Transcriptional Activation of the Human Inducible Nitric-oxide Synthase Promoter by Krüppel-like Factor 6*

Vishal G. WarkeDagger §, Madhusoodana P. NambiarDagger , Sandeep KrishnanDagger , Klaus TenbrockDagger , David A. Geller||, Nicolas P. Koritschoner**, James L. AtkinsDagger , Donna L. FarberDagger Dagger , and George C. TsokosDagger §§§

From the Dagger  Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, the § Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, the  Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, the Dagger Dagger  Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, the || Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, and the ** Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba 5000, Argentina

Received for publication, January 23, 2003, and in revised form, February 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide is a ubiquitous free radical that plays a key role in a broad spectrum of signaling pathways in physiological and pathophysiological processes. We have explored the transcriptional regulation of inducible nitric-oxide synthase (iNOS) by Krüppel-like factor 6 (KLF6), an Sp1-like zinc finger transcription factor. Study of serial deletion constructs of the iNOS promoter revealed that the proximal 0.63-kb region can support a 3-6-fold reporter activity similar to that of the full-length 16-kb promoter. Within the 0.63-kb region, we identified two CACCC sites (-164 to -168 and -261 to -265) that bound KLF6 in both electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutation of both these sites abrogated the KLF6-induced enhancement of the 0.63-kb iNOS promoter activity. The binding of KLF6 to the iNOS promoter was significantly increased in Jurkat cells, primary T lymphocytes, and COS-7 cells subjected to NaCN-induced hypoxia, heat shock, serum starvation, and phorbol 12-myristate 13-acetate/A23187 ionophore stimulation. Furthermore, in KLF6-transfected and NaCN-treated COS-7 cells, there was a 3-4-fold increase in the expression of the endogenous iNOS mRNA and protein that correlated with increased production of nitric oxide. These findings indicate that KLF6 is a potential transactivator of the human iNOS promoter in diverse pathophysiological conditions.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric-oxide synthases are key proteins that produce NO and thereby regulate many important biological processes. NO is generated during the oxidation of L-arginine to L-citrulline by at least three different isoforms of nitric-oxide synthase. Endothelial and neuronal nitric-oxide synthases are constitutively expressed, and their activity is Ca2+- and calmodulin-dependent, whereas the third isoform is transcriptionally inducible (iNOS),1 and its activity is independent of Ca2+ and calmodulin and can produce very high levels of nitric oxide over a sustained period of time (1, 2). It has been shown that iNOS is transcriptionally up-regulated in pathophysiologic conditions such as hypoxia, ischemia-reperfusion injury, and trauma and by reactive oxygen species (3, 4).

NO is a key central molecule in cellular biochemical processes, as it is freely diffusible and traverses cell membranes to reach different targets, alters signaling networks by redox-sensitive modifications, and transcriptionally regulates multiple gene families (5-10). NO production following iNOS up-regulation is associated with increased wound healing and repair in tissue injury (11, 12). NO is also known to activate multiple gene and cell signaling pathways through processes such as nitrosation and cGMP production (3, 9). Furthermore, numerous studies have shown that NO has antitumor effects and that forced expression of iNOS causes regression of tumors (13-16).

Krüppel-like factor 6 (KLF6) is a ubiquitously expressed member of the Krüppel-like family of transcription factors, which have characteristic Cys2/His2 zinc finger motifs and bind very similar "GC box" or "CACCC element" sites on DNA (17, 18). KLF6 is an immediate-early gene that regulates the expression of multiple genes and is involved in tissue differentiation (19-23). KLF6 is rapidly induced in cells after acute injury and directly activates collagen alpha 1 and TGF-beta along with TGF-beta receptor I and II genes, thereby mediating wound-healing mechanisms of fibrogenesis and extracellular matrix formation (18, 24). Recently, KLF6 has been shown to function as a tumor suppressor gene that was mutated in prostate cancer (25).

The iNOS promoter defines a number of NF-kappa B and AP-1 sites dispersed through out the 16-kb region. A number of iNOS inducers, including cell injury, heat shock, and various cytokines, have been found to exert their effect by activating either NF-kappa B or AP-1 (1, 26-28). Because KLF6 and iNOS are involved in common processes such as cell injury, wound repair, embryogenesis, tissue differentiation, and suppression of tumorigenesis and because the iNOS promoter defines multiple CACCC sites (KLF6-binding motifs), we hypothesized that KLF6 binds to the iNOS gene and regulates its expression.

In this study, we have identified a novel transcriptional regulator of iNOS. Specifically, we demonstrate that KLF6 binds to CACCC sites within the proximal 0.63-kb region and regulates the expression of the iNOS promoter in various cell types. We also show that in cells exposed to stress conditions, the enhanced expression of KLF6 causes a direct increase in expression of the endogenous iNOS gene and NO.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Cell Culture-- Jurkat cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. Peripheral blood mononuclear cells were obtained from healthy adult volunteers. Primary T lymphocytes were obtained from peripheral blood mononuclear cells using a pan T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions.

