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Originally published In Press as doi:10.1074/jbc.M509378200 on September 16, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38247-38258, November 18, 2005
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Kruppel-like Factor 4 Is a Mediator of Proinflammatory Signaling in Macrophages*

Mark W. Feinberg1, Zhuoxiao Cao, Akm Khyrul Wara, Maria A. Lebedeva, Sucharita SenBanerjee, and Mukesh K. Jain

From the Program in Cardiovascular Transcriptional Biology, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, August 25, 2005 , and in revised form, September 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-{gamma} (IFN-{gamma}), lipopolysaccharide (LPS), or tumor necrosis factor-{alpha} can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-{beta}1 (TGF-{beta}1) can decrease proinflammatory activation. The molecular mediators regulating the balance of these opposing effectors remain incompletely understood. Herein, we identify Kruppel-like factor 4 (KLF4) as being markedly induced in response to IFN-{gamma}, LPS, or tumor necrosis factor-{alpha} and decreased by TGF-{beta}1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-{beta}1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-{gamma}, LPS, or IFN-{gamma} plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-{beta}1 and Smad3 signaling. The KLF4 induction of the iNOS promoter is mediated by two KLF DNA-binding sites at –95 and –212 bp, and mutation of these sites diminished induction by IFN-{gamma} and LPS. We further provide evidence that KLF4 interacts with the NF-{kappa}B family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-{beta}1/Smad3 induction of the PAI-1 promoter independent of KLF4 DNA binding through a novel antagonistic competition with Smad3 for the C terminus of the coactivator p300/CBP. These findings support an important role for KLF4 as a regulator of key signaling pathways that control macrophage activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Macrophage activation is an integral process in the development of atherosclerosis as well as a number of other chronic inflammatory diseases such as emphysema, inflammatory bowel disease, psoriasis, arthritis, and pancreatitis. Once within the site of inflammation, macrophages elaborate a broad range of cytokines, growth factors, and proteolytic enzymes that may participate in the damage and repair that ensues. Identification of mechanisms that may regulate macrophage activation is, thus, of considerable interest.

One of the key events in macrophage response to proinflammatory stimuli is the expression of inducible nitric-oxide synthase (iNOS)2 and the formation of nitric oxide, an important mediator involved in many host defense actions in macrophages. However, increased amounts of leukocyte-derived nitric oxide can be detrimental by promoting tissue damage in a variety of inflammatory disease states (1, 2). Given the importance of iNOS in a variety of pathophysiological conditions, control of its expression has been the subject of considerable investigation (1). Indeed, several studies have shown that induction or inhibition of iNOS in macrophages by pro- or anti-inflammatory stimuli, respectively, can occur at the level of transcription. For example, proinflammatory stimuli such as LPS or IFN-{gamma} involve activation of NF-{kappa}B or interferon-responsive elements, respectively, on the mouse iNOS promoter, whereas anti-inflammatory mediators such as TGF-{beta}1 can inhibit proinflammatory stimuli by coactivator competition (3, 4). Thus, identification of factors that alter such signaling pathways and, in turn, iNOS expression may offer novel strategies to regulate the inflammatory response in macrophages and diverse inflammatory disease states.

TGF-{beta}1 is a pleiotropic growth factor with potent immunomodulating cellular effects. For example, both experimental and clinical data support a role for TGF-{beta}1 as a regulator of immune responses in the development of atherosclerosis. The observation that patients with advanced atherosclerosis have significantly reduced levels of active TGF-{beta}1 by comparison with patients with angiographically normal coronary arteries supports a role for TGF-{beta}1 in atherogenesis (5). Low levels of TGF-{beta}1 may result in an excessive inflammatory milieu accelerating atherogenesis (6). Indeed, inhibition of endogenous TGF-{beta}1 signaling in mice accelerated atherosclerotic lesion formation characterized by increased macrophages and T cells and decreased collagen content (7, 8). Cellular signaling through the TGF-{beta} superfamily occurs via intracellular mediators, termed Smads, which translocate to the nucleus, where they direct transcriptional responses. Three classes of Smads (pathway-restricted, common, and inhibitory) are responsible for propagating the downstream signaling effects. TGF-{beta}/activin receptors phosphorylate the pathway-restricted Smads, Smad2 and Smad3, whereas bone morphogenic protein receptors activate Smad1, Smad5, and Smad8. Pathway-restricted Smads may hetero-oligomerize with the only common Smad, Smad4, before translocating to the nucleus. The inhibitory Smads, Smad6 and Smad7, are structurally divergent from other Smads and function to block TGF-{beta} signaling by preventing ligand-induced receptor phosphorylation of pathway-restricted Smads. We have previously identified a critical role for Smad3 in suppressing macrophage markers of inflammation such as iNOS, matrix metalloelastase (MMP)-12, and chemokine monocyte chemoattractant protein-1 (MCP-1) (3, 4, 9). We have also shown that cardiac allografts transplanted into Smad3-deficient mice develop accelerated intimal hyperplasia with increased infiltration of macrophages in vivo (3). Therefore, perturbation of TGF-{beta}1 and/or Smad3 signaling may significantly regulate vascular inflammation.

Interferon (IFN)-{gamma} is a pleiotropic cytokine produced by a number of cell types found within the atherosclerotic lesion, including macrophages, T cells (Th1 helper cells), and natural killer (NK) cells. In response to IFN-{gamma}, macrophages express HLA-DR, which allows them to present antigens to T lymphocytes and amplify the inflammatory response (10–14). Consequently, IFN-{gamma} is a potent proinflammatory mediator in a variety of vascular inflammatory disease states. For example, both clinical and experimental studies support a role for IFN-{gamma} in promoting atherosclerotic lesion formation and transplantation arteriosclerosis (15, 16). In animal atherosclerotic models, exogenous administration of IFN-{gamma} accelerates atherosclerosis and is associated with an unstable plaque phenotype (17–19). Conversely, mice deficient in IFN-{gamma} (2, 20, 21) or neutralizing antibodies to IFN-{gamma} (20, 22) demonstrate significantly less neointima formation in mouse models of transplantation arteriosclerosis. In most cells, IFN-{gamma} signals through the Jak-Stat pathway to induce transcriptional responses (23, 24). In response to IFN-{gamma}, receptor-bound Stat1{alpha} becomes tyrosine-phosphorylated and translocates to the nucleus as a homodimer, where it may bind directly to palindromic {gamma}-activated sites of IFN-{gamma} target gene promoters or with other interacting proteins. In addition to mediating downstream proinflammatory effects, Stat1{alpha} is also capable of inhibiting anti-inflammatory signaling pathways such as TGF-{beta}1. Thus, downstream targets of Stat1{alpha} may also be important for carrying out this dual role.

Kruppel-like factors (KLFs) are a subclass of the zinc finger family of transcription factors characterized by the DNA binding domain containing the conserved sequence CX2CX3FX5LX2HX3H (underlined cysteine and histidine residues coordinate zinc). The three zinc fingers are usually found at the C terminus of the protein and bind to either a CACCC element or GC-box. The N terminus is involved in transcriptional activation and/or repression as well as protein-protein interaction (25–27). Gene targeting studies demonstrated that KLF proteins typically regulate critical aspects of cellular development, differentiation, and activation. For example, KLF1 (EKL erythroid Kruppel-like factor) has been shown to be essential in red blood cell maturation, and KLF2 (LKLF; lung Kruppel-like factor) is thought to play an important role in programming the quiescent phenotype of single-positive T cells (28, 29). KLF4 (GKLF; gut Kruppel-like factor) was first identified in the epithelial lining of the gut and skin, and subsequent studies have shown it to play a role in the regulation of cellular growth and differentiation in these tissues (30, 31). In vitro studies suggest that KLF4 is also an IFN-{gamma} target gene in colonic carcinoma cell lines, an effect mediated by Stat1{alpha} binding to a {gamma}-activated site element of the KLF4 promoter (32). Recently, Noti et al. (33) found that KLF4 can regulate the macrophage differentiation marker CD11d. However, a role for KLF4 in regulating macrophage activation remains unknown.

