CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line

Ashish K. Gupta and Bruce C. Kone

Departments of Internal Medicine and of Integrative Biology, Pharmacology and Physiology, University of Texas Medical School at Houston, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicated in epithelial cell injury from oxidative and immunologic stress. The NOS2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytokines in medullary thick ascending limb of Henle's loop (MTAL) cells and other cell types. The 5'-flanking region of the NOS2 gene contains a consensus element for CCAAT/enhancer binding proteins (C/EBP) at -150 to -142 that we hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon-gamma (IFN-gamma ) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBPbeta , but not C/EBPalpha , C/EBPdelta , or C/EBPepsilon . NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN-gamma compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-gamma activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBPbeta stimulated basal NOS2 promoter activity. Thus C/EBPbeta appears to be an important trans-activator of the NOS2 gene in the MTAL.

gene transcription; kidney; cell signaling; promoter; transcription factors


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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NITRIC OXIDE is a potent effector molecule involved in numerous physiological processes, including neurotransmission, the control of vascular tone, inflammation, and immunity. In the kidney, NO participates in the regulation of glomerular and medullary hemodynamics, the tubuloglomerular feedback response, renin release, and the extracellular fluid volume (24). NO production from its substrate L-arginine is catalyzed by the multigene NO synthase (NOS) family, which includes three principal members. NOS1 and NOS3 are constantly expressed in selected tissues (although changes in the expression of these genes have been documented under several conditions, see Refs. 23 and 24 for review) but are inactive until intracellular Ca2+ levels increase sufficiently to maintain calmodulin binding (5). NOS2, which binds calmodulin at resting intracellular Ca2+ levels, is quiescent in most tissues until it is transcriptionally activated by immune stimuli to produce large amounts of NO (32). Although this large production of NO by NOS2 promotes host defense, it also contributes to septic shock and injury to tissues, including epithelial cells of the respiratory tract (37), gastrointestinal tract (26), and renal tubules (23). Indeed, high-output NO production has been linked to several forms of glomerular and renal tubular injury.

In virtually all nucleated cells of mammals, bacterial lipopolysaccharide (LPS) and certain cytokines transcriptionally activate the NOS2 gene. Structure-function studies of the murine NOS2 promoter have demonstrated that the region -48 to -209 (region I), serves as a core promoter module, whereas a more distal region (region II, -780 to -1588; Ref. 4) serves as an LPS- and cytokine-responsive enhancer (4, 29, 41, 49). Numerous response elements reside within the two regions, and several have been shown to be active. These include kappa B sites in both regions I and II, a hypoxia-responsive enhancer element in region I (31), a novel LPS response element in region I to which an Oct-1-like protein binds (47), interferon (IFN) regulatory factor-1 (IRF-1) (30, 42), two sequential IFN-stimulated response elements (ISREs), and an IFN-gamma -activated site (GAS) in region II (13). The responsiveness to LPS and cytokines and the specific mechanisms involved in NOS2 transcriptional regulation vary depending on cell type. For example, our studies in ST-1 cells, a well-characterized cell line derived from murine medullary thick ascending limb of Henle (MTAL) cells, established that nuclear factor for immunoglobulin kappa  chain in B cells (NF-kappa B) proteins p50 and p65, but not c-Rel, are critical for LPS + IFN-gamma inducibility of the NOS2 gene in these cells (25), whereas NF-kappa B p50, c-Rel, and possibly p65 serve this regulatory role in RAW 264.7 macrophage-like cells (48).

The murine NOS2 promoter/enhancer contains a nucleotide sequence -150 TGATGTAAT -142 that conforms to the consensus CCAAT/enhancer binding protein (C/EBP) box TKNNGYAAK (1) and that neighbors the kappa B site (-88 to -74) important for LPS inducibility. In vivo footprinting studies of the 5'-flanking region of the NOS2 gene in RAW 264.7 cells demonstrated LPS-induced protection of guanine -146 within this C/EBP box (15), suggesting transcription factor occupation of this site. In addition, correlative changes in C/EBPbeta [also known as NF-IL6 (2) and LAP, for liver-associated transcriptional activator protein (11)] DNA binding activity and NOS2 gene expression in cultured cardiac myocytes (22) and vascular smooth muscle cells (17) provided inferential evidence to suggest a role for C/EBPbeta in trans-activation of the NOS2 gene. Most recently, work by Eberhardt et al. (12) suggested the involvement of a similarly positioned C/EBP site in the rat NOS2 promoter/enhancer in the cAMP-mediated induction of NOS2 in cultured mesangial cells.

The C/EBP proteins comprise a family of bZIP (basic region leucine zipper) transcription factors that participate in the regulation of genes involved in the acute phase response, inflammation, cell growth, and differentiation (3, 7, 46). The C/EBP proteins alpha , beta , gamma , delta , and epsilon  function as activators of transcription, whereas C/EBP homology protein (CHOP) and the alternative translation products liver-enriched transcriptional inhibitory protein (LIP) and C/EBP-30 serve as repressors (46). Of these, C/EBPbeta and C/EBPdelta are known to be inducible by LPS or cytokines. Induction of C/EBPbeta expression has been demonstrated in kidneys from LPS-treated (3) or hypoxic (50) mice and the MTAL of rats subjected to ischemic injury (36), as well as in macrophages (40), cardiac myocytes (22, 50), lung (50), and vascular smooth muscle cells (17) subjected to immune or hypoxic challenge.