Plasmid Constructs-- The human iNOS-luciferase reporter constructs 0.63, 1.3, 3.8, 5.8, 7.2, and 16.0 kb upstream have been previously described (29). The two CACCC sites in the 0.63-kb iNOS promoter were mutated to AAAAA using the QuikChangeTM XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutations were confirmed by sequencing. The promoterless luciferase gene vectors pXP1 and pXP2 were used as control vectors. The iNOS promoter-thymidine kinase-luciferase constructs have been described previously (29). The KLF6 expression vector pXCPBP and the control vector pX (pBluescript) have been described previously (30). The p50 and p65 NF-kappa B vectors were a kind gift from Dr. Barbara Rellahan (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD).

Transient Transfections and Luciferase Activity Assays-- Plasmid DNA transfections of Jurkat T cells and COS-7 cells were carried out in 24-well plates (Corning Inc., Corning, NY) using LipofectAMINETM 2000 reagent (Invitrogen) following the manufacturer's protocol. The day before transfection, 6 × 104 COS-7 cells or 0.6 × 106 Jurkat T cells were plated in 0.5 ml of medium/well. For each well, LipofectAMINE reagent (2-3 µl) was mixed with plasmid DNA (1.5 µg) in serum-free Opti-MEM to allow DNA-LipofectAMINE reagent complexes to form. The complexes were added to respective wells and mixed by gently rocking the plate back and forth. The cells were incubated in a CO2 incubator at 37 °C for 48 h and then lysed with 60 µl of reporter lysis buffer (Promega, Madison, WI). Luciferase activity was assayed with 20 µl of lysate and 80 µl of luciferase assay reagent (Promega) in a TD20/20 luminometer (Promega). Transfection efficiency was determined in all samples by cotransfection with 0.5 µg of plasmid encoding the cytomegalovirus promoter-driven beta -galactosidase gene, and the luciferase activity was normalized to the beta -galactosidase activity.

Electrophoretic Mobility Shift Assay (EMSA)-- COS-7 cells (2 × 106) were transfected with the KLF6 expression vector overnight. Briefly, the transfected cells were detached using trypsin/EDTA and washed with phosphate-buffered saline, and nuclear extracts were prepared as described previously (31). The sequences of the oligonucleotides used for EMSA are as follows: probe 2, 5'-GAT CAG GTC ACC CAC AGG CCC-3', and its complementary sequence, 5'-GGG CCT GTG GGT GAC CTG ATC-3'; and probe 4, 5'-AGC AGC CAC CCT GCT GAT GAA C-3', and its complementary sequence, 5'-GTT CAT CAG CAG GGT GGC TGC T-3'. The oligonucleotides were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). Complementary single-stranded oligonucleotides were annealed, end-labeled with [gamma -32P]ATP (PerkinElmer Life Sciences) and T4 polynucleotide kinase (Roche Molecular Biochemicals, Mannheim, Germany), purified by Centri-Sep columns (Princeton Separations, Adelphia, NJ), and used as probes in EMSA. In all experiments, unlabeled oligonucleotides were used as unlabeled competitors. In each experiment, 5-10 µg of nuclear extracts were incubated for 20 min at room temperature with the labeled oligonucleotide (2-3 ng) in 20 µl of buffer containing 20 mM HEPES (pH 7.4), 1 mM MgCl2, 10 µM ZnSO4, 20 mM KCl, 15% Ficoll, and 2 µg of poly(dI-dC). For supershift experiments, 3 µg each of control anti-KLF4 (clone T-16) and anti-KLF6 (clone R-173) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were added and incubated for 45 min at room temperature prior to the addition of the radiolabeled probe. The DNA-protein complex was separated on a 4% nondenaturing polyacrylamide gel in 0.5× Tris borate/EDTA buffer. The data were analyzed using the PhosphorImager system (Amersham Biosciences) and Quantity One software (Bio-Rad).