In this report, we identified KLF4 in macrophages as being induced by proinflammatory stimuli such as IFN-{gamma}, LPS, and TNF-{alpha} and inhibited by the anti-inflammatory growth factor, TGF-{beta}1. Our studies demonstrate that KLF4 plays a key role in mediating the proinflammatory effects by IFN-{gamma} or LPS on the induction of the macrophage activation marker iNOS, an effect mediated, in part, through a novel interaction with p65 (RelA), a member of the NF-{kappa}B family of transcription factors. In contrast, KLF4 inhibits TGF-{beta}1/Smad3 function by competing with Smad3 for the C terminus of the coactivator p300. These findings demonstrate that KLF4 plays a critical role in mediating proinflammatory responses in macrophages and may be an important target for modulating TGF-{beta}1/Smad3 effects.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
REFERENCES
 
Cell Culture and Reagents—J774a, THP-1, and RAW264.7 cells were grown as recommended (American Type Culture Collection). Human peripheral blood-derived monocytes were isolated by the Ficoll-Paque centrifugation technique and grown in Iscove's modified Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% human serum type AB (Sigma), penicillin, and streptomycin as previously described (3). We obtained LPS (salmonella typhi) from Sigma and active IFN-{gamma}, TNF-{alpha}, and TGF-{beta}1 from R&D Systems. The mouse fibroblast STAT1+/+ and STAT1–/– cell lines derived from wild-type and STAT1 knock-out mice, respectively, were a generous gift from Dr. David Levy (New York University Medical Center). The Drosophila Schneider cell line, SL2, was obtained and grown as recommended from ATCC.

RNA Extraction and RNA Blot Analysis—Total RNA was isolated from cultured cells by Trizol reagent (Invitrogen) as described by the manufacturer. RNA was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose filters. The filters were hybridized with 32P-labeled, random-primed cDNA probes, washed, and exposed as described previously (3). Mouse and human cDNA probes for KLF4 were generated by reverse transcription-PCR. Mouse cDNA probes for iNOS and PAI-1 were generated as previously described (3, 4). The Smad3 and Smad7 cDNA probes were generated from plasmids kindly provided by R. Derynck (University of California, San Francisco).

Western Analyses and Immunoprecipitation Studies—Western blot analyses were performed as described (3). Immunoblots were incubated with the appropriate primary antibodies for KLF4 (active motif), Smad3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-Smad3 (kindly provided by A. Moustakas, Ludwig Institute for Cancer Research, Sweden), or IgG1 control antibody (Sigma) and were detected using horseradish peroxidase secondary antibodies and chemiluminescence. For immunoprecipitation assays, 293T cells were transfected with the indicated expression plasmids and harvested with radioimmune precipitation assay buffer 48 h later. Lysates were subjected to immunoprecipitation with 4 µg of {alpha}-FLAG M2 monoclonal antibody (Sigma) at 4 °C for 2 h followed by incubation with protein A/G-Sepharose overnight at 4 °C. The beads were washed, and proteins were separated by SDS-PAGE as previously described (3).

PAI-1 and Nitrite Measurements—PAI-1 secretion was measured by ELISA according to the recommendation of the manufacturer (American Diagnostica). The generation of nitrite was used as an indicator of NO release by J774a cells and measured by the Griess reaction according to the manufacturer's recommendations (Promega). Briefly, the medium was centrifuged at 2000 rpm for 10 min at 4 °C to remove cellular debris. The nitrite content in the supernatant was measured by incubating medium with Griess reagent (sulfanilamide and N-1-napthylethylenediamine dihydrochloride) under acidic (phosphoric acid) conditions. The concentration of the resultant chromophore was determined spectrometrically at 540 nm and calculated from a sodium nitrite standard reference curve.

Transient Transfections—RAW264.7 cells were transfected with FugeneTM6 Transfection Reagent (Roche Applied Science) as described (3). The total amount of plasmid DNA was kept constant within each experiment. Luciferase activity was normalized to {beta}-galactosidase activity (to correct for differences in transfection efficiency) by cotransfecting the pCMV-{beta}gal plasmid in all experiments. All transfections were performed in triplicate from at least three independent experiments. The expression plasmids for Smad3, p300, constitutively active TGF-{beta} receptor type I, and the 3TP-Lux reporter have been described previously (3, 4). Expression plasmids for full-length KLF4, KLF4-FLAG, KLF4{Delta}ZnF, and ZnF were generated by reverse transcription-PCR and cloned into pcDNA3.1, and authenticity was verified by sequencing and Western analyses (data not shown). The PAI-1 promoter construct was obtained from A. Garzino-Demo (University of Maryland) and D. Vaughan (Vanderbilt), respectively. Generation of the pGL2 iNOS promoter constructs (–1485/+31, –226/+31, and –69/+31) has been described previously (4, 34). Site-directed mutagenesis (Stratagene) was performed to generate the following pGL2 constructs: iNOS (–105/+31), iNOS (MUT–212), iNOS (MUT–95), and iNOS (MUT–212 and –95). The AP-1 concatamer construct was obtained from Stratagene. For transfection of Drosophila Schneider SL2 cells, Cellfectin reagent was used as described by the manufacturer (Invitrogen). Luciferase values were normalized to total protein. Expression plasmids for pPAC-O and pPAC-KLF4 were kindly provided by K. Bomsztyk (University of Washington), and pPAC-p50 and pPAC-p65 were kindly provided by M. Perrella (Harvard Medical School).

Adenoviral and Retroviral Infections—Adenoviral constructs for KLF4 or empty virus control both encode green fluorescent protein (GFP) and were generated by the Harvard Gene Therapy Group. The GFP and KLF4 are expressed as separate proteins driven by a bidirectional cytomegalovirus promoter. For adenoviral infection of J774a cells, cells were seeded at 2 x 106/10-cm2 dish, infected with the adenoviral vectors at 200–1000 multiplicity of infection (MOI), and incubated overnight or as indicated in the presence of IFN-{gamma}. Transduction efficiencies were typically 60–70% as measured by GFP positivity and fluorescence-activated cell sorter analyses. For retroviral studies, the indicated cDNA was cloned into the retroviral vector green fluorescent protein-RV (gift K. Murphy), and retroviruses were generated as described (35). For retroviral infection of target cells, retroviral supernatant and culture medium (10% fetal calf serum/Dulbecco's modified Eagle's medium plus 8 µg/ml Polybrene) were mixed at a 1:1 ratio and added to preconfluent cells as described (36). Transduction efficiencies were typically >95% as measured by GFP positivity and fluorescence-activated cell sorting.

KLF4 Knockdown Experiments—Morpholino antisense oligonucleotides were generated to the KLF4 mRNA (AS-KLF, 5'-ACAGCCATGTCAGACTCGCCAGGTG-3') and prepared as described by the manufacturer (GeneTools, Philomath, OR). A standard control morpholino oligonucleotide (nonspecific control) was used as a negative control and has been described elsewhere, GeneTools (NS, 5'-CCTCTTACCTCAGTTACAATTTATA-3', GeneTools). Briefly, cells were transfected with the ethoxylated polyethyleneimine reagent and AS-KLF4 or nonspecific control morpholino oligonucleotides at equimolar concentrations (1.4 µM) at room temperature for 20 min in serum-free conditions. After 3 h, cells were replaced with medium containing 10% fetal bovine serum. After 24 h, transient transfection of the indicated plasmids was performed.

