-1beta (Fig. 4). As expected, piNOS-luc exhibited negligible promoter activity under basal conditions but robust promoter activity after IL-1beta treatment of the cells. In contrast, the IL-1beta -induced promoter activity of piNOS-delE-box-luc or piNOS-Delta E-box-luc was only ~60% of that of piNOS-luc. These results suggest that the -893/-888 E-box functions to regulate negatively IL-1beta induction of iNOS gene transcription in these cells.


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Fig. 4.   Functional importance of the -893/-888 E-box in control of the iNOS promoter. Mesangial cells were transfected with iNOS promoter luciferase (piNOS-luc), which contains the wild-type iNOS promoter piNOS-delE-box-luc, which contains the iNOS promoter harboring a deletion of the -893/-888 E-box, or piNOS-Delta E-box-luc, which contains the iNOS promoter harboring a mutation of the -893/-888 E-box. A Renilla luciferase expression vector (pSG5) was also cotransfected to normalize for transfection efficiency. After treatment with IL-1beta for 16 h, cell extracts were prepared and processed to measure luciferase activity (n = 5).

Overexpression of USF-1 and/or USF-2 by transient transfection significantly suppressed IL-1beta -induced promoter activity (Fig. 5). In contrast, overexpression of a dominant-negative USF-2 plasmid, which lacks the DNA binding domain but remains competent to dimerize with USF-1 or USF-2, reversed the inhibitory effect and promoted a further increment in suppressed IL-1beta -induced promoter activity of the iNOS gene (Fig. 5). In addition, overexpression of the dominant-negative USF-2 plasmid promoted an approximately twofold increase in basal iNOS promoter activity (Fig. 5). In a complementary assay, transfection of cis-element decoys, but not E-box-mutated decoy oligonucleotides, resulted in a significant decrease in the target nuclear E-box-protein complex (Fig. 6A) and enhanced basal and IL-1beta -induced iNOS mRNA expression (Fig. 6B), in agreement with the dominant-negative USF-2 experiments presented in Fig. 5. Importantly, levels of GAPDH mRNA were not affected by the transfection, further indicating that the effect of the USF decoys on iNOS mRNA expression was specific. These results suggest that USF-1 and USF-2 serve to silence the iNOS gene under basal conditions and after IL-1beta induction.


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Fig. 5.   USF-1 and USF-2 overexpression suppresses iNOS promoter activation in IL-1beta -treated mesangial cells. Mesangial cells were transfected with USF expression plasmids psv-USF1, psv-USF2, the dominant-negative psv-USF2Delta B, or the parent vector pSG5, as well as a Renilla luciferase expression vector. After treatment with vehicle or IL-1beta for 16 h, cell extracts were prepared and processed to measure luciferase activity (n = 6). #P < 0.05 vs. pSG5 (vehicle); * P < 0.05 vs. pSG5 (IL-1beta ).