Chromatin Immunoprecipitation (ChIP) Assay-- COS-7 cells (10 × 106) were transfected with the KLF6 expression vector or were non-transfected (control). Both the transfected and control cells were divided into two subgroups. One group was left untreated, and the other was treated with 20 mM NaCN for 3 h; both subgroups were used for the ChIP assay. Jurkat cells and T lymphocytes (10 × 106) were also treated similarly to COS-7 cells; however, only the COS-7 cells were subjected to KLF6 transfection. Moreover, all the cells were also treated with PMA/A23187 or heat-shocked or serum-starved and subjected to ChIP analysis. The ChIP assay was performed following the recommendations of Upstate Biotechnology, Inc. (Lake Placid, NY) and previously published protocols (32-34). Briefly, KLF6 was cross-linked to DNA by adding formaldehyde to a final concentration of 1%. The chromatin samples for studying phosphorylated KLF6 were incubated with anti-phosphoserine (clone PSR-45, Sigma) and anti-phosphotyrosine (clone 4G10, Santa Cruz Biotechnology) antibodies overnight at 4 °C and immunoprecipitated with salmon sperm DNA-bovine serum albumin-Sepharose beads, followed by treatment with 10 mM phenyl phosphate for 15 min. The supernatants were used for further steps. All the samples (for phosphorylated as well as non-phosphorylated KLF6) were then incubated overnight at 4 °C with anti-KLF6 antibody, followed by incubation with Sepharose beads. The immunocomplexes were treated with DNase- and RNase-free proteinase K, and DNA was purified using a DNA purification kit (QIAGEN Inc., Santa Clara, CA). PCR was performed with primers flanking the proximal as well as distal KLF6-binding sites in the proximal 0.63-kb iNOS promoter (5'-CAG AGA GCT CCC TGC TGA GGA AA-3' and 5'-GAG AGT TGT TTT TGC ATA AAG GTC TC-3') (see Fig. 5). Amplified fragments (321 bp) were analyzed on a 2% agarose gel by staining with SYBR Green (FMC Corp. BioProducts, Rockland, ME). The no-antibody immunoprecipitation samples served as negative controls.

Real-time Quantitative PCR-- Total RNA was isolated from 5 × 106 control cells using an RNeasy minikit (QIAGEN Inc.), treated with DNase I, and reverse-transcribed using avian myeloblastosis virus reverse transcriptase and oligo(dT) primer (Promega). The PCR primers synthesized by Genosys Biotechnologies, Inc. were as follows: KLF6, 5'-AGA GCG AGC CCT GCT ATG TTT CAG-3' (forward) and 5'-CGC TGG TGT GCT TTC AAG TGG GAG-3' (reverse); GAPDH, 5'-CAA CTA CAT GGT TTA CAT GTT CC-3' (forward) and 5'-GGA CTG TGG TCA TGA GTC CT-3' (reverse); and iNOS, 5'-ACC TAC CAC ACC CGA GAT GGC CAG-3' (forward) and 5'-AGG ATG TCC TGA ACA TAG ACC TTG GG-3' (reverse). Quantitative PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on a SmartCyclerTM (Cepheid, Sunnyvale, CA) according to the manufacturer's instructions. The relative expression level of the target gene in KLF6-transfected and NaCN-treated cells was plotted as -fold change compared with control vector-transfected and non-NaCN-treated cells, respectively. GAPDH gene expression was used for normalization. Each real-time quantitative PCR assay was performed twice using triplicate samples.

SDS-PAGE and Immunoblotting-- KLF6-transfected and control COS-7 cells (0.5 × 106 cells) were lysed in cold 1% Nonidet P-40 lysis buffer (Sigma) with protease inhibitors as described previously (35), and protein was assayed using a Bio-Rad protein assay kit. Seventy micrograms of the protein were resolved by 4-12% BisTris-NuPAGE and transferred to polyvinylidene difluoride membrane. The blot was probed with control anti-beta -actin (clone 54, BD Biosciences), anti-KLF6, and anti-iNOS (BD Biosciences) antibodies. The membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Protein bands were detected by ECL enhanced chemiluminescence reagents (Amersham Biosciences). The Western blot bands were quantitated by densitometry using GelProTM software (Media Cybernetics, Silver Spring, MD).

Estimation of Nitrite + Nitrate-- Briefly, 50 µl of cell culture medium (Dulbecco's modified Eagle's medium) were treated with nitrate reductase (N-7265, Sigma) in the presence of NADPH (N-7505, Sigma) to convert nitrate to nitrite (36, 37). Upon the addition of 2,3-diaminonaphthalene (Sigma), which reacts with nitrite under acidic conditions to form a fluorescent product (1H-naphthotriazole), fluorescence intensity was measured with a fluorescence microplate reader with excitation at 365 nm and emission at 450 nm, and nitrite was quantitated by comparison with a standard curve of NaNO2.

Quantitation and Statistical Analysis-- Statistical analysis of the data was carried out with Minitab Version 14 using Student's t test, and p values <0.05 were considered as significant.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

KLF6 Induces iNOS Promoter Activity in COS-7 Cells-- A schematic diagram showing putative KLF6-binding motifs in the 5'-flanking region of the human iNOS gene is shown in Fig. 1A. To investigate the effect of KLF6 on the expression of human iNOS, we performed luciferase reporter assays in COS-7 cells transfected with the full-length 16-kb iNOS promoter construct and the KLF6 expression vector. As shown in Fig. 1B, transfection of COS-7 cells with KLF6 induced the luciferase activity by 3-fold compared with the control vector. To compare the induction of iNOS with a well characterized inducer, we studied the effect of transcription factor NF-kappa B subunits p50 and p65 on the iNOS promoter. NF-kappa B has been reported to bind multiple sites on the 7.3-kb iNOS promoter and to induce its activity by 4.1-fold in AKN-1 and by 3.9-fold in A549 cells following cytokine stimulation (1). Transfection of COS-7 cells with p65 and p50 with the full-length iNOS promoter construct induced promoter activity by 2- and 4-fold, respectively (Fig. 1B). These results indicate that KLF6 can induce the transcription of the iNOS promoter at levels comparable to those induced by NF-kappa B (4, 26, 29, 38, 39).


