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Originally published In Press as doi:10.1074/jbc.M108673200 on November 27, 2001

J. Biol. Chem., Vol. 277, Issue 5, 3109-3116, February 1, 2002
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Integrin-linked Kinase Regulates Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Expression in an NF-kappa B-dependent Manner*

Clara TanDagger , Alice MuiDagger §, and Shoukat DedharDagger §

From the Dagger  Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6H 3Z6 and the § Jack Bell Research Center at Vancouver General Hospital and Health Service Center, Vancouver, British Columbia V6H 3Z6, Canada

Received for publication, September 7, 2001, and in revised form, November 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) and prostaglandins are produced as a result of the stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, respectively, in response to cytokines or lipopolysaccharide (LPS). We demonstrate that the activity of integrin-linked kinase (ILK) is stimulated by LPS activation in J774 macrophages. Inhibition of ILK activity by dominant-negative ILK or a highly selective small molecule ILK inhibitor, in epithelial cells or LPS-stimulated J774 cells and murine macrophages, resulted in inhibition of iNOS expression and NO synthesis. LPS stimulates the phosphorylation of Ikappa B on Ser-32 and promotes its degradation. Inhibition of ILK suppressed this LPS-stimulated Ikappa B phosphorylation and degradation. Similarly, ILK inhibition suppressed the LPS-stimulated iNOS promoter activity. Mutation of the NF-kappa B sites in the iNOS promoter abolished LPS- and ILK-mediated regulation of iNOS promoter activity. Overexpression of ILK-stimulated NF-kappa B activity and inhibition of ILK or protein kinase B (PKB/Akt) suppressed this activation. We conclude that ILK can regulate NO production in macrophages by regulating iNOS expression through a pathway involving PKB/Akt and NF-kappa B. Furthermore, we also demonstrate that ILK activity is required for LPS stimulated cyclooxygenase-2 expression in murine and human macrophages. These findings implicate ILK as a potential target for anti-inflammatory applications.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse macrophages express an inducible form of nitric oxide synthase (iNOS),1 which catalyzes the production of nitric oxide (NO) from L-arginine. Macrophage-derived NO is important for host defense and microbial and tumor cell killing (reviewed in Ref. 1). Activating stimuli such as lipopolysaccharide (LPS) (2), other bacterial cell wall products (3), and cytokines such as interferon-gamma (4) all stimulate iNOS expression in induced macrophages. However, because excess production of NO results in inappropriate tissue injury and septic shock, iNOS expression is subject to stringent regulatory control.

The mouse iNOS promoter has been studied extensively and consists of two clusters of regulatory elements (5, 6). A proximal region (region I or RI, -48 to -209) functions as the basal promoter containing an octamer element and an NF-kappa B binding site, which mediates responsiveness to LPS. The distal region (RII, -913 to -1029) functions as an enhancer element and responds to LPS and interferon-gamma through NF-kappa B, interferon regulatory factor-1, interferon-stimulated response element, and gamma -interferon activation site binding sites. The NF-kappa B sites are essential for LPS-mediated NO production (7).

Protein kinase B (PKB/Akt) has been shown to phosphorylate and activate Ikappa B kinase (8, 9), which in turn phosphorylates Ikappa B. Phosphorylated Ikappa B is targeted for ubiquitin-mediated degradation, thus releasing active NF-kappa B and allowing its translocation into the nucleus (10).

Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine/threonine protein kinase that can interact with the cytoplasmic domain of the beta 1 integrin and regulates integrin-dependent functions (11, 12). It has been demonstrated to regulate the activity of transcription factors such as beta -catenin-TCF/LEF-1 (T-cell/lymphoid enhancer factor) (13, 14), AP-1 (15), and CREB (16). ILK activity is regulated in a phosphatidylinositol 3-kinase-dependent manner (17-20), and ILK can regulate the phosphorylation and activation of PKB/Akt (17-20). Because the transcription factor NF-kappa B has been shown to be activated by PKB/Akt, which is known to lead to activation of iNOS in mice (7), we wanted to determine whether ILK could also regulate NF-kappa B activity. To examine a physiologically relevant system for the regulation of NF-kappa B by ILK, we examined the role of ILK in LPS-stimulated expression of iNOS and NO. We found that ILK is an upstream regulator of LPS-mediated phosphorylation of Ikappa B and of NF-kappa B-dependent expression of iNOS.