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Fig. 6.   Blockade of USF binding to the E-box element augments basal and IL-1beta -stimulated iNOS mRNA expression. A: mesangial cells were mock transfected (no DNA added, -) or transfected with double-stranded oligonucleotides containing a 4-repeat palindrome of the wild-type (WT) -904/-883 element or with an identical concatamer, except the -893/-888 E-box was mutated (oligonucleotide AEboxm; see Table 1, designated M for mutant). After transfection, cells were stimulated with IL-1beta for 4 h, and nuclear extracts were prepared for EMSA with the radiolabeled -904/-883 probe. Transfection with the wild-type -904/-883 concatamer oligonucleotides significantly diminished the E-box gel shift band compared with mock-transfected or mutant oligonucleotide-transfected cells (n = 3). B: cells were transfected as described in A and treated with vehicle or IL-1beta for 16 h. RNA was harvested, and Northern blots were prepared for analysis of iNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression (n = 3).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the course of glomerular injury, mesangial cells respond to circulating cytokines or those produced by invading inflammatory cells by increasing iNOS gene expression and NO production. Because of the potential for excessive NO production to prove toxic to the host cells, complex layers of regulatory control have been placed on the iNOS gene. In this report, we showed that site-directed mutation of the -893/-888 E-box of the iNOS promoter abolished the USF binding to this region in vitro and disrupted the IL-1beta regulation of the piNOS-luc reporter constructs in transfection assays of mesangial cells. The supershift results indicate that the DNA-protein complexes principally contain USF-1/USF-2 heterodimers. When cotransfected with the iNOS promoter constructs, USF-1 and USF-2 inhibited and dominant-negative USF-2 could further activate IL-1beta -stimulated iNOS promoter activity. In agreement with these findings, transfection of cis-element decoy oligonucleotides of the -893/-888 E-box enhanced IL-1beta induction of the iNOS gene in these cells. Interestingly, blockade of USF action by overexpression of a dominant-negative USF-2 construct (Fig. 5) or transfection of the E-box cis-element decoy (Fig. 6B) resulted in an increase in basal iNOS promoter activity and mRNA abundance, respectively, suggesting a constitutive role of USF-1 and USF-2 to silence basal iNOS expression. Collectively, these data demonstrate that USF binding to the E-box at -893/-888 suppresses iNOS transcription and its induction and, thereby, may serve to constrain excessive production of NO by this enzyme. Given the limited transfection efficiencies of mesangial cells and the fact that only a fraction of the cells are successfully transfected, these results may underestimate the effects within the transfected cells. Because NO has been shown to be antiproliferative in mesangial cells (4, 29), our findings suggest yet another mechanism by which USF may regulate cellular proliferation.

USFs belong to the bHLH leucine zipper family of transcription factors characterized by a highly conserved COOH-terminal domain responsible for their dimerization and DNA binding. Structurally, USF-1 and USF-2 are highly related, except at the NH2 terminus (19). USF-1 and USF-2 are structurally related to the Myc family of proteins and normally bind to an E-box as dimers (homodimers as well as heterodimers) (37). Because of the demonstration of USF involvement in the transcriptional activation of the adenovirus major late promoter, USFs have been reported as potential regulators of numerous cellular genes involved in different important cellular processes. The effect on cell proliferation is in part related to the involvement of USF in regulation of p53 (26), cdc2 (5), and the cyclin B1 gene (5). USF is also involved in modulation of ras and c-myc transformation (1, 18).

Although the USF-1 and USF-2 genes are ubiquitously expressed in mammalian cells, the relative abundance of USF-1 and USF-2 gene product varies among cell types (36). It has recently been shown that the function of USFs is modulated in a cell-specific manner. This regulation depends on a short sequence stretch between the NH2-terminal transactivation domain and the DNA-binding domain known as the USF-specific region, which is critical for transactivation and nuclear localization (25). The cell specificity of USF proteins within the kidney is unknown, and there have been no reports of USF expression in glomerular mesangial cells. Given their ability to regulate a number of genes involved in cell proliferation and now iNOS, these proteins may prove to have important roles in modulating proliferative and inflammatory glomerular diseases.

The array of pathological conditions in which iNOS is maximally expressed is derived from different signals inducing iNOS and the involvement of different transcriptional activators to control its transcription. Recent work suggests that combinatorial interactions among transcription factors, and perhaps accessory proteins, may be important for specificity in the responsiveness of the iNOS gene to various stimuli in different cells types (30). USFs are known to interact with other transcription factors to alter gene transcription, (28, 35); whether USF-1 and USF-2 exert their inhibitory effects on iNOS transcription by interfering with other transactivators remains to be examined. In this regard, a number of cis elements neighboring the -893/-888 E-box have been shown to be functionally important in enhancing LPS- or cytokine-mediated iNOS induction in other cell types, including IFN-gamma -activated site elements at -942/-934 and -879/-871 and an NF-kappa B element (-971/-962). However, the fact that iNOS mRNA was expressed basally, without IL-1beta induction, when USF binding to the E-box element was blocked with cis-element decoys suggests that at least the basal effect of USF to limit iNOS transcription does not involve interference with inducible transcription factors, such as NF-kappa B or STAT-1, known to transactivate the iNOS gene.

The rodent and human iNOS promoters differ substantially in sequence and regulatory control. The proximal promoter of the human iNOS gene contains two CATGTG E-box elements at -358/-353 and -1832/-1827. However, the context of these elements with regard to neighboring consensus binding elements differs from the functional E-box element in the mouse iNOS promoter reported here. Thus it remains to be established whether USF proteins exert regulatory control on human iNOS transcription in a manner similar to that of the murine gene.