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Fig. 1.   KLF6 induces iNOS expression in COS-7 cells. A, shown is a schematic diagram of the full-length 16-kb human iNOS promoter. The positions of the KLF6-binding sites (CACCC) identified in the 8-kb sequenced region are indicated by arrows. The position of the beginning nucleotide of each CACCC site according to Spitsin et al. (41) is indicated. B, the 16-kb iNOS-luciferase construct was transfected with KLF6 or the p50 or p65 subunit of NF-kappa B into COS-7 cells, and the luciferase activity was determined. Data are representative of three independent experiments performed in triplicate.

The Proximal 0.63-kb iNOS Promoter Is Sufficient for the Induction of Activity by KLF6-- After ascertaining that KLF6 can activate the full-length 16-kb iNOS promoter, we performed transfection experiments to define the region of the iNOS promoter that is required for the induction of transcription by KLF6 (29). COS-7 and Jurkat cells transfected with serial deletion constructs of the 16-kb iNOS promoter-luciferase reporter compared with cells transfected with the control vector demonstrated that the induction of iNOS promoter activity remained similar in all constructs compared with the full-length 16-kb iNOS promoter (Fig. 2, A and B). Previous studies have shown that KLF6 binds to the CACCC motifs of DNA (40). The 0.63-kb iNOS construct has two CACCC motifs, and there are a total of 10 CACCC binding sites in the first 8 kb of the 16-kb iNOS promoter. Optimum induction (3-fold) of the 0.63-kb iNOS promoter by KLF6 (Fig. 2, A and B) suggests that additional upstream KLF6-binding sites do not further enhance the iNOS promoter activity in the presence of CACCC sites in the proximal 0.63-kb region. However, when the iNOS core promoter with the CACCC sites (within 0.63 kb) was replaced with the thymidine kinase (thymidine kinase-luciferase) core promoter, a 20% increase in the luciferase activity with the KLF6 expression vector (compared with transfection with the control pX vector) was observed with the 3.8-5.8-kb iNOS- and 7.2-16-kb iNOS-thymidine kinase-luciferase constructs, whereas there was no luciferase induction with the iNOS promoter region from 5.8 to 7.0 kb (Fig. 2C). The significance of this finding is presently unknown.


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Fig. 2.   Effect of KLF6 on iNOS promoter activity in cells transfected with various deletion constructs of the iNOS promoter. Shown is a schematic view of the iNOS promoter indicating the restriction enzymes used to generate various deletion constructs with luciferase. A and B, COS-7 and Jurkat cells were transfected with the KLF6 expression vector (pXCPBP, 1.5 µg) and iNOS promoter deletion constructs (1.5 µg) or with the empty KLF6 vector (1.5 µg) and iNOS promoter deletion constructs (1.5 µg) along with a beta -galactosidase-expressing vector (0.5 µg; as a transfection efficiency control). pXP1 and pXP2 are empty vectors of iNOS transfected with KLF6 or the empty KLF6 vector (pX). The luciferase activity was determined 24-48 h post-transfection. The effect of KLF6 on the respective constructs is indicated as -fold induction of luciferase activity over that of the control pX vector. C, COS-7 cells were transfected with the KLF6 expression vector (pXCPBP, 1.5 µg) and iNOS promoter-thymidine kinase (TK)-luciferase (Luc) deletion constructs or with the empty KLF6 vector (1.5 µg) and iNOS promoter deletion constructs (1.5 µg) along with a beta -galactosidase-expressing vector (0.5 µg; as a transfection efficiency control). The luciferase activity was determined 24-48 h post-transfection. The effect of KLF6 on the respective constructs is indicated as percent increase in luciferase activity over that of the control pX vector. Data are representative of five independent experiments performed in triplicate.

Mutational Analysis of the Two CACCC Sites in the 0.63-kb iNOS Promoter-- To understand the contribution of KLF6 to the activation of the 0.63-kb iNOS promoter by the two CACCC sites, we created single mutation constructs in which we mutated the proximal and distal CACCC sites individually and a dual mutation construct with both CACCC sites mutated. Transfection studies with these mutants revealed that the two sites, which are separated by 92 bases, had an additive effect on the stimulation of the 0.63-kb iNOS promoter. The distal CACCC site contributed to 1.6- and 3.2-fold induction of luciferase activity in COS-7 cells (Fig. 3A) and Jurkat cells (Fig. 3B), respectively. Similarly, the proximal CACCC site contributed to 1.3- and 2-fold induction in COS-7 cells (Fig. 3A) and Jurkat cells (Fig. 3B), respectively. Mutation of both CACCC sites completely abolished the KLF6-induced iNOS promoter activity, suggesting that the CACCC motifs are necessary for the interaction of KLF6 with the 0.63-kb iNOS promoter. These studies demonstrate that the proximal and distal CACCC sites in the 0.63-kb iNOS promoter are necessary for optimum basal promoter activity.