Mouse and human macrophages have different iNOS promoters (21). To determine a similar role of NF-kappa B in human macrophages, we analyzed the expression of cyclooxygenase-2 (COX-2), a protein that regulates the production of proinflammatory prostaglandins by catalyzing arachidonic acid into prostaglandins (22, 23). There are two isoforms of COX, COX-1 and COX-2, which are the products of two different genes. COX-1 is constitutively expressed in most tissues and is a housekeeping gene (24). COX-2 is not detectable in most normal tissues or resting immune cells, but cytokines, growth factors, and endotoxins can induce its expression (25-27). The role of NF-kappa B has been demonstrated to be important in mouse and human macrophage/monocytes in the induction of COX-2 (27, 28-33). The AP-1 and CREB transcription factors, in addition to NF-kappa B, have been demonstrated to be important in the regulation of COX-2 expression (34-36). We demonstrate here that in addition to regulating iNOS gene expression in an NF-kappa B-dependent manner, ILK activity is also required for LPS-mediated COX-2 expression in murine and human macrophages.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Cell Culture-- Rat intestinal epithelial cells, IEC-18, were obtained from American Type Culture Collections (ATCC). IEC-18 cell clones (ILK-13 Ala3 and A1C3) stably overexpressing wild-type sense ILK cDNA, ILK-14 clones stably expressing antisense ILK cDNA, and dominant-negative ILK (DN-ILK (GH31RH)) stably expressing ILK with a 359 glutamate to lysine mutation were all prepared as described previously (13, 37). IEC-18 cells and stably transfected cell clones were routinely cultured in alpha -minimal essential medium (alpha -MEM, Invitrogen) supplemented with 5% fetal bovine serum (FBS, Invitrogen), glucose (3.6 mg/ml), and insulin (10 µg/ml). Stably transfected derivatives were grown in the presence of G418 (80 µg/ml) to maintain selection pressure. Mouse monocyte/macrophage J774 cells (ATCC) and platinum E packaging cells from Dr. T. Kitamura's laboratory (University of Tokyo, Tokyo) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Primary mouse macrophages were a gift from Dr. Urs Steinbrecher's laboratory (University of British Columbia, Vancouver, Canada), and they were isolated as previously described by (38-40). Human monocyte-derived macrophages were a gift from Dr. Anthony Chow's laboratory (University of British Columbia, Vancouver, Canada), and they were isolated as described by Sly et al. (41, 42). All cells were grown at 37 °C in a 99% humidified atmosphere of 5% CO2 in air.

Plasmids-- The ILK wild-type and dominant-negative plasmids are described by Persad et al. 18, 20, 45). The NF-kappa B response-element reporter construct conjugated to a luciferase reporter gene was a kind gift from Dr. Nathan Yoganathan's laboratory (Kinetek Pharmaceuticals, Vancouver, Canada). The iNOS promoters were a kind gift from Dr. W. J. Murphy's laboratory (University of Kansas Medical Center, Kansas City). Retrovirus constructs were a kind gift from Dr. Gerald Krystal's laboratory (British Columbia Cancer Agency, Vancouver, Canada).

Transient Transfection-- Cells were seeded into 6-well dishes for 24 h prior to transfection so that they would be ~60% confluent on the day of transfection. Transfection of IEC-18 cells was carried out using Lipofectin (Invitrogen) according to the manufacturer's guidelines, and 2-3 µg of plasmid/well of a 6-well dish. Transfection of J774 cells was carried out using LipofectAMINE or LipofectAMINE Plus (Invitrogen) according to manufacturer's instructions and 1-3 µg of plasmid/well as described by Pierce et al. (43). Transfections were carried out for 4 to 6 h in Opti-MEM (Invitrogen) medium. The transfection medium was then replaced by serum-containing medium for 6 h prior to the beginning of an experiment.

Expression of cDNAs in J774 Cells by Retroviral Transduction-- cDNAs, which are V5-tagged, were cloned into the retroviral vector pCMVpuro and introduced into J774 cells by retroviral infection. Packaging cells (Platinum E cells) were transfected with constructs using Lipofectin according to manufacturer's instructions. Platinum E cells were irradiated (2000 rads) 24 h after transfection and co-cultured with J774 cells for an additional 24 h. Once the cell clones were infected with the virus, they were selected for drug resistance with 6 µg/ml puromycin (Sigma). Independent puromycin-resistant colonies were isolated, and high-expressing clones identified by anti-V5 antibody (Invitrogen).