In the kidney, physiological amounts of NO have an important role in the regulation of renal hemodynamics, as well as sodium and water excretion (13). On the other hand, NO release as a result of cytokine-mediated activation of iNOS in mesangial cells can be sustained and lead to oxidative injury in various forms of glomerular inflammation. Accordingly, inhibition of iNOS expression and/or activity could be an effective anti-inflammatory strategy. Through their ability to suppress iNOS activation, USF-1 and USF-2 appear to serve this function in mesangial cells in vivo. However, given the large amounts of NO generated by maximally activated iNOS, the specific biological responses to a partial (~40% in the case of the USF protein reported here) inhibition of iNOS transcription are difficult to predict. In this regard, the ability of the USF proteins to suppress iNOS gene expression under basal conditions may serve as an important constitutive brake on the expression of the iNOS gene and the production of NO.


    ACKNOWLEDGEMENTS

We thank Dr. Q.-W. Xie (Cornell University) for helpful technical suggestions.


    FOOTNOTES

B. C. Kone was supported by National Institutes of Health Grants RO1 DK-50745 and P50 GM-20529. A. K. Gupta was supported by a National Kidney Foundation Research Fellowship.

Address for reprint requests and other correspondence: B. C. Kone, Dept. of Internal Medicine and Dept. of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030 (E-mail: Bruce.C.Kone{at}uth.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

May 22, 2002;10.1152/ajpcell.00100.2002

Received 5 March 2002; accepted in final form 22 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Aperlo, C, Boulukos KE, and Pognonec P. The basic region/helix-loop-helix/leucine repeat transcription factor USF interferes with Ras transformation. Eur J Biochem 241: 249-253, 1996[Abstract].

2.   Babior, BM. Phagocytes and oxidative stress. Am J Med 109: 33-44, 2000[ISI][Medline].

3.   Boyd, KE, and Farnham PJ. Myc versus USF: discrimination at the cad gene is determined by core promoter elements. Mol Cell Biol 17: 2529-2537, 1997[Abstract].

4.   Chin, TY, Lin YS, and Chueh SH. Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase. Eur J Biochem 268: 6358-6368, 2001[Abstract/Free Full Text].

5.   Cogswell, JP, Godlevski MM, Bonham M, Bisi J, and Babiss L. Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter. Mol Cell Biol 15: 2782-2790, 1995[Abstract].

6.   Furusu, A, Miyazaki M, Abe K, Tsukasaki S, Shioshita K, Sasaki O, Miyazaki K, Ozono Y, Koji T, Harada T, Sakai H, and Kohno S. Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis. Kidney Int 53: 1760-1768, 1998[ISI][Medline].

7.   Gao, J, Morrison DC, Parmely TJ, Russell SW, and Murphy WJ. An interferon-gamma -activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon-gamma and lipopolysaccharide. J Biol Chem 272: 1226-1230, 1997[Abstract/Free Full Text].

8.   Goldring, CE, Reveneau S, Algarte M, and Jeannin JF. In vivo footprinting of the mouse inducible nitric oxide synthase gene: inducible protein occupation of numerous sites including Oct and NF-IL6. Nucleic Acids Res 24: 1682-1687, 1996[Abstract/Free Full Text].

9.   Grandori, C, Cowley SM, James LP, and Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu Rev Cell Dev Biol 16: 653-699, 2000[ISI][Medline].

10.   Gregor, PD, Sawadogo M, and Roeder RG. The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer. Genes Dev 4: 1730-1740, 1990[Abstract].

10a.   Gupta, AK, Diaz RA, Higham S, and Kone BC. alpha -Melanocyte-stimulating hormone inhibits induction of C/EBPbeta -DNA binding acitivity and NOS2 gene transcription in macrophages. Kidney Int 57: 2239-2248, 2000[ISI][Medline].

11.   Kee, BL, Quong MW, and Murre C. E2A proteins: essential regulators at multiple stages of B-cell development. Immunol Rev 175: 138-149, 2000[ISI][Medline].