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Fig. 3.   CACCC motifs in the 0.63-kb iNOS promoter are necessary for activation by KLF6. The two CACCC sites in the 0.63-kb iNOS promoter were mutated to AAAAA by PCR-based mutagenesis. A and B, the wild-type and mutant reporter genes (1.5 µg) were cotransfected with either the empty vector (pX, 1.5 µg) or the KLF6 expression vector (pXCPBP, 1.5 µg) in the presence of a beta -galactosidase-expressing vector (0.5 µg) in COS-7 or Jurkat cells. The luciferase activity was measured 24-48 h post-transfection and normalized to beta -galactosidase levels. Data are presented as -fold induction over that of the empty vector. Data are representative of three similar experiments performed in triplicate. N, no mutations; *, mutation.

KLF6 Binds to the CACCC Sites in the 0.63-kb iNOS Promoter-- To demonstrate that KLF6 binds to the human iNOS promoter, we designed two oligonucleotides, each defining the CACCC motif regions at positions -164 to -168 and -261 to -265 in the 0.63-kb iNOS promoter, respectively (41). The primers were end-labeled, and EMSA was performed using nuclear extracts from KLF6-transfected COS-7 cells. Nuclear extracts from these cells bound to both oligonucleotides defined by the 0.63-kb iNOS promoter (Fig. 4, A and B). To demonstrate the specificity of the binding, supershift assays were performed using an antibody specific to KLF6. As shown in Fig. 4 (A and B, lanes 4), the shifted band in lane 1 was supershifted by anti-KLF6 antibody, demonstrating that KLF6 directly interacts with the iNOS promoter in vitro. The absence of a supershifted band with anti-KLF4 antibody (Fig. 4, A and B, lanes 2) further confirmed the specificity of KLF6 for the CACCC binding sites.


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Fig. 4.   KLF6 binds to oligonucleotides containing the two CACCC motifs in the 0.63-kb iNOS promoter. Nuclear extracts were prepared from KLF6-transfected COS-7 cells and incubated with labeled oligonucleotides from the iNOS promoter, followed by EMSA as described under "Materials and Methods." A, EMSA was done using the oligonucleotide defining the proximal CACCC site in lane 1. A nonspecific antibody (Ab; anti-KLF4) was added in lane 2, and a hundredfold excess of specific unlabeled competitor was added in lane 3. Lane 4 depicts a supershifted band with anti-KLF6 antibody. Antibody (2 µg) was added to the oligonucleotide and nuclear extract. B, EMSAs were repeated as described for A with an oligonucleotide defining the distal CACCC site.

In Vivo Binding of KLF6 to the iNOS Promoter-- Next, to determine whether KLF6 interacts with iNOS in vivo, we performed ChIP analysis using primary T, Jurkat, and COS-7 cells. The DNA-KLF6 complexes were immunoprecipitated with anti-KLF6 antibody, followed by reversal of cross-linking and PCR amplification using primers flanking the proximal and distal CACCC binding sites in the 0.63-kb iNOS promoter (Fig. 5A). In transfected COS-7 cells, the intensity of the PCR product was significantly higher compared with the non-transfected cells (Fig. 5B, lane 2 versus lane 4), suggesting that increased expression of KLF6 increases its binding to the iNOS promoter.


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Fig. 5.   In vivo binding of KLF6 to the iNOS promoter in COS-7 and Jurkat cells and T lymphocytes. COS-7 cells (10 × 106/sample) were treated as per protocol, fixed with formalin, washed, lysed, and sonicated. The DNA-protein complexes were immunoprecipitated (IP) with anti-KLF6 antibody (Ab) and extracted with protein A-agarose beads. The DNA was purified and amplified with primers flanking the iNOS promoter. A, a schematic view of the 0.63-kb iNOS promoter depicting the positions of the primers flanking the CACCC sites that were used for PCR of the ChIP DNA. Primer sequences are indicated by arrows. The two KLF6-binding CACCC sites are shown. B, ChIP analysis of the iNOS promoter in KLF6-transfected COS-7 cells and non-transfected COS-7, Jurkat, and T cells after treatment with sodium cyanide. Lane 1, the PCR product from the input (positive control) DNA; lanes 2 and 3, KLF6-transfected COS-7 cells; lanes 4 and 5, 6 and 7, and 8 and 9, non-transfected COS-7, Jurkat, and primary T cells, respectively. Cells in lanes 3, 5, 7, and 9 were treated with NaCN. PCR products were resolved on a 2% agarose gel. C, ChIP analysis of control and treated Jurkat, primary T, and COS-7 cells. D, ChIP analysis of serine- and tyrosine-phosphorylated KLF6 in PMA/A23187-treated, sodium cyanide-treated, and control primary T cells. Data are representative of two similar experiments.