Generation of Glutathione S-Transferase Fusion Protein and Pull-down Assays—Glutathione S-transferase (GST) fusion protein for KLF4 was generated using the pGEX-4T-1 vector according to the manufacturer's recommendations (Amersham Biosciences). GST pull-down assays were performed as described previously by incubating whole cell extract from 293T cells expressing full-length KLF4 or pcDNA3 (37). GST fusion proteins containing different domains of p300 were obtained from Y. Shi (Harvard Medical School, Boston, MA).

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift analyses were performed using nuclear extracts from 293T cells or GST-KLF4 as previously described (3). Radiolabeled DNA probes were generated to the Smad binding element at positions –688 to –660 bp of the PAI-1 promoter as doublestranded oligonucleotides. Supershift antibodies for Smad3 (sc-8332), FLAG, or IgG1 were incubated with nuclear extracts for 1 h at 4 °C prior to adding the radiolabeled oligonucleotide (38). DNA probes were also generated to the KLF sites at positions –95 and –212 bp of the mouse iNOS promoter as double-stranded, radiolabeled oligonucleotides corresponding to the wild-type sequences (5'-TGCACACCCA-3' and 5'-CTGCCTAGGGGCCACTG-3', respectively) and mutant sequences (5'-TGCATACTTA-3' and 5'-CTGCCTAGTGTCCACTG-3', respectively). The radiolabeled DNA probes to the NF-{kappa}B site at positions –85 to –76 bp of the iNOS promoter were generated as described (39).

Statistics—Data are expressed as mean ± S.E. For comparison between two groups, the unpaired Student's t test was used. For multiple comparisons, analysis of variance followed by unpaired Student's t test was used. A value of p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
REFERENCES
 
Identification of KLF4 in Monocytes/Macrophages and Responsiveness to IFN-{gamma} and Cytokines—In light of the acknowledged role for KLFs in red blood cell and T cell biology, we hypothesized that members of this family may also play a role in macrophage biology. Using the conserved zinc finger region of KLF1 as a probe, we performed a low stringency homology screen of a rat monocyte/macrophage cDNA library. Four clones encoded the partial sequence of a previously identified KLF termed KLF4. This factor has been shown to regulate epithelial cell differentiation and inhibit cellular growth (30, 31, 40). A role for this factor in hematopoietic biology has only recently been appreciated (33). One of the hallmarks of an activated macrophage is its ability to respond to inflammatory cytokines. To assess the expression of KLF4 in response to cytokines such as IFN-{gamma}, several macrophage cell lines and primary human macrophages were examined. As shown in Fig. 1, KLF4 mRNA is markedly induced after only 1 h of stimulation with IFN-{gamma} in J774a or THP-1 cells and in a dose-dependent manner in peripheral blood monocyte-derived human macrophages (Fig. 1, A–C). IFN-{gamma} also induced KLF4 protein after only 1 h of stimulation in J774a cells (Fig. 1A, right). To verify that the IFN-{gamma} mediated induction of KLF4 was Stat1-dependent, we examined Stat1+/+ and Stat1–/– cells. As shown in Fig. 1D, IFN-{gamma} increased KLF4 mRNA expression in Stat1+/+ cells but was severely reduced in Stat1–/– cells. KLF4 is also induced after 1 h of stimulation with TNF-{alpha} or LPS (Fig. 1E). In contrast, treatment with the anti-inflammatory growth factor, TGF-{beta}1, inhibits KLF4 basal expression (Fig. 1F). Taken together, these data indicate that KLF4 expression may be regulated by opposing signaling pathways, which modulate the inflammatory response in macrophages.

KLF4 Induces the Macrophage Activation Marker iNOS—Because KLF4 was induced by IFN-{gamma}, LPS, and TNF-{alpha}, we considered the possibility that this transcription factor may regulate the expression of cytokine-responsive genes. In cells overexpressing KLF4, we observed a marked induction on the expression of iNOS, a cytokine-responsive gene that is transcriptionally regulated and a key marker of macrophage activation (Fig. 2, A and B). Furthermore, in the presence of IFN-{gamma}, KLF4 strongly augmented the iNOS induction (Fig. 2A). To determine whether this induction of iNOS translates into an effect on NO production, we assessed for production of nitrite, as a measure of NO synthesis. As shown in Fig. 2C, in comparison with J774a cells adenovirally infected with EV (Ctrl), cells overexpressing KLF4 induced nitrite formation by ~3.5-fold.



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  		FIGURE 1.
KLF4 expression in macrophages in response to cytokines. A, J774a cells were treated with IFN-{gamma} (400 IU/ml) for 1 h, and Northern (10 µg of total RNA/lane) and Western analyses (50 µgof total protein/lane) were performed. B, induction of KLF4 mRNA in THP-1 cells. Total RNA was harvested for Northern analysis after THP-1 cells were treated with IFN-{gamma} (400 IU/ml) at the indicated time points. C, induction of KLF4 mRNA in primary macrophages. Human peripheral blood-derived macrophages were isolated and stimulated with IFN-{gamma} for 1 h with the indicated concentration of IFN-{gamma}. D, KLF4 is not induced by IFN-{gamma} in STAT1–/– cells. Northern analysis was performed from total RNA from STAT1+/+ and STAT1–/– cells after stimulation for 1 h with IFN-{gamma} (400 IU/ml). E and F, differential effects of cytokines and TGF-{beta}1 on KLF4 mRNA expression. THP-1 cells were treated with IFN-{gamma} (400 IU/ml), TNF-{alpha} (10 ng/ml), LPS (25 ng/ml), or TGF-{beta}1 (10 ng/ml) as indicated for 6 h, and total RNA was harvested for Northern analyses. KLF4 expression is induced by IFN-{gamma}, TNF-{alpha}, or LPS, whereas TGF-{beta}1 inhibits KLF4 expression. EtBr or {alpha}-tubulin is shown to verify equal loading on Northern or Western analyses, respectively.

 



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  		FIGURE 2.
KLF4 induces iNOS expression. A and B, Northern analysis of total RNA derived from cells that were either retrovirally infected with EV control (GFP-RV) or KLF4 (KLF4-GFP) in NIH 3T3 fibroblasts (A) or adenovirally infected with control (Ad-GFP (EV); 200 MOI) or Ad-KLF4 (200 MOI) in J774a cells (B) for 24 h. Induction of iNOS mRNA is observed in KLF4-overexpressing cells and is markedly enhanced in response to IFN-{gamma} (400 IU/ml) for 8 h (A). KLF4 (exo), exogenous KLF4 expression. C, KLF4 induces nitrite activity. J774a cells were adenovirally infected as described above, and a Griess reaction was performed to assay for nitrite production in conditioned medium. n = 3/group; *, p < 0.005. D, KLF4 transactivates the iNOS promoter. Transient transfection studies using the –1.5 kb-iNOS-Luc promoter and the indicated KLF. In contrast to other KLFs, KLF4 is able to transactivate the iNOS promoter. A representative of three independent experiments is shown for all transient transfection experiments. n = 3–6/group; *, p < 0.0001. E, structural basis for KLF4 induction of iNOS promoter. The various KLF4 expression plasmids were tested for their ability to transactivate the iNOS promoter. The induction of iNOS requires a fully intact KLF4, since neither the DNA-binding domain (ZnF) nor the non-DNA-binding domain (KLF4{Delta}ZnF) alone is capable of affecting promoter activity. n = 3–6/group; *, p < 0.0001. DBD, DNA binding domain.