12.   Kone, BC. Nitric oxide in renal health and disease. Am J Kidney Dis 30: 311-333, 1997[ISI][Medline].

13.   Kone, BC, and Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol Renal Physiol 272: F561-F578, 1997[Abstract/Free Full Text].

14.   Kone, BC, Schwobel J, Turner P, Mohaupt MG, and Cangro CB. Role of NF-kappa B in the regulation of inducible nitric oxide synthase in an MTAL cell line. Am J Physiol Renal Fluid Electrolyte Physiol 269: F718-F729, 1995[Abstract/Free Full Text].

15.   Kraner, SD, Rich MM, Sholl MA, Zhou H, Zorc CS, Kallen RG, and Barchi RL. Interaction between the skeletal muscle type 1 Na+ channel promoter E-box and an upstream repressor element. Release of repression by myogenin. J Biol Chem 274: 8129-8136, 1999[Abstract/Free Full Text].

16.   Levonen, AL, Patel RP, Brookes P, Go YM, Jo H, Parthasarathy S, Anderson PG, and Darley-Usmar VM. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases. Antioxid Redox Signal 3: 215-229, 2001[ISI][Medline].

17.   Liang, M, and Knox FG. Production and functional roles of nitric oxide in the proximal tubule. Am J Physiol Regul Integr Comp Physiol 278: R1117-R1124, 2000[Abstract/Free Full Text].

18.   Luo, X, and Sawadogo M. Antiproliferative properties of the USF family of helix-loop-helix transcription factors. Proc Natl Acad Sci USA 93: 1308-1313, 1996[Abstract/Free Full Text].

19.   Luo, X, and Sawadogo M. Functional domains of the transcription factor USF2: atypical nuclear localization signals and context-dependent transcriptional activation domains. Mol Cell Biol 16: 1367-1375, 1996[Abstract].

20.   Martin, E, Nathan C, and Xie QW. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J Exp Med 180: 977-984, 1994[Abstract].

21.   Medvedev, AV, Snedden SK, Raimbault S, Ricquier D, and Collins S. Transcriptional regulation of the mouse uncoupling protein-2 gene. Double E-box motif is required for peroxisome proliferator-activated receptor-gamma -dependent activation. J Biol Chem 276: 10817-10823, 2001[Abstract/Free Full Text].

22.   Meier, JL, Luo X, Sawadogo M, and Straus SE. The cellular transcription factor USF cooperates with Varicella zoster virus immediate-early protein 62 to symmetrically activate a bidirectional viral promoter. Mol Cell Biol 14: 6896-6906, 1994[Abstract].

23.   Moon, YS, Latasa MJ, Kim KH, Wang D, and Sul HS. Two 5'-regions are required for nutritional and insulin regulation of the fatty-acid synthase promoter in transgenic mice. J Biol Chem 275: 10121-10127, 2000[Abstract/Free Full Text].

24.   Norton, JD. ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J Cell Sci 113: 3897-3905, 2000[Abstract/Free Full Text].

25.   Qyang, Y, Luo X, Lu T, Ismail PM, Krylov D, Vinson C, and Sawadogo M. Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation and transcriptional activation. Mol Cell Biol 19: 1508-1517, 1999[Abstract/Free Full Text].

26.   Reisman, D, and Rotter V. The helix-loop-helix-containing transcription factor USF binds to and transactivates the promoter of the p53 tumor suppressor gene. Nucleic Acids Res 21: 345-350, 1993[Abstract].

27.   Rippe, RA, Umezawa A, Kimball JP, Breindl M, and Brenner DA. Binding of upstream stimulatory factor to an E-box in the 3'-flanking region stimulates alpha 1(I) collagen gene transcription. J Biol Chem 272: 1753-1760, 1997[Abstract/Free Full Text].

28.   Roy, AL, Du H, Gregor PD, Novina CD, Martinez E, and Roeder RG. Cloning of an inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1. EMBO J 16: 7091-7104, 1997[Abstract/Free Full Text].

29.   Rupprecht, HD, Akagi Y, Keil A, and Hofer G. Nitric oxide inhibits growth of glomerular mesangial cells: role of the transcription factor EGR-1. Kidney Int 57: 70-82, 2000[ISI][Medline].