Since we observed residual LPS + IFN-gamma induction of NOS2 in ST-1 cells even when NF-kappa B activity was completely inhibited by pyrrolidine dithiocarbamate (25), we hypothesized that transcription factors other than NF-kappa B must be important for NOS2 induction in this setting. Given the emerging evidence that C/EBP family proteins might be important for NOS2 induction in other cell types (12) and the preliminary report of C/EBPbeta induction in the postischemic MTAL of the rat (36), we hypothesized that C/EBPbeta contributes to NOS2 induction in ST-1 cells. In this report, using ST-1 cells as a model epithelium, we show that LPS + IFN-gamma induces nuclear expression of C/EBPbeta and that binding of C/EBPbeta to the -150 to -142 C/EBP box in the NOS2 promoter is necessary for maximal LPS + IFN-gamma -mediated induction of the NOS2 gene in these cells.


    MATERIALS AND METHODS
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Reagents. L-Glutamine, fetal bovine serum (FBS), penicillin-streptomycin, and DMEM were from Life Technologies (Grand Island, NY). LPS from Escherichia coli O111:B4 was from Sigma Chemical (St. Louis, MO). Mouse recombinant IFN-gamma was from Genzyme (Cambridge, MA). Radiochemicals were purchased from Amersham (Arlington Heights, IL). RNAzol II was acquired from TEL-TEST "B" (Friendswood, TX). Rabbit polyclonal IgGs specific for C/EBPalpha , C/EBPbeta , C/EBPdelta , and C/EBPepsilon were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Other oligonucleotides were custom synthesized by Genosys (The Woodlands, TX). Poly(dI-dC)-poly(dI-dC) was purchased from Pharmacia-LKB Biotech. The Dual-Luciferase Reporter Assay System and the luciferase vectors pGL3-Basic and pRL-TK were from Promega. The bicinchoninic acid (BCA) protein estimation kit was from Pierce Chemical.

Plasmids and site-directed mutagenesis. The C/EBPbeta expression plasmid pMSV-C/EBPbeta was kindly provided by Dr. Steven McKnight (Tularik). This plasmid was designed to express selectively C/EBPbeta -LAP under the control of the Moloney murine sarcoma virus long terminal repeat (7, 51). Excision of the encoding DNA for C/EBPbeta -LAP from this plasmid, followed by religation of the ends, produced the recombinant molecule pMSV-BS, which was used as a vector control in some experiments. pRG-GBF-F, a C/EBP-specific, dominant-negative expression plasmid (34), was a gift from Drs. Charles Vinson and Michelle Olive (National Cancer Institute). The GBF-F protein contains the basic DNA binding region of the plant bZIP protein GBF-1 joined to a leucine zipper that preferentially heterodimerizes with the leucine zippers of C/EBP family members (34). GBF-F inhibits the activity of all known C/EBP proteins (34). The murine NOS2 promoter/enhancer and a portion of exon 1 (nucleotides -1486 to +145) were amplified from 100 ng mouse genomic DNA by PCR using oligonucleotides based on the published sequence (49). The forward primer (P1) was 5'-TCAGCTCTTGTTTCCCAGGTT-3' (-1486 to -1466) and the reverse primer (P3) was 5'-GGAGTGAACAAGACCCAAGCGTG-3' (+133 to +145). The product was cloned into pCR2.1 (Invitrogen) and sequenced (Sequenase, Amersham). The sequence was identical to that previously reported (29). The NOS2 promoter fragment was then excised with Hind III and Xho I and cloned into these sites in pGL3-Basic to create pNOS2-luc.

Deletion and site-directed mutation of the -150 to -142 C/EBP box in pNOS2-luc was accomplished by PCR splicing by overlap extension (18), using the wild-type NOS2 promoter/enhancer cDNA as template. For deletion of the C/EBP box, forward primer P1 was used with mutagenic reverse primer P3 (5'-TACTCCTATGTGGTGTCTCGTTCGTGTGTCTGATCC-3') in the upstream reaction, and mutagenic forward primer P4 (5'-ATGAGGATACACCACAGAGCAAGCACACAGACTAGG-3') and reverse primer P2 were used in the downstream PCR. For mutation of the C/EBP box (-150 TGATGTAAT -142 replaced with <UNL>GC</UNL>A<UNL>CT</UNL>T<UNL>GC</UNL>T), the upstream reaction contained forward primer P1 and mutagenic reverse primer P5 (5'-GTGTGCTTG<UNL>AGCAAGTGC</UNL>CTCTGTGGTGTATCC-3'), whereas mutagenic forward primer P6 (5'-GGATACACCACAGAG<UNL>GCACTTGCT</UNL>CAAGCACAC-3') and reverse primer P2 were used in the downstream reaction. The full-length, site-deleted or -mutated NOS2 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 fragments 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 pNOS2-C/EBPdel-luc (C/EBP box deleted) and pNOS2-Delta C/EBP-luc (C/EBP box mutated).