It has been demonstrated in independent studies that iNOS and KLF6 are up-regulated during cell stress (3, 4, 18, 24). To examine whether up-regulation of iNOS is mediated by KLF6 under conditions of stress, we treated COS-7 and Jurkat cells and primary T lymphocytes with NaCN. NaCN blocks mitochondrial respiration and induces cellular hypoxia (42). As shown in Fig. 5B (lanes 3, 5, 7, and 9), treatment with NaCN strongly increased the binding of KLF6 to the iNOS promoter as evidenced by the increased intensity of the PCR product.

We also subjected COS-7, Jurkat, and primary T cells to heat stress, serum starvation, and PMA/A23187 and analyzed the binding of KLF6 to the iNOS promoter by ChIP assay. As shown in Fig. 5C, compared with the control cells, KLF6 binding to the iNOS promoter was increased in a similar fashion in all cell types subjected to these conditions. These data conclusively show that the association of KLF6 with the iNOS promoter is increased in cells subjected to conditions of stress and stimulation.

Next, we investigated whether the phosphorylation status of KLF6 could play a role in its binding to the iNOS promoter. To ascertain whether serine and tyrosine phosphorylation of KLF6 plays a role in binding to the iNOS promoter, we performed ChIP analysis involving a two-step immunoprecipitation process using anti-phosphoserine or anti-phosphotyrosine antibody, followed by treatment with phenyl phosphate (to separate the phosphorylated protein from the bound antibody) and further immunoprecipitation with anti-KLF6 antibody. The data show that the KLF6 that bound to the iNOS promoter was serine-phosphorylated in resting T cells, which was increased in PMA/A23187- and NaCN-treated cells (Fig. 5D). Interestingly, however, we observed that tyrosine-phosphorylated KLF6 bound to the iNOS promoter only in NaCN-treated cells. The absence of any PCR products for the no-antibody and mouse IgG immunoprecipitation controls further confirmed the specificity of our findings (data not shown). Thus, differential phosphorylation of KLF6 mediates differential binding to iNOS in various conditions.

Induction of iNOS mRNA and Protein and NO in KLF6-transfected Cells-- To establish the functional association between KLF6 and iNOS expression, we measured the iNOS mRNA and protein and NO production in KLF6-transfected COS-7 cells. We performed real-time PCR to estimate the -fold induction of mRNA based on the fluorescence cycle threshold differences between various time points compared with controls. As shown in Fig. 6A, the production of KLF6 mRNA was induced by up to 14-fold 24 h post-transfection and by 13-fold 48 h post-transfection. In tandem with the KLF6 mRNA, there was a corresponding increase in the iNOS mRNA, which showed a steady increase ranging from 3.5-fold (24 h) to 6-fold (48 h) (Fig. 6A). There was no increase in the control (GAPDH) mRNA.


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Fig. 6.   KLF6 transfection up-regulates iNOS mRNA and protein and NO in COS-7 cells. A, COS-7 cells were transfected with KLF6 and incubated for various time periods. Total RNA was isolated; cDNA was synthesized; and KLF6, iNOS, and GAPDH (control) mRNAs were amplified by real-time quantitative PCR. B and C, KLF6, iNOS, and beta -actin protein levels were estimated by Western blotting. D, NO production was estimated by assay for NO metabolites (nitrite + nitrate) from the same samples. Data are representative of three similar experiments.

Next, to study the kinetics of iNOS protein induction by KLF6 following KLF6 expression vector transfection, we performed Western blot experiments at earlier time points (0-18 h) (Fig. 6B) and pursued the kinetics of KLF6 and iNOS expression over 72 h (Fig. 6C). KLF6 protein levels increased starting at 6 h post-transfection, whereas iNOS protein levels increased starting at 12 h post-transfection (Fig. 6B), suggesting that KLF6 is able to initiate transcription and to produce iNOS protein within 6 h. It was previously observed that an increase in KLF6 mRNA in culture-activated cells is accompanied by an even greater increase in KLF6 protein; moreover, the rate of degradation of KLF6 protein is also lower (18). In light of these data, we believe that the induction of KLF6 protein seen beyond 48 h could be attributed to accumulation of KLF6 as a result of decreased protein degradation (Fig. 6C). Furthermore, NO production, as measured by assay for NO metabolites (nitrite and nitrate), indicated a significant increase in cells transiently transfected with KLF6 (Fig. 6D). There was no increase in either KLF6 or iNOS mRNA or protein (data not shown) or NO in control vector-transfected cells (Fig. 6D) and in cells subjected to LipofectAMINE transfection agent (data not shown), which were incubated under similar culture conditions as the KLF6-transfected cells. These data, taken together with the ChIP results (Fig. 5), demonstrate that KLF6 acts as a transactivator of the iNOS gene.