In Vitro Kinase Assay-- ILK assays were carried out as described previously (17, 37). The immunoprecipitates were washed, and the reactions were carried as described by Hannigan et al. (37). Myelin basic protein was used as a substrate for ILK activity. Phosphorylated myelin basic protein was resolved by 15% SDS-PAGE and visualized by autoradiography. The ILK inhibitor KP-392 (previously known as KP-SD-1) was obtained from Kinetek Pharmaceuticals and was used as described previously (14, 18, 20, 44, 45). All experiments were done with equivalent amounts of vehicle, Me2SO.

Western Blot Analysis-- Cells were harvested in Nonidet P-40 lysis buffer (1% Nonidet P-40, 50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaV04, 1 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and stored at -70 °C. Protein concentrations were measured using a Bradford protein assay kit (Bio-Rad). Equivalent amounts of protein were resolved in SDS-PAGE, transferred onto polyvinylidene fluoride (Immobilon-P, Millipore) membranes, and probed with antibody. The protein of interest was visualized with enhanced chemiluminescent (ECL; Amersham Biosciences, Inc.) reagents. The following antibodies were used in the experiments: anti-ILK (rabbit polyclonal; Upstate Biotechnology Inc.), anti-Ikappa B and anti-phosphoSer-32 Ikappa B (rabbit polyclonal; New England Biolabs), anti-Akt and anti-phosphoSer-473 Akt (rabbit polyclonal; New England Biolabs), anti-iNOS (rabbit polyclonal; New England Biolabs), anti-COX-2 (mouse monoclonal; Transduction Laboratories), and anti-beta -actin (mouse monoclonal; Transduction Laboratories).

Luciferase Assays-- Luciferase assays were performed on transiently transfected cells according to the manufacturer's instructions (Promega Corp.). All assays were normalized for transfection efficiency by measuring a modified luciferase activity (pRenilla, dual luciferase; Promega). Triplicate samples were assayed for each trial of each condition in these experiments.

Detection of Nitric Oxide-- LPS (Escherichia coli 055:b5; DIFCO, Detroit, MI) was added at the indicated concentrations at 500 ng/ml for 24-h experiments or at 1 µg/ml for 1-h experiments. It is also important to note that the experiments done with human monocytes/macrophages were stimulated with 500 ng/ml LPS and 100 units/ml interferon-gamma (a kind gift from Dr. Bill Sahl (University of British Columbia, Vancouver, Canada) who purchased this from Calbiochem). Supernatants were removed after incubation times of 24 h, and nitric oxide concentrations were determined using the Greiss reagent (2.5% v/v phosphoric acid, 0.1% w/v naphthylethylene, and 1.0% w/v sulfanilamide in distilled water) and the Greiss method as described by O'Farrell et al. (46). Triplicate samples were assayed for each trial of each condition in these experiments.