30.   Saura, M, Zaragoza C, Bao C, McMillan A, and Lowenstein CJ. Interaction of interferon regulatory factor-1 and nuclear factor-kappa B during activation of inducible nitric oxide synthase transcription. J Mol Biol 289: 459-471, 1999[ISI][Medline].

31.   Sawadogo, M. Multiple forms of the human gene-specific transcription factor USF. II. DNA binding properties and transcriptional activity of the purified HeLa USF. J Biol Chem 263: 11994-12001, 1988[Abstract/Free Full Text].

32.   Sawadogo, M, and Roeder RG. Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell 43: 165-175, 1985[ISI][Medline].

33.   Sawadogo, M, Van Dyke MW, Gregor PD, and Roeder RG. Multiple forms of the human gene-specific transcription factor USF. I. Complete purification and identification of USF from HeLa cell nuclei. J Biol Chem 263: 11985-11993, 1988[Abstract/Free Full Text].

34.   Shimano, H. Sterol regulatory element-binding protein-1 as a dominant transcription factor for gene regulation of lipogenic enzymes in the liver. Trends Cardiovasc Med 10: 275-278, 2000[ISI][Medline].

35.   Sieweke, MH, Tekotte H, Jarosch U, and Graf T. Cooperative interaction of ets-1 with USF-1 required for HIV-1 enhancer activity in T cells. EMBO J 17: 1728-1739, 1998[Abstract/Free Full Text].

36.   Sirito, M, Lin Q, Maity T, and Sawadogo M. Ubiquitous expression of the 43- and 44-kDa forms of transcription factor USF in mammalian cells. Nucleic Acids Res 22: 427-433, 1994[Abstract].

37.   Sirito, M, Walker S, Lin Q, Kozlowski MT, Klein WH, and Sawadogo M. Members of the USF family of helix-loop-helix proteins bind DNA as homo- as well as heterodimers. Gene Expr 2: 231-240, 1992[Medline].

38.   Timchenko, N, Wilson DR, Taylor LR, Abdelsayed S, Wilde M, Sawadogo M, and Darlington GJ. Autoregulation of the human C/EBP-alpha gene by stimulation of upstream stimulatory factor binding. Mol Cell Biol 15: 1192-1202, 1995[Abstract].

39.   Vallet, VS, Casado M, Henrion AA, Bucchini D, Raymondjean M, Kahn A, and Vaulont S. Differential roles of upstream stimulatory factors 1 and 2 in the transcriptional response of liver genes to glucose. J Biol Chem 273: 20175-20179, 1998[Abstract/Free Full Text].

40.   Wang, D, and Sul HS. Upstream stimulatory factors bind to insulin response sequence of the fatty acid synthase promoter. USF1 is regulated. J Biol Chem 270: 28716-28722, 1995[Abstract/Free Full Text].

41.   Wang, JS, Tseng HH, Shih DF, Jou HS, and Ger LP. Expression of inducible nitric oxide synthase and apoptosis in human lupus nephritis. Nephron 77: 404-411, 1997[ISI][Medline].

42.   Weihua, X, Lindner DJ, and Kalvakolanu DV. The interferon-inducible murine p48 (ISGF3gamma ) gene is regulated by protooncogene c-myc. Proc Natl Acad Sci USA 94: 7227-7232, 1997[Abstract/Free Full Text].

43.   Wice, BM, and Gordon JI. Forced expression of Id-1 in the adult mouse small intestinal epithelium is associated with development of adenomas. J Biol Chem 273: 25310-25319, 1998[Abstract/Free Full Text].

44.   Wilcox, CS, Deng X, and Welch WJ. NO generation and action during changes in salt intake: roles of nNOS and macula densa. Am J Physiol Regul Integr Comp Physiol 274: R1588-R1593, 1998[Abstract/Free Full Text].

45.   Xie, Q. A novel lipopolysaccharide-response element contributes to induction of nitric oxide synthase. J Biol Chem 272: 14867-14872, 1997[Abstract/Free Full Text].

46.   Xie, QW, Kashiwabara Y, and Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem 269: 4705-4708, 1994[Abstract/Free Full Text].


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