NaCN-induced Hypoxia Up-regulates KLF6 and iNOS mRNAs and Proteins as Well as NO in COS-7 Cells-- ChIP analysis (Fig. 5A) demonstrated increased binding of KLF6 to the iNOS promoter in cells exposed to NaCN. Therefore, to establish a functional association between the expression of KLF6 and iNOS in pathophysiological conditions, we measured the KLF6 and iNOS mRNA and protein and NO production in NaCN-treated COS-7 cells. The cells were transiently treated with 20 mM NaCN for 4 h and then incubated in fresh medium for various time periods. Following exposure of cells to NaCN, the production of KLF6 mRNA was analyzed by real-time PCR. As shown in Fig. 7A, a significant induction of KLF6 mRNA was seen, with levels reaching 4.5- and 8-fold over control levels by 24 and 48 h, respectively. Consistent with the KLF6 mRNA results, there was an increase in KLF6 protein by 24 h, and the high amounts were sustained over a 72-h period. iNOS protein were also induced by 12 h, and the levels reached a peak by 48 h (Fig. 7B). Furthermore, NO production, as assayed by its metabolites (nitrite and nitrate), also indicated a significant increase in the nitrite + nitrate levels starting at 6 h, with a steady and sustained increase over 72 h (Fig. 7C). Thus, even though we cannot rule out the additional effect of other transcription factors in the induction of iNOS following NaCN treatment, the above data, combined with our ChIP findings (Fig. 5B) and increased iNOS mRNA and protein expression data following KLF6 transfection (Fig. 6, A and B), strongly suggest that KLF6 plays a role in iNOS induction following treatment of cells with NaCN.


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Fig. 7.   NaCN exposure up-regulates KLF6 and iNOS mRNAs and proteins and NO in COS-7 cells. A, COS-7 cells were exposed to 20 mM NaCN for 4 h, washed, and incubated for various time periods in normal medium. Total RNA was isolated; cDNA was synthesized; and KLF6, iNOS, and GAPDH (control) mRNAs were amplified by real-time quantitative PCR. B, KLF6, iNOS, and beta -actin protein levels were estimated by Western blotting. C, NO production was estimated by assay for NO metabolites (nitrite + nitrate) from the same samples. Data are representative of three similar experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our study provides the first evidence for the regulation of the human iNOS promoter by a member of the Krüppel-like family of transcription factors. Using luciferase reporter gene assays, EMSA, and ChIP analysis, we have demonstrated that KLF6, a member of the Krüppel-like family of transcription factors, directly interacts with the iNOS promoter in resting cells and with greater intensity following cell stress, injury, and stimulation. Furthermore, we have shown that up-regulated KLF6 increases both iNOS mRNA and protein expression, which correlates functionally with the concomitant nitric oxide expression. The presented evidence strongly suggests that KLF6 is a major transcriptional regulator of iNOS under conditions of hypoxia and cell stress.

The human iNOS promoter is 16 kb long and is one of the largest known promoters (1). Its regulation is complex and occurs at multiple levels, orchestrated by multiple transcription factors, in response to diverse conditions in a tissue-specific context. Multiple transcription factors such as NF-kappa B, AP-1, STAT1alpha , and interferon regulatory factor are known to regulate the iNOS promoter (reviewed in detail in Ref. 1). NF-kappa B is ubiquitously expressed and regulates iNOS in multiple cell types, and the regulation of iNOS by NF-kappa B is well characterized (4, 26, 29, 38, 39). Our data demonstrate that the induction of iNOS by KLF6 is comparable to that by the NF-kappa B subunits, suggesting that KLF6 could be an equally important regulator of iNOS.

Despite the presence of at least 10 CACCC sites in the 16-kb iNOS promoter, the KLF6-induced transcriptional activation of the 0.63-kb construct was similar to that of the 16-kb construct in COS-7 and Jurkat cells (Fig. 2). The CACCC sites in the 0.63-kb construct are placed in close proximity at positions -164 and -261 compared with the other CACCC sites, the nearest of which is much farther upstream at position -2736. Previous studies have reported similar binding of KLF6 to either tandem sites or sites in close proximity to each other in other promoters and placed in close proximity to the basal promoter elements. KLF6 is known to bind the leukotriene C4 synthase promoter at two tandem CACCC sites located between positions -135 and -149 close to the basal promoter elements (40). In yet another study, KLF6 was shown to interact with TGF-beta 1 and both TGF-beta receptor I and II promoters at multiple sites. KLF6 strongly transactivates TGF-beta 1 by binding to two tandem Sp1-binding sites between positions -239 and -209 compared with much lesser interactions with promoter regions including single Sp1-binding sites (24). The transactivation of the TGF-beta receptor II promoter requires the presence of closely placed GC-rich regions from positions -152 to -127 and -118 to -85 (24). Thus, it is very likely that the two closely placed CACCC sites, in proximity to the basal promoter elements, in the 0.63-kb iNOS promoter are sufficient for induction of the iNOS gene by KLF6. Our data suggest that CACCC sites located between 3.8 and 5.8 kb and 7.0 and 16.0 kb upstream in the iNOS promoter may play a role in KLF6-mediated regulation of the iNOS gene only in the absence of proximal (0.63 kb) CACCC sites.