 
KLF4 Regulates the iNOS Promoter—KLFs are transcription factors that bind DNA and thereby regulate the expression of various target genes. As a first step toward understanding how KLF4 can induce iNOS, we assessed its effect on iNOS promoter activity. For these studies, we used a –1.5 kb fragment of the mouse iNOS promoter. This promoter contains all of the regulatory elements necessary for cytokine responsiveness in vivo (41). As shown in Fig. 2D, this effect was specific, since KLF4 but not two other family members (KLF5 and KLF15) potently transactivated the iNOS promoter. Furthermore, neither the DNA-binding domain (ZnF) nor the non-DNA-binding region (KLF4{Delta}ZnF) alone was able to induce the iNOS promoter, suggesting that this induction required intact KLF4 (Fig. 2E).

To determine the region within the iNOS promoter that is responsible for KLF4-mediated induction, we assessed effects of KLF4 on the –1.5 kb, –226 bp, and the –69 bp iNOS promoter. As demonstrated in Fig. 3A, KLF4 was able to potently induce both the full-length and –226 bp iNOS promoter. However, this transactivation was almost completely lost upon further deletion up to the –69 bp construct. These data suggest that the critical regulatory elements necessary for mediating KLF4 effects on the iNOS promoter lie within a 157-base pair region. Examination of this region reveals potential KLF DNA binding sites at –95 and –212 bp (Fig. 3A). To assess the importance of these sites, we performed site-directed mutagenesis and examined the ability of KLF4 to induce the iNOS promoter. As shown in Fig. 3B, mutation of the KLF site at either –95 or –212 partially attenuated the ability of KLF4 to induce the iNOS promoter by ~36 and ~58%, respectively. However, mutation of both KLF sites markedly prevented KLF4 induction of the iNOS promoter by ~78% (Fig. 3B). To determine whether these sites are capable of binding KLF4, we performed gel shift studies using a GST-KLF4 fusion protein. As shown in Fig. 3C, KLF4 bound to each of these sites, and specificity was verified by using mutant radiolabeled oligonucleotides to which KLF4 did not bind and by supershift studies. In addition, the induction of the iNOS promoter bearing mutation of these two KLF sites was attenuated in response to IFN-{gamma}, LPS, or IFN-{gamma} plus LPS (~74, ~76, or ~80%, respectively) (Fig. 3D). Conversely, transient transfection with KLF4 enhanced iNOS responsiveness to IFN-{gamma}, LPS, and IFN-{gamma}+LPS (8.1-, 2.7-, and 2.0-fold, respectively) (Fig. 3E) in comparison with pcDNA3. Finally, to examine the relative contribution of endogenous KLF4 to cytokine induction of the iNOS promoter, we performed knockdown experiments using morpholino antisense oligonucleotides to KLF4 (AS-KLF4). We first verified knockdown of endogenous KLF4 in RAW cells transfected with nonspecific KLF4 or AS-KLF4 (Fig. 3F, inset); AS-KLF4 resulted in a ~45% knockdown of endogenous KLF4 mRNA expression. In comparison with the nonspecific control, AS-KLF4 reduced the induction of the iNOS promoter by IFN-{gamma}, LPS, or IFN-{gamma} plus LPS (~64, ~63, or ~65%, respectively) (Fig. 3F). Taken together, these data indicate that KLF4 can induce iNOS expression through KLF sites in the proximal iNOS promoter, and mutation of these sites or knockdown of KLF4 markedly decreases IFN-{gamma} and LPS responsiveness.



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  		FIGURE 3.
Identification and function of KLF4 DNA-binding sites in the iNOS promoter. A, identification of KLF4 binding sites within the iNOS promoter. The indicated iNOS promoter deletion constructs were used, and the ability of KLF4 to transactivate was assessed by transient transfection studies in RAW cells. KLF4 transactivation is lost with deletion from –226 to –69 bp, n = 3–6/group; *, p < 0.0002. B, site-directed mutagenesis of the KLF sites alone or together markedly reduced the ability of KLF4 to induce the iNOS promoter. n = 3–6/group; *, p < 0.00007; **, p < 0.00009; #, p < 0.000008. C, assessment of KLF4 binding. The potential KLF sites at –95 and –212 were assessed for the ability to bind KLF4 by gel shift studies using purified GST-KLF4 protein. KLF4 is able to bind to a radiolabeled wild-type (WT) iNOS KLF sites at –95 (left) and –212 (right) but not a mutated (MUT) site. D, effect of mutation of the KLF4 DNA-binding sites on cytokine responsiveness of the iNOS promoter. RAW cells were transiently transfected with the wild-type –226 iNOS promoter or the mutant (MUT) iNOS promoter bearing mutations of the KLF sites followed by stimulation with control (Ctrl), IFN-{gamma}, LPS, or IFN-{gamma} + LPS for 18 h. Mutation of the KLF4 DNA-binding sites markedly reduced induction by each of these cytokines. n = 3–6/group; *, p < 0.0004; **, p < 0.000016; #, p < 0.00001; ##, p < 0.00000003. E, the iNOS promoter is more cytokine responsive in the presence of KLF4. RAW cells were transiently transfected with pcDNA3 or KLF4 in the presence or absence of control, IFN-{gamma}, LPS, or IFN-{gamma} + LPS. n = 3–6/group; *, p < 0.0004; **, p < 0.0003; #, p < 0.000004; ##, p < 0.00003. F, effect of knockdown studies of endogenous KLF4 on cytokine responsiveness of the iNOS promoter. RAW cells were transiently transfected with nonspecific KLF4 (NS-KLF4) or AS-KLF4 morpholino oligonucleotides for 24 h followed by cytokine stimulation for 18 h. KLF4 knockdown markedly reduced cytokine responsiveness of the iNOS promoter. n = 3–6/group; *, p < 0.002; **, p < 0.0009; #, p < 0.007. Inset, KLF4 expression in RAW cells by Northern analysis in the presence of nonspecific KLF4 or AS-KLF4. EtBr is shown to verify equal loading.

 
Cooperative Induction of the iNOS Promoter by KLF4 and p65—The proximal NF-{kappa}B site of the iNOS promoter has been shown to be critical for LPS inducibility (1). Because the KLF4 DNA-binding sites are in close proximity to this NF-{kappa}B site and KLF4 knockdown resulted in a decrease in LPS induction of the iNOS promoter, we examined whether a relationship existed between KLF4 and p65. To assess this, we first used an iNOS promoter construct bearing a mutation of this NF-{kappa}B site (iNOS pro-NF-{kappa}B-Mut) and co-transfected pcDNA3, KLF4, or p65 in the presence or absence of LPS. As expected, in the presence of the iNOS pro-NF-{kappa}B-Mut, the p65 induction was potently repressed (Fig. 4A). Interestingly, there was also a marked reduction in the ability of KLF4 to induce the iNOS pro-NF-{kappa}B-Mut by ~60% in the presence or absence of LPS. Conversely, the induction by p65 on the iNOS promoter bearing mutation of the KLF sites (iNOS pro-Mut KLF) was also significantly impaired by ~40% in the presence or absence of LPS, suggesting that both sites are important for optimal iNOS responsiveness to LPS (Fig. 4B). One possible explanation for these observations is that p65 and KLF4 may interact with each other. To test this, we co-transfected 293T cells and performed co-immunoprecipitation studies with pcDNA3, KLF4-Myc, or p65-FLAG. As shown in Fig. 4C, an {alpha}-Myc antibody immunoprecipitated p65 from lysates transfected with FLAG-p65 and KLF4-Myc but not with FLAG-p65 + pcDNA3 or KLF4-Myc + pcDNA3. Conversely, an {alpha}-FLAG antibody immunoprecipitated KLF4 from lysates transfected with FLAG-p65 and KLF4-Myc as well (Fig. 4D). To assess whether KLF4 could functionally augment the p65 induction of the iNOS promoter, we performed co-transfection studies in the Drosophila Schneider SL2 cells, a heterologous cell system deficient in p50, p65, and KLF4. Indeed, we found that KLF4 and p50/p65 cooperatively induced the iNOS promoter (Fig. 4E). Finally, to assess whether KLF4 affects NF-{kappa}B DNA-protein binding, we performed gel shift studies using J774a cells and adenovirus for EV (control) or KLF4. As shown in Fig. 4F, stimulation of J774a cells with LPS markedly induced an NF-{kappa}B DNA-protein complex containing p50 and p65; however, overexpression of KLF4 had no effect on this induction. Taken together, these results suggest that KLF4 and p65 interact to cooperatively induce the iNOS promoter.