Immunohistochemistry-- Human biopsy specimens were kind gifts from Dr. John English (Department of Pathology, University of British Columbia and Vancouver General Hospital). They were fixed in formalin and imbedded into paraffin (Sigma). 5-µm sections were prepared, and the sections were placed on Silane (Sigma)-coated slides. Conventional deparaffinization and rehydration techniques were employed. The sections were heated in a pH 5 acetate buffer bath at 95 °C for 10 min. They were treated with 3% hydrogen peroxide and 0.02% Triton X-100 in 150 mM Tris-buffered saline (TBS; Sigma) followed by three washes with 500 mM TBS. The slides were then blocked with 5% milk in 150 mM TBS. The sections were then incubated with ILK antibody (1:500 dilution, rabbit polyclonal; Upstate Biotechnology Inc.) in 5% milk in 150 mM TBS overnight at 4 °C. This was followed by three washes with 500 mM TBS and an incubation with biotin-conjugated secondary antibody (rabbit IgG and IgM, 1:1000; Jackson Immunoresearch) in 5% bovine serum albumin in 150 mM TBS. The slides were then washed three times with 500 mM TBS and incubated with streptavidin-conjugated horseradish peroxidase (1:1000, Jackson Immunoresearch) in 150 mM TBS followed by three washes with 500 mM TBS. The detection was performed with 0.03% diaminobenzidine (Sigma) as the chromogen in TBS containing 0.015% hydrogen peroxide. Gill's hematoxylin (Sigma) counter-staining was done by soaking the slides in the dye for 2 min followed by soaking the slides in distilled water. Slides were mounted with Permount (Sigma) and 1.5-mm micro-glass cover glasses (VWR Scientific). Micrographs were generated using the Nikon Eclipse TE300 microscope and Nikon D1 digital camera.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ILK Up-regulates NF-kappa B Activity-- To determine whether ILK regulates NF-kappa B activity, reporter assays were performed. These experiments were carried out by transfecting the NF-kappa B response-element reporter construct coupled to a luciferase gene reporter into IEC-18 intestinal epithelial cells and stably transfected clones overexpressing sense or antisense ILK, characterized previously (13, 17). Overexpression of wild-type ILK, but not dominant-negative ILK, stimulates NF-kappa B response-element reporter construct activity. As shown in Fig. 1A, NF-kappa B activity is substantially higher in two independent ILK-overexpressing cell lines relative to the control cell lines. Transfection of dominant-negative ILK cDNA (Fig. 1B), as well as incubation with highly selective, small molecule inhibitor of ILK (KP-392, previously known as KP-SD-1) (14, 18, 20, 44, 45) (Fig. 1D), both inhibit NF-kappa B response-element reporter construct activity in the ILK overexpressing IEC-18 cells (ILK-13 clone) in a dose-dependent manner. This indicates that the observed stimulation of NF-kappa B activity in this cell line is ILK-dependent. Furthermore, transfection of the ILK-overexpressing cells with a potent dominant-negative PKB/Akt cDNA (PKB/Akt(AAA)) (17, 18) also resulted in the inhibition of NF-kappa B activity, suggesting that the ILK-induced stimulation of NF-kappa B in these cells involved PKB/Akt (Fig. 1C).


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  		Fig. 1.   A, ILK up-regulates NF-kappa B activity. IEC-18 cells and stable clones were transfected with the NF-kappa B response-element reporter construct (pGL3NF-kappa B response-element reporter with a luciferase reporter (open bars)) or a control plasmid (pGL3Basic, no promoter with a luciferase reporter (filled bars)). 48 h post-transfection, cells were harvested and assessed for luciferase activity. Samples were normalized with pRenilla, and activity is expressed as relative light units (RLU). Data are the mean ± standard deviation of six independent trials. B, dominant-negative ILK decreases NF-kappa B activity in epithelial cells in a dose-dependent manner. ILK wild-type overexpressing IEC-18 cells (clone A1a3 ILK-13) were co-transfected with increasing amounts of DN-ILK:V5, and the total amount of plasmid was kept constant by supplementing it with appropriate amounts of Empty:V5 and 2 µg of pGL3NF-kappa B response-element reporter construct. After 48 h, cells were assayed for luciferase activity. All samples were normalized with pRenilla. Data are the mean ± standard deviation of four independent trials. Increased expression of DN-ILK:V5 was monitored by Western blot using anti-V5 antibody. C, transfection of dominant-negative PKB/Akt inhibits NF-kappa B activity in epithelial cells in a dose-dependent manner. IEC-18 cells overexpressing WT-ILK (clone A1a3 ILK-13) were co-transfected with increasing amounts of dominant-negative PKB/Akt(AAA):HA and pGL3NF-kappa B response-element construct. After 48 h, cells were assayed for luciferase activity. All samples were normalized with pRenilla. Data are the mean ± standard deviation of four independent trials. Increasing PKB/Akt(AAA):HA protein expression was monitored with Western blot analysis with anti-HA antibody. D, pharmacological inhibition of ILK with ILK inhibitor KP-392 decreases NF-kappa B activity in a dose-dependent manner. ILK-13 IEC-18 cells were transfected with equivalent amounts of either pGL3NF-kappa B or pGL3Basic and pRenilla. Cells were incubated with complete medium for 6 h post-transfection. Cells were then treated with increasing concentrations of KP-392 for an additional 24 h and were then assessed for luciferase activity. All samples were normalized with pRenilla. Data are the mean ± standard deviation of three independent trials.