Cell culture and transfection. ST-1 cells, a gift from Drs. Adam Sun and Steve Hebert, were maintained in DMEM supplemented with 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine (complete medium). These cells express phenotypic properties of the MTAL in vivo, including expression of Tamm-Horsfall glycoprotein, and the 5-HT1A receptor (25). Vehicle or LPS (100 ng/ml) + IFN-gamma (0.5 U/ml) were added to the cells as indicated in the text and legends to Figs. 1-3. These concentrations have been shown to activate maximally NOS2 transcription in ST-1 cells (25).

Plasmid preparations were made using the Endotoxin-free Plasmid Maxi-prep kit (Qiagen, Santa Clarita, CA). For transient transfections, ST-1 cells were seeded in 6-well plates and grown to 70-90% confluence in DMEM + 10% FBS without antibiotics and transfected the following day using the LipoFectamine PLUS reagent following the manufacturer's protocol and a total of 5 µg/well of plasmid DNAs. For comparative purposes between reporter gene constructs, transfection efficiencies were normalized by cotransfection with 0.5 µg/well of the Renilla luciferase expression plasmid pRL-TK. Trans-activation/trans-repression experiments used 1 µg of pNOS2-luc and 3 µg of pMSV-C/EBPbeta , pRG-GBF-F, or insertless expression vector. Twenty-four hours after transfection, the medium was replaced with complete medium and vehicle or LPS + IFN-gamma . Sixteen hours later, cell lysates for measurement of firefly and Renilla luciferase activities were prepared using Passive Lysis Buffer (Promega) according to the manufacturer's directions. Protein content of the lysates was determined using the BCA Assay kit (Pierce). Firefly and Renilla luciferase activities in 100-µl lysate samples were measured in a Turner Systems 20/20 luminometer using the Dual-Luciferase Reporter Assay System according to the manufacturer's protocol. After background subtraction, firefly luciferase activities were normalized for Renilla luciferase activity and protein content of the lysates and recorded as "normalized firefly luciferase activity." The value for the vector-transfected cells (see pGL3-Basic and pMSV-BS in Figs. 2 and 3, respectively) was arbitrarily set at 1.0. Each observation represents the mean of duplicate determinations using a new plasmid preparation.

Electrophoretic mobility shift assays (EMSA). Nuclear extracts were prepared from time-paired control and LPS + IFN-gamma -treated (4 h) ST-1 cells as detailed in our earlier work (25). Double-stranded oligonucleotides (1.75 pmol) corresponding to nucleotides -157 to -136 (sense strand, 5'-CACAGAG<UNL>TGATGTAAT</UNL>CAAGCA-3', C/EBP box is underscored) of the native murine NOS2 promoter were end labeled with [gamma -32P]ATP (3,000 Ci/mmol) using T4 polynucleotide kinase. Binding reactions were performed in 20 µl of solution for 30 min at room temperature by incubating 20 µg nuclear extract protein with 1.75 pmol of duplex DNA probe (~2 × 105 cpm) in 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)] in the presence or absence of a 50-fold molar excess of nonradiolabeled competitor oligonucleotides. For supershift assays, antibodies specific for individual transcription factors were added to the binding reaction and incubated at room temperature for 30 min. Aliquots of the reactions were resolved on 5% native polyacrylamide gels in 0.5× Tris borate-EDTA buffer. The gels were dried and exposed to X-ray film with an enhancing screen at -70°C to detect the DNA-protein and DNA-protein-antibody complexes. Each observation represents a binding reaction performed on a new nuclear extract preparation. Experiments were replicated a minimum of three times as indicated in the legends to Figs. 1-3.

Data analysis. The number of independent observations (n) for each experiment is indicated in the legends to Figs. 1-3. Quantitative data are presented as means ± SE and were analyzed by analysis of variance. Significance was assigned at P < 0.05.


    RESULTS
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LPS and IFN-gamma promote C/EBPbeta binding activity in ST-1 cells. To determine whether LPS + IFN-gamma induces expression of C/EBP family proteins, EMSAs with double-stranded oligonucleotides containing the -150 to -142 C/EBP box of the native murine NOS2 promoter and nuclear extracts prepared from control and LPS + IFN-gamma -treated ST-1 cells were performed. As seen in Fig. 1A, a major gel shift band was evident in nuclear extracts prepared from both control and LPS + IFN-gamma -treated cells. The approximate abundance of the gel shift band was consistently greater in LPS + IFN-gamma -treated cells compared with the controls (n = 4). Sequence specificity of the protein-DNA complex was verified in competition experiments: the gel shift band was not evident in the presence of a 50-fold molar excess of unlabeled C/EBP box oligomers but was apparent when a 50-fold molar excess of unlabeled activator protein-2 (AP-2) site oligomers were included in the reaction (Fig. 1A).