KLF4 Inhibits TGF-{beta}1 Induction of PAI-1 mRNA and Promoter Activity—The above data indicate that KLF4 is important for promoting expression of proinflammatory genes such as iNOS. As shown in Fig. 1F, KLF4 mRNA expression is inhibited by the anti-inflammatory growth factor, TGF-{beta}1. To determine whether KLF4 can regulate this signaling pathway, we first assessed effects of KLF4 overexpression on the TGF-{beta} target gene, PAI-1. As shown in Fig. 5, A and B, overexpressing KLF4 cells potently blocked both basal and TGF-{beta}1-induced PAI-1 mRNA expression and activity. Similar KLF4 inhibitory effects were verified on the –800 bp PAI-1 promoter, a promoter well characterized for its ability to respond to TGF-{beta} effectors, Smads. As shown in Fig. 5C, KLF4 potently repressed the induction by TGF-{beta}1, Smad3, or the combination of TGF-{beta}1 and Smad3. In addition, we found that this inhibition is not dependent on DNA binding, since both the full-length and non-DNA binding domain constructs (KLF4{Delta}ZnF) are capable of inhibiting promoter activity (Fig. 5D). However, inhibition was lost with the DNA-binding domain alone (ZnF). Consistent with this observation, using the 3TP-Lux promoter, which contains three Smad binding elements from the PAI-1 promoter and lacks any KLF binding sites, KLF4 still potently repressed induction by Smad3 and TGF-{beta}1 (Fig. 5E). Finally, because overexpression of KLF4 inhibited TGF-{beta}1 and Smad3 function, we hypothesized that KLF4 knock-down would enhance TGF-{beta}1/Smad3 induction of the PAI-1 promoter. As shown in Fig. 5F, in comparison with the nonspecific control oligonucleotide, AS-KLF4 markedly enhanced the ability of TGF-{beta}1 and Smad3 to induce the PAI-1 promoter. Taken together, these data indicate that KLF4 is an important negative regulator of TGF-{beta}1/Smad3 signaling.

Effect of KLF4 on Smad3 DNA Binding, Expression, and Phosphorylation—Inhibition of TGF-{beta}1/Smad3 signaling may occur on several levels such as by affecting receptor-activated Smad phosphorylation, Smad expression, or DNA binding. To examine whether overexpression of KLF4 affects Smad3 DNA binding, we performed gel shift studies in 293T cells. As shown in Fig. 5G, increasing amounts of KLF4 had no effect on Smad3 DNA binding. We also assessed whether KLF4 overexpression affected Smad3 or Smad7 expression in the presence or absence of TGF-{beta}1. Consistent with its known effects (42–44), TGF-{beta}1 increased Smad7 and decreased Smad3 expression (Fig. 5H); however, KLF4 overexpression had no effect on the expression of these factors (Fig. 5H). To examine whether KLF4 overexpression had any effect on Smad3 phosphorylation, we performed co-immunoprecipitation studies in 293T cells. As shown in Fig. 5I, an {alpha}-FLAG antibody immunoprecipitated phospho-Smad3 in the presence of a constitutively active TGF-{beta} type I receptor; however, the presence of KLF4 had no effect on the TGF-{beta} receptor type I-induced Smad3 phosphorylation. Taken together, these data indicate that KLF4 had no effect on Smad3 DNA binding, expression, or phosphorylation and indicate that KLF4 may inhibit the downstream effects of TGF-{beta}1 by modulating Smad3 co-activators.

Recruitment of the Co-activator p300/CBP by KLF4—The studies above demonstrate that KLF4 is able to induce iNOS and inhibit TGF-{beta}1 activity. The former requires direct DNA binding, whereas the latter does not (Figs. 3 and 5). Furthermore, the inhibition occurs in the absence of any effect on Smad3 expression, DNA binding, or phosphorylation (Fig. 5, G–I). To reconcile these observations, we considered the possibility that KLF4 may interfere with Smad3 activity by interacting with a critical Smad3 co-activator. One possible consequence of this interaction is that KLF4 may recruit away from Smad3 a critical co-activator, perhaps to its target genes such as iNOS. Smad3 is critically modulated by several co-activators such as p300/CBP and P/CAF (45, 46). We chose to focus on p300/CBP because of its critical role in regulating transcriptional activity and the fact that KLF4 and several other KLF members have been found to interact with this co-activator (37, 47–49). Consistent with the proposed hypothesis, the addition of p300 in transient transfection studies rescued the KLF4-mediated repression of the PAI-1 promoter (Fig. 6A). Furthermore, co-transfection of KLF4 and p300 augmented iNOS promoter activity (Fig. 6B). In addition, induction of iNOS by KLF4 is inhibited by the E1A oncoprotein (Fig. 6C), a viral protein that modulates several cellular proteins including p300/CBP (50, 51). Moreover, the E1A inhibition of KLF transactivation is lost with deletion of the conserved region 1 (E1A{Delta}CR1) (Fig. 6C), the region previously shown to bind p300/CBP (52–54). To examine which domain of p300 interacts with KLF4, GST binding studies were undertaken. In Fig. 6D, we found that KLF4 can bind to GST-p300C. In contrast, KLF4 was unable to bind GST-p300N, GST-p300M, or GST alone. Because Smad3 can also bind to the C terminus of p300 (55–57), we hypothesized that KLF4 may compete with Smad3 for this region of p300. Consistently, increasing amounts of KLF4 reduced the ability of Smad3 to bind GST-p300C (Fig. 6E). Interestingly, increasing amounts of Smad3 failed to decrease the binding of KLF4 to GST-p300C (Fig. 6E), an effect suggesting that the affinity of KLF4 for p300C is probably higher than the affinity of Smad3 for p300C. To determine whether the Smad3-mediated inhibition of the iNOS promoter is attenuated in the presence of KLF4, we co-transfected the iNOS promoter along with pcDNA3, KLF4, or Smad3 in the presence of IFN-{gamma} or TGF-{beta}1. Whereas TGF-{beta}1 and Smad3 could potently repress the induction of the iNOS promoter in the presence of pcDNA3, this inhibition was abolished in the presence of KLF4 or KLF4 + IFN-{gamma} (Fig. 6F). In contrast, KLF4 and IFN-{gamma} repressed the TGF-{beta}1 and Smad3 induction of the PAI-1 promoter (Fig. 6G). Taken together, these data support the hypothesis that the KLF4 interaction with the co-activator p300 may be an important mechanism by which KLF4 is able to inhibit certain target genes and induce others.