The facts that KLF is very rich in serines (30) and that its transactivation domain contains serines as well as tyrosines (30) suggest that the activity and binding of KLF6 to the iNOS promoter could also be regulated by serine and tyrosine phosphorylation. There are several examples of transcription factors being regulated by their phosphorylation status. For example, the activation and intermolecular interactions of KLF1 are known to be dependent on its serine phosphorylation (43). Similarly, studies have indicated that two distinct phosphorylation events, i.e. phosphorylation of tyrosine 701 and serine 727, are necessary for full activation of STAT1 by interferon-gamma (44, 45). Thus, our observations that serine-phosphorylated KLF6 binds to the iNOS promoter in resting, stimulated (PMA/A23187), and stressed (NaCN-treated) cells and that serine- as well as tyrosine-phosphorylated KLF6 binds to the iNOS promoter in NaCN-treated cells indicate that differential phosphorylation of KLF6 could play a very important role in gene regulation. The precise mechanism of this interaction is currently under investigation.

Induction of iNOS by KLF6 may have important implications in the prevention of apoptosis, tissue injury repair, and cancer. Previous studies have addressed the protective role of NO in apoptosis. NO blocks apoptosis by multiple mechanisms. First, NO inhibits caspases (46) by inhibiting interleukin-1beta -converting enzyme-like and cysteine protease protein-32-like proteases (47). Second, NO protects the mitochondria, lowers cytochrome c release, and inhibits calcium fluxes (48). Third, NO prevents an increase in Bcl-2 and induces the expression of heat shock proteins such as HSP70 that have an anti-apoptotic role. NaCN triggers apoptosis of cells predominantly by inducing cytotoxic hypoxia by inhibiting cytochrome c oxidase, the terminal enzyme of the respiratory chain, and by causing activation of voltage-sensitive calcium channels and calcium fluxes (49). In NaCN-treated cells, we observed increased binding of KLF6 to the iNOS promoter (Fig. 5B), in addition to increased levels of iNOS protein and concomitant NO production (Fig. 7, B and C). In a similar study, it has been shown that NO protects NaCN-treated chick embryonic neurons from cyanide-induced apoptosis (42).

NO plays a role in wound healing and tissue repair. In several studies on colon anastomosis, bone fracture, and cutaneous wound healing, the reparative role of NO through up-regulation of iNOS has been well demonstrated (10, 50, 51). Similarly, KLF6 plays a direct anti-apoptotic role in conditions of acute injury. KLF6 was found to be responsible for healing of acutely injured hepatic stellate cells (18) and aortic endothelial cells (52). Our ChIP data (Fig. 5B) demonstrate that in heat-shocked and NaCN-treated cells, there is a strong binding of KLF6 to the iNOS promoter, thereby suggesting that KLF6 can orchestrate its anti-apoptotic and protective effects by up-regulating NO through iNOS (Fig. 6).

Recently, KLF6 was demonstrated to be a tumor suppressor gene that was found mutated in 77% of prostate cancer patients, and it was shown to act in a p53-independent manner through the p21WAF1/CIP1 pathway (25). The tumor-suppressive role of iNOS is also well documented (13, 15, 16). NO production by iNOS through its transcriptional up-regulation by KLF6 could serve as another mechanism for the antitumor effect of KLF6. In conclusion, our findings provide evidence that KLF6 binds to the human iNOS promoter and regulates its expression under conditions of cell stress, injury, and stimulation with possible implications in the treatment of organ injury and cancer.

    ACKNOWLEDGEMENT

We gratefully acknowledge Dr. Barbara Rellahan for the p50 and p65 NF-kappa B vectors.

    FOOTNOTES

* This work was supported by Medical Research Materiel Command of the United States Army STEP C and by United States Public Health Service Grant RO1 49954.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.

§§ To whom correspondence should be addressed: Dept. of Cellular Injury, Walter Reed Army Inst. of Research, Bldg. 503, Rm. 1A32, Robert Grant Ave., Silver Spring, MD 20910. Tel.: 301-319-9911; Fax: 301-319-9133; E-mail: gtsokos@usa.net.

Published, JBC Papers in Press, February 16, 2003, DOI 10.1074/jbc.M300787200

    ABBREVIATIONS

The abbreviations used are: iNOS, inducible nitric-oxide synthase; KLF, Krüppel-like factor; TGF, transforming growth factor; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; PMA, phorbol 12-myristate 13-acetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; STAT, signal transducers and activators of transcription.

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