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Fig. 1.   Bacterial lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma ) promotes increased beta -isoform of CCAAT/enhancer binding protein (C/EBPbeta ) DNA binding activity in nuclear protein extracts of ST-1 cells. A: nuclear proteins extracted from ST-1 cells that had been exposed to vehicle (Ctrl) or LPS (100 ng/ml) + IFN-gamma (0.5 U/ml) for 4 h were subjected to electrophoretic mobility shift assay with a 32P-labeled oligomer containing the -150 to -142 C/EBP box of the murine nitric oxide synthase 2 gene (NOS2) promoter. "N" indicates a negative control in which nuclear extract was omitted from the binding reaction; "-" indicates that the binding reaction was carried out in the absence of unlabeled specific or heterologous oligomers. To demonstrate binding specificity, reactions were also conducted in the presence of a 50-fold molar excess of unlabeled murine NOS2 C/EBP box oligomer (S) or heterologous (H) oligomers. Autoradiogram is representative of 4 independent experiments performed on separate preparations of nuclear extracts. B: polyclonal IgGs specific for C/EBPalpha , -beta , -delta , and -epsilon and NF-kappa B p50, p65, and c-Rel were used in supershift experiments with nuclear extracts from LPS + IFN-gamma -treated (4 h) ST-1 cells and the 32P-labeled murine NOS2 C/EBP box oligomer. Autoradiograms are representative of 3 independent experiments performed on separate preparations of nuclear extracts, and they have been cropped so that free probe is not seen. NF-kappa B, nuclear factor for immunoglobulin kappa  chain in B cells.

To determine which of the C/EBP proteins contributed to the gel shift complex, supershift assays with antibodies specific for C/EBPalpha , -beta , -delta , and -epsilon were performed. Nuclear extracts from LPS + IFN-gamma -treated ST-1 cells were preincubated with IgGs specific for each of these C/EBP proteins before reaction with the 32P-labeled oligomers containing the -150 to -142 C/EBP box of the native murine NOS2 promoter. Only the IgG specific for C/EBPbeta significantly supershifted the complex (n = 3; Fig. 1B), indicating that the complex primarily contained this C/EBP family member. Since 1) the C/EBP box neighbors the kappa B site in region I of the NOS2 promoter, 2) C/EBPbeta is capable of forming heterodimers with NF-kappa B proteins, and 3) our previous work demonstrated LPS + IFN-gamma induction of NF-kappa B proteins in ST-1 cells, supershift assays including the NOS2 promoter C/EBP box probe, ST-1 nuclear extracts, and antibodies specific for NF-kappa B p50, p65, and c-Rel were conducted. In three separate experiments, no supershift was observed when the binding reaction included the NF-kappa B p50, p65, or c-Rel antibodies (Fig. 1B), indicating that C/EBPbeta -NF-kappa B heterodimers are not formed under these conditions.

Mutation of the -150 to -142 C/EBP box limits LPS + IFN-gamma induction of NOS2 promoter activity. To determine whether the -150 to -142 C/EBP box is functional within the structural context of the NOS2 promoter, structure-function analyses were performed. Three constructs were made: one contained the wild-type NOS2 promoter (pNOS2-luc), a second contained the NOS2 promoter lacking the -150 to -142 C/EBP box (pNOS2-C/EBPbeta del-luc), and a third contained the NOS2 promoter bearing a six-nucleotide mutation in the -150 to -142 C/EBP box (pNOS2-Delta C/EBPbeta -luc). These constructs or the parent vector pGL3-Basic were transfected together with pTK-RL (to control for transfection efficiency) into ST-1 cells, and the cells were later stimulated with LPS + IFN-gamma . Multiple plasmid preps were used to verify reproducibility of the findings. After vehicle treatment alone, cells transfected with each vector exhibited comparable, background-level activity of the luciferase reporter gene (Fig. 2). ST-1 cells transfected with pNOS2-luc together with pTK-RL and treated with LPS + IFN-gamma exhibited roughly an eightfold increase in normalized firefly luciferase activity (Fig. 2). In contrast, cells transfected with pNOS2-C/EBPdel-luc or pNOS2-Delta C/EBP-luc together with pTK-RL generated only fourfold increases in normalized firefly luciferase activities after LPS + IFN-gamma stimulation (Fig. 2). These results indicated that the -150 to -142 C/EBP box is functional in regulating murine NOS2 gene expression in response to these stimuli.


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Fig. 2.   Contribution of the C/EBP box to the activity of the murine NOS2 promoter. ST-1 cells were transiently cotransfected with pTK-RL (to normalize for transfection efficiency) and either the promoterless vector pGL3-Basic or one of the following three promoter-luciferase constructs: pNOS2-luc, containing the wild-type NOS2 promoter; pNOS2-C/EBPbeta del-luc, containing a mutated NOS2 promoter lacking the C/EBP box; or pNOS2-Delta C/EBPbeta -luc, containing the NOS2 promoter bearing a 6-nucleotide mutation in the C/EBP box. Forty-eight hours after transfection, cells were stimulated with vehicle (open bars) or LPS (100 ng/ml) + IFN-gamma (0.5 U/ml) (solid bars) for 16 h. Firefly and Renilla luciferase activities in lysates of the cells were then determined in a luminometer and normalized to cell protein content. Bars are means (error lines are +SE) of 4 separate experiments. * P < 0.05 vs. pGL3-Basic-transfected, LPS + IFN-gamma -treated cells. # P < 0.05 vs. pNOS2-luc-transfected, LPS + IFN-gamma -treated cells.