LPS Stimulates NO Production in an ILK-dependent Manner-- We next wanted to determine the physiological relevance of ILK-mediated stimulation of NF-kappa B. Because LPS is known to stimulate iNOS expression and NO production in murine macrophages in a NF-kappa B-dependent manner, we examined the role of ILK in this pathway. As shown in Fig. 2A, LPS stimulates both NO production and NF-kappa B activity in the J774 macrophage cell line. We next wanted to examine whether LPS had any effect on ILK activity in the cell line. As shown in Fig. 2B, ILK activity is rapidly and transiently stimulated by LPS, as is the phosphorylation of PKB/Akt on Ser-473 and Ikappa B on Ser-32. The subsequent degradation of Ikappa B (Fig. 2B) allows the release and translocation of NF-kappa B to the nucleus. There is no significant change in ILK and PKB/Akt protein levels within the time course of activation of ILK and PKB/Akt phosphorylation (1 h).


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  		Fig. 2.   A, LPS stimulates the production of nitric oxide in J774 cells and up-regulates the NF-kappa B activity. J774 cells were transfected with equivalent amounts of pGL3NF-kappa B (filled bars) and pRenilla. After transfection, cells were incubated in complete medium for 6 h. Cells were then stimulated with increasing amounts of LPS in complete medium for an additional 24 h. After 24 h, nitric oxide production was measured according to the Greiss method, and cells were harvested, lysed, and assessed for luciferase activity. Data are the mean ± standard deviation of six independent samples. RLU, relative light units. B, LPS stimulates ILK activity and Ikappa B Ser-32 phosphorylation. Cells were exposed to 1 µg/ml LPS for 60 min. ILK activity was determined in J774. PKB/Akt Ser-473 and Ikappa B Ser-32 phosphorylation and protein levels were determined by Western blot analysis.

Inhibition of ILK Suppresses LPS-stimulated iNOS Expression and NO Production in J774 Cells and in Primary Murine Macrophages-- To determine whether LPS-stimulated NO production is ILK-dependent, we exposed J774 cells to LPS and increasing doses of a recently identified, highly selective ILK-inhibitor, KP-392, previously called KP-SD-1 (14, 18, 20, 44, 45). We observed a parallel dose-dependent inhibition of NO production and iNOS expression (Fig. 3A). As seen in Fig. 3A, incubation with the KP-392 also inhibits LPS-stimulated ILK activity in these cells in a dose-dependent manner. Although we have previously shown that the ILK inhibitor (KP-392, previously known as KP-SD-1) is highly selective for ILK compared with other kinases (20), it is always possible that pharmacological inhibitors could have nonspecific effects in cells. We therefore carried out experiments to determine whether inhibition of ILK by dominant-negative ILK would also suppress iNOS expression and NO production in J774 cells treated with LPS. We isolated J774 cells stably expressing either wild-type ILK (WT-ILK:V5) or dominant-negative ILK (DN-ILK:V5) after retrovirus infection, as described in "Experimental Procedures." As shown in Fig. 3B, stable expression of DN-ILK:V5 resulted in a much lower stimulation of NO production and iNOS expression (at both the transcriptional regulation and protein expression levels) in response to LPS compared with J774 clones expressing the empty vector. In contrast J774 clones expressing wild-type ILK:V5 exhibited elevated responses to LPS as compared with the empty vector clones and the DN-ILK:V5-expressing clones. Furthermore, the elevated iNOS expression and NO production in the WT-ILK:V5 J774 clones were significantly inhibited by the ILK inhibitor in a dose-dependent manner, as was phosphorylation of PKB/Akt on Ser-473 (Fig. 3C). These results suggest that ILK is an important regulator of iNOS expression and NO production in response to LPS.