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  		FIGURE 4.
KLF4 and p65 interact to cooperatively induce the iNOS promoter. A, mutation of the NF-{kappa}B site attenuates KLF4 induction of the iNOS promoter. RAW cells were transiently transfected with the wild-type (WT) iNOS promoter or NF-{kappa}B mutant (Mut NF-{kappa}B) iNOS promoter and either pcDNA3, p65, or KLF4 in the presence or absence of LPS. n = 3–6/group; *, p < 0.00005; **, p < 0.0000008; ***, p < 0.0001; #, p < 0.00002; ##, p < 0.0002; ###, p < 0.0002. B, mutation of the KLF sites attenuates p65 induction of the iNOS promoter. RAW cells were transiently transfected with the wild type or the –212/–95 KLF mutant (Mut KLF) iNOS promoter and either pcDNA3, p65, or KLF4 in the presence or absence of LPS. n = 3–6/group; *, p < 0.004; **, p < 0.00003; ***, p < 0.03; #, p < 0.0005; ##, p < 0.02; ###, p < 0.002. C and D, KLF4 associates with p65. 293T cells were transfected with the indicated expression plasmids. Whole-cell lysates were immunoprecipitated (IP) with either an {alpha}-Myc (C)oran {alpha}-FLAG (D) antibody followed by immunoblotting (IB) with the indicated antibodies. Immunoblotting was also performed on prelysates to verify relative expression levels of p65-FLAG and KLF4-Myc. E, KLF4 and p65 cooperatively induce the iNOS promoter. Drosophila Schneider SL2 cells were transfected with either pPAC-p50, pPAC-p65, or pPAC-KLF4 along with the iNOS promoter. Total DNA was kept constant by transfecting the empty vector pPAC-O. KLF4 augments p50, p65, or p50 + p65 iNOS induction. n = 3/group; *, p < 0.0004; **, p < 0.00002; #, p < 0.0008; ##, p < 0.0006. F, overexpression of KLF4 has no effect on NF-{kappa}B DNA-protein binding. Nuclear extracts were harvested from J774a cells overexpressing Ad-GFP (control) or KLF4 at 1000 MOI in the presence or absence of LPS (250 ng/ml) for 1 h. Specificities of bands were verified by supershift studies with {alpha}-p50, {alpha}-p65, or {alpha}-IgG antibodies.

 



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  		FIGURE 5.
KLF4 inhibits TGF-{beta}1 signaling. A, Northern analysis of retrovirally infected NIH 3T3 fibroblasts (A) or adenovirally infected J774a cells (B) with EV control or KLF4 for 24 h demonstrates a decrease in PAI-1 expression at base line or in response to TGF-{beta}1 (10 ng/ml). B, KLF4 inhibits PAI-1 secretion. KLF4 was overexpressed by retroviral infection of NIH 3T3 fibroblasts or adenoviral infection of J774a macrophages followed by stimulation with TGF-{beta}1 (10 ng/ml). n = 3–6/group; *, p < 0.002; #, p < 0.03. C, KLF4 inhibits PAI-1 promoter activity. Transient transfection studies were performed using the –800 bp PAI-1 promoter along with Smad3 or KLF4 in the presence or absence of TGF-{beta}1 (10 ng/ml) in RAW cells. KLF4 potently inhibits TGF-{beta}1-, Smad3-, or TGF-{beta}1 + Smad3-mediated induction on the PAI-1 promoter. #, p < 0.001 versus pcDNA3; *, p < 0.001 versus TGF-{beta}1, n = 3–6/group; **, p < 0.0001 versus Smad3; ##, p < 0.00001 versus Smad3 + TGF-{beta}1. D, structural basis for KLF4 inhibition of PAI-1. Transient transfection studies using the –800 bp PAI-1 promoter in RAW cells demonstrate that both KLF4 and the KLF4{Delta}ZnF potently inhibit promoter activity, whereas the ZnF domain alone does not. *, p < 0.0005; **, p < 0.0001. E, KLF4 inhibits the TGF-{beta}1/Smad3-mediated induction of the 3TP-Lux promoter. Transient transfection studies were performed with Smad3 or TGF-{beta}1 in the presence and absence of KLF4. n = 3–6/group; #, p < 0.001 versus pcDNA3; *, p < 0.0001 versus Smad3; **, p < 0.0001 versus TGF-{beta}1; ***, p < 0.0005 versus Smad3 + TGF-{beta}1. F, KLF4 knockdown enhances TGF-{beta}1/Smad3 induction of the PAI-1 promoter. Nonspecific KLF4 (NS-KLF4) or AS-KLF4 morpholino oligonucleotides were transfected in RAW cells followed by transient transfection of the PAI-1 promoter with pcDNA3 or Smad3 + TGF-{beta}1. n = 3–6/group; *, p < 0.0001. G, KLF4 overexpression does not affect Smad3 DNA binding to PAI-1 Smad binding sites. Nuclear extracts were harvested, and gel shift was performed after 293T cells were transiently transfected with pcDNA3, KLF4, or Smad3 in the presence of TGF-{beta}1. Specificity of the Smad3 band was verified by using antibodies to Smad3, FLAG, or IgG. NS, nonspecific. H, KLF4 does not affect Smad3 or Smad7 expression. Northern analysis was performed from total RNA of NIH3T3 fibroblasts that were retrovirally infected with EV or KLF4 as described in A and treated with either control (Ctrl) or TGF-{beta}1 (10 ng/ml) for 6 h. KLF4 (exo), exogenous KLF4 expression. EtBr is shown to verify equal loading. I, overexpression of KLF4 has no effect on Smad3 phosphorylation. Whole-cell lysates were immunoprecipitated (IP) with an {alpha}-FLAG antibody followed by immunoblotting (IB) with either an anti-Smad3-phospho antibody to detect phosphorylated-Smad3 or an {alpha}-FLAG antibody to detect total Smad3 protein. Immunoblotting was also performed on prelysates to verify relative expression levels of KLF4.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Infiltration of activated macrophages is a critical hallmark in the pathogenesis of a variety of chronic inflammatory diseases. Our laboratory and others have demonstrated that TGF-{beta}1/Smad signaling can have profound anti-inflammatory effects on vascular inflammation. Specifically, our previous studies have identified that the TGF-{beta}1 downstream effector Smad3 directly mediates the anti-inflammatory effects in macrophages in vitro and in vivo. In contrast, IFN-{gamma} is a potent proinflammatory cytokine that has profound atherosclerotic-promoting properties. Thus, modulation of either of these two pathways may impact vascular inflammation.