Effects of overexpression of C/EBPbeta or a C/EBP dominant-negative mutant on the NOS2 promoter. To determine whether overexpression or inhibited expression of C/EBPbeta in ST-1 cells could alter basal or LPS + IFN-gamma -induced NOS2 promoter activity, transient transfections were performed with the wild-type NOS2 promoter construct pNOS2-luc together with pTK-RL and either empty expression vector or expression plasmids for C/EBPbeta (pMSV-C/EBPbeta ) or the C/EBP-specific, dominant-negative mutant (pRG-GBF-F). ST-1 cells cotransfected with pMSV-C/EBPbeta exhibited normalized NOS2 promoter activity that was approximately twofold greater than that of vector-transfected controls after exposure to vehicle alone (Fig. 3). In the presence of LPS + IFN-gamma , comparable levels of induction of normalized NOS2 promoter activity were observed in the pMSV-C/EBPbeta and vector-transfected controls, suggesting that induction of endogenous C/EBPbeta was sufficient to promote maximal NOS2 promoter activity. In contrast, cells cotransfected with pRG-GPF-F exhibited basal, normalized NOS2 promoter activity that was comparable to that of controls and LPS + IFN-gamma -stimulated levels that were roughly two- to threefold less than the vector controls treated with LPS + IFN-gamma (Fig. 3).


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Fig. 3.   C/EBPbeta trans-activates the NOS2 promoter. ST-1 cells were transfected with the pNOS2-luc reporter construct in presence of expression vector for C/EBPbeta (pMSV-C/EBPbeta ), a C/EBP-specific dominant-negative inhibitor (pRG-GBF-F), or an insertless vector (pMSV-BS) containing the MSV promoter. Forty-eight hours after transfection, cells were stimulated with vehicle (open bars) or LPS (100 ng/ml) + IFN-gamma (0.5 U/ml) (solid bars) for 16 h, and cell lysates were prepared. Firefly and Renilla luciferase activities in lysates of the cells were assayed and normalized to cell protein content. Bars are means (error lines are +SE) of 4 separate experiments. * P < 0.05 vs. pMSV-BS-transfected, vehicle-treated cells. # P < 0.05 vs. pMSV-BS-transfected, LPS + IFN-gamma -treated cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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The MTAL is the principal nephron segment responsible for urinary concentration and dilution (38), a major site for the action of diuretics (14), and an important site for NO-mediated injury related to toxins, inflammation, and ischemia (6, 23, 33). In previous work, we demonstrated that LPS + IFN-gamma transcriptionally induces NOS2 in ST-1 cells by both NF-kappa B-dependent and -independent mechanisms (25). In this study, we provide evidence for the involvement a C/EBP response element (C/EBP-RE) and C/EBPbeta in LPS + IFN-gamma -induced trans-activation of the murine NOS2 gene in ST-1 cells. The present results extend our earlier work, as well as previous in vivo footprinting, EMSA, and NOS2 gene expression studies in different cell types that suggested, but did not confirm, the involvement of C/EBPbeta in NOS2 gene activation (12, 15, 17, 22). Taken together, these data suggest that C/EBPbeta and perhaps other C/EBP family members participate in NOS2 transcriptional activation in a number of cell types and in response to disparate immune stimuli.

In addition to NOS2, C/EBPbeta is known to trans-activate several other genes involved in the inflammatory response, including cell adhesion molecules (8, 39), integrins (28), proinflammatory cytokines (10, 35), prostaglandin endoperoxide synthase 2 (20), and manganese superoxide dismutase (21). C/EBPbeta is also induced in the kidneys and lungs of mice subjected to hypoxia (50). The hypoxic induction of C/EBPbeta , and consequently or coordinately (31) NOS2, may be particularly relevant to epithelial cells like those of the MTAL (6) and airways (27) that may be more frequently exposed to hypoxic stress. Since C/EBPbeta has also been implicated in the transcriptional activation of arginase I (16), an enzyme that degrades the substrate for NOS, L-arginine, C/EBPbeta appears to play a pivotal role in coordinating NO biosynthesis. Indeed, coinduction of NOS2 and arginase I in vivo has been demonstrated in response to LPS (40) and immune-mediated injury (9, 45). Thus C/EBPbeta may contribute in some tissues not only to induction of NOS2 expression but also to inhibition of NOS2 activity by limiting substrate availability. The latter effect may be important in limiting cytotoxicity to host cells (45).

The NOS2 promoter fragment used in the present study contains two other potential C/EBP boxes (-474 TGGGGAAAT -466, 9/9 nucleotide match; -387 TGTTGGAAT -379, 8/9 nucleotide match) that we did not specifically examine. We restricted our focus to the -150 to -142 C/EBP-RE, because the other two elements do not reside within the regions I and II documented in previous deletion analyses to contain functionally important promoter and enhancer elements (4, 29, 41, 49), and they were not footprinted after immune challenge (15). The fact that overexpression of the C/EBP-specific dominant-negative mutant GBF-F (Fig. 3) produced a reduction in NOS2 promoter activity that was roughly equivalent to the decrement observed with the NOS2 promoter constructs bearing mutation or deletion of the -150 to -142 C/EBP-RE (Fig. 2) suggests that the -474 to -466 and -387 to -379 C/EBP boxes did not contribute significantly to NOS2 induction in our study. It is also possible that C/EBP-REs located at sites upstream of the 1.6-kb fragment we examined contribute to C/EBPbeta trans-activation of the murine NOS2 gene. Studies of the human NOS2 promoter, for example, indicated that cis elements upstream of -4.7 kb contribute to cytokine induction (44). This latter result highlights important differences in transcriptional regulation of the human and murine NOS2 genes. Whereas the first 1.5 kb of the murine NOS2 promoter/enhancer contains important LPS- and cytokine-enhancer elements (as detailed earlier), the first 4.7 kb of the human NOS2 5'-flanking region appears to be devoid of apparent cytokine-enhancer elements (44). Moreover, deletional and mutational analysis of the human NOS2 promoter/enhancer revealed that the proximal (-115 to -106) NF-kappa B element in the human NOS2 promoter did not limit cytokine-induced promoter activity in human liver and lung epithelial cell lines (44), whereas the comparably positioned NF-kappa B element in the murine NOS2 promoter was found to be essential for LPS-inducibility in macrophages (48). Four NF-kappa B motifs upstream of 5 kb were, however, found to be important for maximal cytokine-induced activity of the human NOS2 promoter in these human cell lines (44). Interestingly, the human NOS2 promoter contains the sequence -191 TGATGTAA<UNL>C</UNL> -183 that differs at a single nucleotide (underscored) compared with the similarly positioned -150 TGATGTAA<UNL>T</UNL> -142 C/EBP box in the murine NOS2 promoter. The human sequence does not conform to the consensus C/EBP box TKNNGYAA<UNL>K</UNL> (1) (where K represents G or T), which might provide at least one explanation why cytokine responsiveness was not conferred by the proximal 4.7 kb of the human NOS2 promoter in reporter gene assays (44). Formal tests of the possible functionality of the -191 to -183 sequence in the human NOS2 promoter are clearly needed.

The fact that DNA binding activities for C/EBPalpha , -delta , and -epsilon were not observed in nuclear extracts prepared from LPS + IFN-gamma -treated ST-1 cells indicates that these isoforms were not involved in the NOS2 promoter response. This result does not exclude the possibility that one or more of these isoforms, or C/EBPgamma , which was not examined, might contribute to NOS2 regulation in other tissues. For example, redundancy in the activities of C/EBPalpha , -beta , and -delta in inducing the expression of IL-6 and monocyte chemoattractant protein-1 was observed in cultured lymphoblasts (19). Such redundancy might also account for the finding that peritoneal macrophages harvested from mice with targeted disruption of the C/EBPbeta gene exhibited LPS + IFN-gamma -induced nitrite production and NOS2 mRNA levels that were interpreted to be comparable to those of wild-type mice (43). In addition, close inspection of the data obtained from the knockout mice reveals that the nitrite levels of the stimulated cells from the null mice were numerically lower (statistical analysis was not reported) than those from the wild-type mice, and the NOS2 mRNA levels, which were not shown, were only qualitatively analyzed by RT-PCR (43). Thus more detailed and quantitative analyses of NOS2 induction in tissues from these knockout mice may reveal deficiencies in NOS2 induction.

Our findings add C/EBPbeta to the growing list of regulatory factors that contribute to NOS2 biosynthesis and high-output NO generation in epithelial cells. Given its ability to form functional heterodimers with other C/EBP family members, cAMP-response element binding protein (CREB), and NF-kappa B (46), C/EBPbeta likely contributes versatility in the magnitude and cell-specificity of NOS2 induction, at least in mouse and rat. We did not observe heterodimers consisting of C/EBPalpha , -delta , or -epsilon or NF-kappa B proteins, nor is NOS2 induced by cAMP in ST-1 cells (16). However, cAMP and C/EBP-CREB heterodimers have been implicated in NOS2 induction in other tissues (22), and thus different cell types likely exploit distinct signaling cascades to stimulate NOS2 expression.


    ACKNOWLEDGEMENTS

We thank Drs. Steven McKnight, Charles Vinson, and Michelle Olive for the gifts of plasmids. We thank Drs. Adam Sun and Steve Hebert for the gift of the ST-1 cell line.


    FOOTNOTES

This work was supported by National Institutes of Health Grants RO1-DK-50745 and P50-GM-20529 (B. C. Kone) and was conducted during his tenure as an Established Investigator of the American Heart Association. A. K. Gupta was supported by a National Kidney Foundation Research Fellowship.

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. §1734 solely to indicate this fact.

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

Received 8 September 1998; accepted in final form 23 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akira, S., H. Isshiki, T. Nakajima, S. Kinoshita, Y. Nishio, S. Hashimoto, S. Natsuka, and T. Kishimoto. A nuclear factor for the IL-6 gene (NF-IL6). Chem. Immunol. 51: 299-322, 1992[Medline].

2.   Akira, S., H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, and T. Kishimoto. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9: 1897-1906, 1990[Abstract].