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  		FIGURE 6.
KLF4 interacts directly with p300: antagonistic competition with Smad3 for p300C. A, p300 rescues KLF4-mediated inhibition of PAI-1 promoter. Transient transfection studies were performed with the indicated plasmids in RAW cells. Co-transfection studies demonstrate that KLF4-mediated inhibition can be rescued by exogenous addition of p300. n = 3–6/group; *, p < 0.0001. B, KLF4 and p300 cooperate to induce the iNOS promoter. Co-transfection studies were performed in RAW cells. Co-transfection of KLF4 and p300 induces iNOS in a greater than additive fashion. *, p < 0.001. C, KLF4 induction of iNOS is suppressed by the p300 inhibitory protein E1A, an effect lost in the absence of the E1A-p300 interacting domain CR1. Co-transfection studies were performed in RAW cells with the indicated plasmids. n = 3–6/group; *, p < 0.0001; **, p < 0.0005. D, KLF4 interacts with the C terminus of p300 (p300C). GST fusion proteins were generated for p300, and pull-down studies were performed with 293T whole cell lysate containing pcDNA3 or KLF4. KLF4 interacts specifically with the C terminus of p300. E, KLF4 and Smad3 compete for the C terminus of p300. GST pull-down studies were performed using GST-p300C and 293T whole cell lysate containing pcDNA3, KLF4-myc, or Smad3-FLAG as indicated. Whereas a 3-fold molar excess of KLF4 effectively competed away Smad3 binding to GST-p300C, there was no effect of a 3-fold molar excess of Smad3 on KLF4 binding to p300C. E and F, functional competition of KLF4 and Smad3. RAW cells were transiently transfected with the indicated plasmids in the presence or absence of IFN-{gamma} or TGF-{beta}1. Co-transfection studies demonstrate that whereas TGF-{beta}1 and Smad3 can potently repress the induction of the iNOS promoter, KLF4 or KLF4 + IFN-{gamma} can effectively rescue the TGF-{beta}1/Smad3 inhibition (F). n = 3/group; *, p < 0.000006; **, p < 0.0001; #, p < 0.00002. Co-transfection studies also demonstrate that KLF4 and IFN-{gamma} can overcome the TGF-{beta}1/Smad3 induction of the PAI-1 promoter (G). n = 3/group; *, p < 0.001; **, p < 0.0002.

 
In this report, we provide evidence that links KLF4 as a key regulator of macrophage activation through dual mechanisms: 1) by mediating the induction of IFN-{gamma}- and LPS-responsive gene expression such as iNOS and 2) by negatively regulating TGF-{beta}1/Smad3 function. In support, we found that KLF4 is induced by IFN-{gamma}, LPS, or TNF-{alpha} in a variety of macrophage cell lines (Fig. 1). Overexpression of KLF4 induced iNOS expression, an effect enhanced in the presence of IFN-{gamma} (Fig. 2). In contrast, mutation of the KLF DNA binding sites at –212 and –95 bp of the iNOS promoter or knockdown of KLF4 attenuated the responsiveness to IFN-{gamma} or LPS (Fig. 3). Furthermore, we find that KLF4 and p65 interact to cooperatively induce the iNOS promoter (Fig. 4). In contrast, overexpression of KLF4 potently inhibited the expression and function of TGF-{beta}1 and Smad3 target genes, such as PAI-1 (Fig. 5). Consistently, KLF4 knockdown markedly enhanced TGF-{beta}1- and Smad3-induced PAI-1 promoter activity (Fig. 5F). Finally, we suggest that the underlying mechanism for these effects of KLF4 is through a novel competition with Smad3 for the coactivator p300 (Fig. 6). As such, these studies identify KLF4 as a novel mediator of proinflammatory effects in macrophages.

The mechanisms by which activated macrophages produce nitric oxide have been extensively examined (for a review, see Ref. 1). Formation of large amounts of free radical nitric oxide (NO) is involved in many host defense actions in macrophages, an effect that in excess may be detrimental and promote tissue damage. Indeed, accumulating studies have demonstrated a deleterious role for iNOS in the context of atherosclerosis (58, 59), sepsis (60), inflammatory bowel disease (61), and carcinogenesis and tumor progression (62–64) among others. The mechanisms governing the induction of the iNOS promoter by inflammatory cytokines have been the subject of considerable study (1). Transgenic studies highlight the importance of the proximal –1.3 kb of the iNOS promoter to confer responsiveness to inflammatory stimuli IFN-{gamma} and LPS in vivo (41). Using macrophage cell lines, previous studies have found that the induction of iNOS is primarily regulated at the transcriptional level in response to IFN-{gamma} and LPS (39, 65, 66). Analyses of the iNOS promoter revealed that the downstream NF-{kappa}B site (–85 to –76) is important for the LPS induction of iNOS, whereas a distal region (–951 to –911) is responsible for the synergistic induction by IFN-{gamma} and LPS (66). However, mutation of this distal region still allowed for IFN-{gamma} alone to induce the iNOS promoter and only partially blocked the synergistic induction by IFN-{gamma} and LPS (65–67), raising the possibility of other IFN-{gamma}-responsive sites in the iNOS promoter. Consistent with this, IFN-{gamma} alone induced the –226 bp iNOS promoter by ~3-fold in RAW264.7 macrophages (Fig. 3). Through a combination of promoter deletion analyses, site-directed mutagenesis, and gel shift studies, we found that the KLF sites at –95 bp and –212 bp conferred the ability of KLF4 to induce the iNOS promoter (Fig. 3). Our loss-of-function studies using morpholino antisense oligonucleotides to knock down KLF4 in RAW macrophages also verified that the IFN-{gamma} and LPS induction of the –226 bp iNOS promoter was dependent, in part, on KLF4. This effect may be explained, in part, by the interaction of p65 and KLF4 to cooperatively induce the iNOS promoter (Fig. 4). Because KLF4 interacts with the C terminus of p300 (Fig. 6) and p65 interacts with the N terminus of p300 (68), both KLF4 and p65 may help recruit p300 to the iNOS promoter. In support, the p300 inhibitor, E1A, potently blocked the ability of KLF4 to induce the iNOS promoter (Fig. 6C). Indeed, this type of recruitment of p300 by nearby transcription factors has been well described in other promoter contexts, such as the assembly of the IFN-{beta} enhanceosome complex (47). Whether KLF4 also cooperates with other factors within this 157-bp region important for cytokine inducibility such as CCAAT/enhancer-binding protein or Oct-1 has yet to be determined. However, a cooperative interaction between KLFs and members of the CCAAT/enhancer-binding protein families has been reported in other cell types (36, 69, 70). Finally, we cannot rule out the possibility that other KLFs may participate with KLF4 in mediating the iNOS induction. For example, KLF6 has recently been shown to induce the human iNOS promoter in the context of T cell activation (71). However, it remains unknown whether KLF6 is also expressed in macrophages and is capable of inducing the mouse iNOS promoter. Because some KLFs may interact with each other (26, 27, 72), the possibility also remains that KLF4 may cooperate or compete with other yet unidentified KLFs that are expressed in macrophages for similar DNA-binding sites.