3.   Alam, T., M. R. An, and J. Papaconstantinou. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response. J. Biol. Chem. 267: 5021-5024, 1992[Abstract/Free Full Text].

4.   Alley, E. W., W. J. Murphy, and S. W. Russell. A classical enhancer element responsive to both lipopolysaccharide and interferon-gamma augments induction of the iNOS gene in mouse macrophages. Gene 158: 247-251, 1995[Medline].

5.   Bredt, D. S., and S. H. Snyder. Nitric oxide: a physiologic messenger molecule. Annu. Rev. Biochem. 63: 175-195, 1994[Medline].

6.   Brezis, M., and S. Rosen. Hypoxia of the renal medulla: its implications for disease. N. Engl. J. Med. 332: 647-655, 1996[Free Full Text].

7.   Cao, Z., R. M. Umek, and S. L. McKnight. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 5: 1538-1552, 1991[Abstract].

8.   Chini, B. A., M. A. Fiedler, L. Milligan, T. Hopkins, and J. M. Stark. Essential roles of NF-kappa B and C/EBP in the regulation of intercellular adhesion molecule 1 after respiratory syncytial virus infection of human respiratory epithelial cell cultures. J. Virol. 72: 1623-1626, 1998[Abstract/Free Full Text].

9.   Cook, H. T., A. Jansen, S. Lewis, P. Largen, M. O'Donnell, D. Reaveley, and V. Cattell. Arginine metabolism in experimental glomerulonephritis: interaction between nitric oxide synthase and arginase. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F646-F653, 1994[Abstract/Free Full Text].

10.   Davydov, I. V., P. H. Krammer, and M. Li Weber. Nuclear factor IL6 activates the human IL-4 promoter in T cells. J. Immunol. 155: 5273-5279, 1995[Abstract].

11.   Descombes, P., and U. Schibler. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67: 569-579, 1991[Medline].

12.   Eberhardt, W., C. Pluss, R. Hummel, and J. Pfeilschifter. Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells. J. Immunol. 160: 4961-4969, 1998[Abstract/Free Full Text].

13.   Gao, J., D. C. Morrison, T. J. Parmely, S. W. Russell, and W. J. Murphy. 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].

14.   Giebisch, G., and G. Klein-Robbenhaar. Recent studies on the characterization of loop diuretics. J. Cardiovasc. Pharmacol. 22: S1-S10, 1993[Medline].

15.   Goldring, C. E., S. Reveneau, M. Algarte, and J. F. Jeannin. 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].

16.   Gotoh, T., S. Choudhury, M. Takiguchi, and M. Mori. The glucocorticoid responsive gene cascade. Activation of the rat arginase gene through induction of C/EBPbeta . J. Biol. Chem. 272: 3694-3698, 1997[Abstract/Free Full Text].

17.   Hecker, M., C. Preiss, and V. B. Schini Kerth. Induction by staurosporine of nitric oxide synthase expression in vascular smooth muscle cells: role of NF-kappa B, CREB and C/EBPbeta . Br. J. Pharmacol. 120: 1067-1074, 1997[Abstract].

18.   Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51-59, 1991.

19.   Hu, H. M., M. Baer, S. C. Williams, P. F. Johnson, and R. C. Schwartz. Redundancy of C/EBP-alpha , -beta , and -delta in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1. J. Immunol. 160: 2334-2342, 1998[Abstract/Free Full Text].

20.   Inoue, H., C. Yokoyama, S. Hara, Y. Tone, and T. Tanabe. Transcriptional regulation of human prostaglandin endoperoxide synthase 2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin 6 expression site and cAMP response element. J. Biol. Chem. 270: 24965-24971, 1995[Abstract/Free Full Text].

21.   Jones, P. L., D. Ping, and J. M. Boss. Tumor necrosis factor alpha and interleukin 1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBPbeta and NF-kappa B. Mol. Cell. Biol. 17: 6970-6981, 1997[Abstract].

22.   Kinugawa, K., T. Shimizu, A. Yao, O. Kohmoto, T. Serizawa, and T. Takahashi. Transcriptional regulation of inducible nitric oxide synthase in cultured neonatal rat cardiac myocytes. Circ. Res. 81: 911-921, 1997[Abstract/Free Full Text].

23.   Kone, B. C. Nitric oxide in renal health and disease. Am. J. Kidney Dis. 30: 311-333, 1997[Medline].

24.   Kone, B. C., and C. Baylis. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am. J. Physiol. 272 (Renal Physiol. 41): F561-F578, 1997[Abstract/Free Full Text].

25.   Kone, B. C., J. Schwobel, P. Turner, M. G. Mohaupt, and C. B. Cangro. Role of NF-kappa B in the regulation of inducible nitric oxide synthase in an MTAL cell line. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F718-F729, 1995[Abstract/Free Full Text].

26.   Lamarque, D., J. Kiss, J. Tankovic, J. F. Flejou, J. C. Delchier, and B. J. Whittle. Induction of nitric oxide synthase in vivo and cell injury in rat duodenal epithelium by a water soluble extract of Helicobacter pylori. Br. J. Pharmacol. 123: 1073-1078, 1998[Abstract].

27.   Le Cras, T. D., C. Xue, A. Rengasamy, and R. A. Johns. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L164-L170, 1996[Abstract/Free Full Text].