One of the hallmarks of IFN-{gamma} is to endow the macrophage with the capacity to execute a variety of effector functions, an effect largely mediated through the signal transduction of a number of immunologically relevant downstream factors that are members of the Jak-Stat family (23, 24). Jak-Stat signaling involves receptor activation of Jaks (Jaks 1–3 and Tyk2) and Stats (Stats 1–6) to regulate transcription of target genes through IFN-{gamma} response elements ({gamma}-activated site or ISRE). This propagation of signal transduction typically occurs within 30–60 min of IFN-{gamma} treatment. Consistent with this effect, we found that KLF4 induction was quite rapid in several monocyte/macrophage cell lines (Fig. 1). IFN-{gamma} signaling may cross-talk with several other pathways by virtue of the ability of its downstream effectors to form a variety of protein-protein interactions. Consistently, the ability of KLF4 to interact with p65 may underlie some of the synergistic induction of the iNOS promoter by IFN-{gamma} and LPS. Indeed, we found that site-directed mutagenesis of the KLF4 DNA-binding sites or knockdown of KLF4 markedly impaired the IFN-{gamma} and LPS synergism on the iNOS promoter (Fig. 3). In response to IFN-{gamma}, downstream effectors may also antagonize other signaling pathways. Two studies have examined the importance of such antagonistic cross-talk between IFN-{gamma} and TGF-{beta}1 signaling pathways. Whereas Ulloa et al. (73) demonstrated that IFN-{gamma} may inhibit TGF-{beta}1 by inducing the inhibitory Smad, Smad7, and thereby blocking the downstream effects of Smad3, Ghosh et al. (74) found that IFN-{gamma} may prevent TGF-{beta}1/Smad3 signaling independent of Smad7 through coactivator competition for p300/CBP between Stat1{alpha} and Smad3. The discrepancy of these two studies may reflect cell type-specific effects, since the latter was performed in dermal fibroblasts, whereas the former was performed in epithelial cell types. On the basis of these observations, it has been suggested that IFN-{gamma} may have distinct cell type-specific inhibitory effects on TGF-{beta}1 signaling. However, these studies could not rule out the possibility that additional factor(s) downstream to Stat1{alpha} may also participate in the IFN-{gamma} inhibition of TGF-{beta}1/Smad3 signaling. Our data indicate that in response to IFN-{gamma}, KLF4 is induced and inhibits TGF-{beta}1 signaling by modulating Smad3 function through competing for its coactivator p300/CBP. In support, we show that KLF4 can augment the IFN-{gamma} inhibition of the TGF-{beta}1-induced target gene PAI-1 (Fig. 6F). Conversely, the addition of KLF4 markedly enhanced the IFN-{gamma} induction of the iNOS promoter (Fig. 6E). Moreover, loss of KLF4 enhanced the TGF-{beta}1 induction of the PAI-1 promoter (Fig. 5F). Thus, in response to IFN-{gamma}, KLF4 may be able to differentially regulate target genes such as iNOS and PAI-1.



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  		FIGURE 7.
Schema. In response to cytokines, KLF4 may inhibit TGF-{beta}1-responsive gene expression through competition for the Smad3 coactivator, p300. The interaction of KLF4 with the C terminus of p300 (p300C) is stronger than that of Smad3 with the C terminus of p300. Consequently, KLF4 may allow for induction of some promoters (e.g. iNOS) and inhibition of Smad3-responsive promoters (e.g. PAI-1). The KLF4 induction of the iNOS promoter is mediated, in part, through two KLF DNA-binding sites that are adjacent to the proximal NF-{kappa}B site and through a cooperative interaction with the NF-{kappa}B family member p65. Because KLF4 interacts with the C terminus of p300 and p65 interacts with the N terminus of p300, both KLF4 and p65 may help recruit p300 to the iNOS promoter.

 
Inhibition of TGF-{beta}1/Smad signaling can occur on multiple levels, including modulation of receptor-activated Smad phosphorylation, translocation, or DNA-binding (45). Whereas in response to TGF-{beta}1, there can be non-Smad-dependent effects, such as through mitogen-activated protein kinase pathways, we found no evidence that the mitogen-activated protein kinase inhibitor, U0126, could affect KLF4 expression or the KLF4 induction of the iNOS promoter (data not shown). Several factors have been linked with inhibiting Smad transduction through the induction of the inhibitory Smad, Smad7. For example, signals emanating from TNF-{alpha} or IL-1{beta} activate NF-{kappa}B/RelA that, in turn, can induce Smad7 expression (75). In contrast, other factors (e.g. co-repressors TGIF, c-Ski, SnoN, or HDACs) interrupt Smad3 signaling by directly interacting with Smad3 to form a repressor complex (45). Whereas KLF4 potently inhibited Smad3 function, we found no effect of KLF4 on Smad7 expression or Smad3 expression, phosphorylation, or DNA binding (Fig. 5). To reconcile these findings and the observation that KLF4 can simultaneously inhibit PAI-1 and induce iNOS, we assessed whether KLF4 may compete with a coactivator important for Smad3 function. The fact that Smad3 function is critically dependent on the coactivator p300/CBP and the observation that other KLFs can interact with p300/CBP to augment their transcriptional activity (26, 27) led us to consider that competition for this coactivator may underlie the contrasting effects of KLF4. A similar mechanism has been implicated in a variety of other signaling pathways, such as the functional inhibition between nuclear receptors and AP-1, IFN-{gamma}/Stat1 and Ras/AP-1, and NF-{kappa}B/p65 and the glucocorticoid receptor, among others (76–78). Indeed, our GST pull-down assays demonstrated that KLF4 strongly bound to the C terminus of p300. Whereas increasing amounts of KLF4 directly inhibited Smad3 binding to GST-p300C (Fig. 6E), increasing amounts of Smad3 failed to prevent the KLF4 interaction with GST-p300C, suggesting a higher binding affinity of KLF4 to this area (Fig. 6E). Whereas, in theory, KLF4 may also interact with Smad3, rendering it incapable of binding to p300, we found no direct interaction between Smad3 and KLF4 by co-immunoprecipitation studies (data not shown), indicating that KLF4 probably competes for p300 as the operative mechanism. Consistent with a competitive effect between Smad3 and KLF4 for p300, our transient transfection experiments demonstrated that p300 could rescue the KLF4-mediated inhibition of TGF-{beta}1-induced PAI-1 promoter activity, whereas it augmented the KLF4 induction of the iNOS promoter (Fig. 6, A and B). Indeed, KLF4 induction of the iNOS promoter was blocked by E1A, an oncoprotein that inhibits p300. Collectively, these data indicate that KLF4 alters Smad3 function by disrupting the Smad3-p300 interaction and support the hypothesis that the KLF4 interaction with the coactivator p300 may be an important mechanism by which KLF4 is able to inhibit certain target genes and induce others (Fig. 7). This mechanistic paradigm is somewhat analogous to the observation that KLF2 can compete with NF-{kappa}B for p300/CBP in endothelial cells (37). However, in contrast to KLF4, KLF2 was found to interact with a separate region of p300, the N terminus. Thus, through binding to specific domains of p300, KLFs may be able to differentially regulate gene expression or function.

In summary, we found that KLF4 is an IFN-{gamma}- and cytokine-responsive transcription factor that regulates key pro- and anti-inflammatory signaling pathways in macrophages through distinct mechanisms. These observations support the possibility that induction of KLF4 may be important for optimal proinflammatory gene responses that, in turn, may accelerate the development of a variety of vascular inflammatory pathologies. Future studies overexpressing KLF4 in a macrophage-specific manner will help to elucidate this possibility further.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL-67755 (to M. W. F.), HL-69477 (to M. K. J.), HL-72952 (to M. K. J.), HL-75427 (to M. K. J.), and HL-76754 (to M. K. J.), American Heart Association Awards 0355691T (to M. W. F.) and 0250030N (to M. K. J.), and American Diabetes Association Award 1–02-JF-40 (to M. K. J.). This work was also supported by a Carl J. and Ruth Shapiro Scholar Award (to M. W. F.) and a Lerner Junior Faculty Scholar Award (to M. W. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115. Tel.: 617-732-7820; Fax: 617-732-5132; E-mail: mfeinberg{at}rics.bwh.harvard.edu.

2 The abbreviations used are: iNOS, inducible nitric-oxide synthase; MMP, matrix metalloproteinase; MCP, monocyte chemoattractant protein; IFN, interferon; NK, natural killer; KLF, Kruppel-like factor; LPS, lipopolysaccharide; GFP, green fluorescent protein; MOI, multiplicity of infection; GST, glutathione S-transferase; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; EV, empty vector; AS-KLF4, antisense KLF4; TNF-{alpha}, tumor necrosis factor-{alpha}; TGF-{beta}1, transforming growth factor-{beta}1. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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