28.   Lopez, Rodriguez, C., L. Botella, and A. L. Corbi. CCAAT enhancer binding proteins (C/EBP) regulate the tissue specific activity of the CD11c integrin gene promoter through functional interactions with Sp1 proteins. J. Biol. Chem. 272: 29120-29126, 1997[Abstract/Free Full Text].

29.   Lowenstein, C. J., E. W. Alley, P. Raval, A. M. Snowman, S. H. Snyder, S. W. Russell, and W. J. Murphy. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon-gamma and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90: 9730-9734, 1993[Abstract].

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

31.   Melillo, G., T. Musso, A. Sica, L. S. Taylor, G. W. Cox, and L. Varesio. A hypoxia responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J. Exp. Med. 182: 1683-1693, 1995[Abstract].

32.   Nathan, C., and Q. W. Xie. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269: 13725-13728, 1994[Free Full Text].

33.   Noiri, E., T. Peresleni, F. Miller, and M. S. Goligorsky. In vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia. J. Clin. Invest. 97: 2377-2383, 1996[Abstract/Free Full Text].

34.   Olive, M., S. C. Williams, C. Dezan, P. F. Johnson, and C. Vinson. Design of a C/EBP-specific, dominant-negative bZIP protein with both inhibitory and gain-of-function properties. J. Biol. Chem. 271: 2040-2047, 1996[Abstract/Free Full Text].

35.   Pope, R. M., A. Leutz, and S. A. Ness. C/EBPbeta regulation of the tumor necrosis factor alpha gene. J. Clin. Invest. 94: 1449-1455, 1994[Medline].

36.   Price, P. M., J. Megyesi, N. Udvarhelyi, and R. Safirstein. Increased renal expression of CCAAT/enhancer binding protein beta  (C/EBPbeta ) transcription factor in acute renal failure and its effect on renal gene transcription (Abstract). J. Am. Soc. Nephrol. 6: 987, 1995.

37.   Saleh, D., P. J. Barnes, and A. Giaid. Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 155: 1763-1769, 1997[Abstract].

38.   Sands, J. M., and J. P. Kokko. Current concepts of the countercurrent multiplication system. Kidney Int. Suppl. 57: S93-S99, 1996[Medline].

39.   Seo, S. J., S. S. Kang, G. Cho, H. M. Rho, and G. Jung. C/EBPalpha and C/EBPbeta play similar roles in the transcription of the human Cu/Zn SOD gene. Gene 203: 11-15, 1997[Medline].

40.   Sonoki, T., A. Nagasaki, T. Gotoh, M. Takiguchi, M. Takeya, H. Matsuzaki, and M. Mori. Coinduction of nitric oxide synthase and arginase I in cultured rat peritoneal macrophages and rat tissues in vivo by lipopolysaccharide. J. Biol. Chem. 272: 3689-3693, 1997[Abstract/Free Full Text].

41.   Spink, J., J. Cohen, and T. J. Evans. The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase. Activation by nuclear factor-kappa B. J. Biol. Chem. 270: 29541-29547, 1995[Abstract/Free Full Text].

42.   Spink, J., and T. Evans. Binding of the transcription factor interferon regulatory factor-1 to the inducible nitric-oxide synthase promoter. J. Biol. Chem. 272: 24417-24425, 1997[Abstract/Free Full Text].

43.   Tanaka, T., S. Akira, K. Yoshida, M. Umemoto, Y. Yoneda, N. Shirafuji, H. Fujiwara, S. Suematsu, N. Yoshida, and T. Kishimoto. Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. Cell 80: 353-361, 1995[Medline].

44.   Taylor, B. S., M. E. de Vera, R. W. Ganster, Q. Wang, R. A. Shapiro, S. M. Morris, Jr., T. R. Billiar, and D. A. Geller. Multiple NF-kappa B enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene. J. Biol. Chem. 273: 15148-15156, 1998[Abstract/Free Full Text].

45.   Waddington, S. N., K. Mosley, H. T. Cook, F. W. Tam, and V. Cattell. Arginase AI is upregulated in acute immune complex-induced inflammation. Biochem. Biophys. Res. Commun. 247: 84-87, 1998[Medline].

46.   Wedel, A., and H. W. Ziegler Heitbrock. The C/EBP family of transcription factors. Immunobiology 193: 171-185, 1995[Medline].

47.   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].

48.   Xie, Q.-W., Y. Kashiwabara, and C. Nathan. 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].

49.   Xie, Q. W., and C. Nathan. Promoter of the mouse gene encoding calcium independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. Trans. Assoc. Am. Phys. 106: 1-12, 1993[Medline].

50.   Yan, S. F., Y. S. Zou, M. Mendelsohn, Y. Gao, Y. Naka, S. Du Yan, D. Pinsky, and D. Stern. Nuclear factor interleukin 6 motifs mediate tissue specific gene transcription in hypoxia. J. Biol. Chem. 272: 4287-4294, 1997[Abstract/Free Full Text].

51.   Yeh, W.-C., Z. Cao, M. Classon, and S. L. McKnight. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev. 9: 168-181, 1995[Abstract].


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