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  		Fig. 3.   A, inhibition of ILK activity decreases iNOS expression. J774 cells were incubated with increasing concentrations of KP-392 and 500 ng/ml of LPS for 24 h in 5% serum. Nitric oxide production was measured according to the Greiss method. Cells were harvested, and iNOS expression was measured by Western blot analysis. Parallel experiments of ILK activity were measured as described under "Experimental Procedures." Data are the mean ± standard deviation of four independent trials. B, ILK up-regulates iNOS expression and NO production in LPS-stimulated J774 cells. J774 clones, stably expressing Empty:V5, dominant-negative ILK:V5 (DN-ILK), or wild-type ILK:V5 (WT-ILK) were incubated with 500 ng/ml LPS and 2.5% FBS. After 24 h, nitric oxide production was quantified according to the Greiss method. The expression of iNOS, Empty:V5, DN-ILK:V5, WT-ILK:V5, and beta -actin were assessed by pooling three independent experiments and performing Western blot analysis with the lysates. 5 of 10 clones of each variety were assessed. Data are the mean ± standard deviation of three independent experiments. RLU, relative light units. C, decreased ILK activity decreases iNOS expression, NO production, and PKB/Akt phosphorylation in LPS-stimulated J774 cells stably over-expressing wild-type ILK. J774 clones expressing WT-ILK:V5 were incubated with 500 ng/ml LPS, 2.5% FBS, and the indicated concentrations of KP-392. After 24 h, NO production was quantified according to the Greiss method, and the expression of iNOS, PKB/Akt, and WT-ILK:V5 and the phosphorylation of PKB/Akt on the Ser-473 site were assessed by Western blot. D, inhibition of LPS-stimulated NO production in primary mouse macrophages by KP-392. Primary mouse macrophages were incubated with 500 ng/ml LPS and increasing amounts of KP-392 as indicated. After 24 h, nitric oxide production was assessed by the Greiss method. Data are the mean ± standard deviation of three independent trials.

To determine whether this effect of ILK inhibitor on LPS-stimulated NO production could be demonstrated in primary murine macrophages, murine monocytes were differentiated into macrophages using macrophage colony-stimulating factor as described under "Experimental Procedures." The macrophages were maintained for 24 h without macrophage colony-stimulating factor prior to the experiment. The macrophages were then exposed to LPS in the presence of increasing amounts of ILK inhibitor (KP-392). As shown in Fig. 3D, the ILK inhibitor decreased LPS-stimulated NO production in a manner similar to that observed in the J774 cell line after 24 h.

Inhibition of ILK Suppresses LPS-stimulated NF-kappa B Expression and NF-kappa B-dependent iNOS Gene Expression-- To examine the mechanism of the effect of ILK on NO production, we first determined whether inhibition of ILK would also suppress LPS-stimulated NF-kappa B transcription. J774 cells were co-transfected with increasing amounts of the dominant-negative ILK:V5 (DN-ILK:V5) plasmid and the NF-kappa B response-element reporter construct. As can be seen from Fig. 4A, NF-kappa B activity is inhibited in a dose-dependent manner by increased expression of the dominant-negative ILK. Because it is well known that macrophages have an extremely low transfection efficiency (43, 48, 49, 51, 52), we were unable to detect the levels of DN-ILK:V5 expression on a Western blot. Therefore, J774 cells were transfected with the NF-kappa B response-element reporter construct, and reporter activity was measured in LPS-stimulated J774 cells exposed to increasing concentrations of KP-392 ILK inhibitor. As shown in Fig. 4B, inhibition of ILK decreases LPS-stimulated NF-kappa B response-element reporter construct activity. The mouse iNOS promoter possesses two NF-kappa B sites, which have been shown to be essential for LPS-mediated NO production (2, 4). To determine whether LPS-stimulated iNOS expression could also be inhibited by the ILK inhibitor and whether this ILK-dependent iNOS expression was dependent on NF-kappa B, the J774 cells were transfected with either the full-length wild-type iNOS promoter (7) or the iNOS promoter with point mutations in the NF-kappa B binding sites (7). The cells were then stimulated with LPS and treated with increasing concentrations of KP-392 ILK inhibitor. LPS stimulated the full-length iNOS promoter but showed only minimal stimulation of iNOS promoter activity containing point mutations in the NF-kappa B sites (Fig. 4C). In addition, the KP-392 ILK inhibitor reduced the LPS-stimulated iNOS promoter activity in a dose-dependent manner, similar to that observed with the NF-kappa B response-element promoter (Fig. 4C).


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  		Fig. 4.   A, dominant-negative ILK decreases NF-kappa B activity in J774 macrophage cells in a dose-dependent manner. J774 macrophages cells were co-transfected with increasing amounts of DN-ILK:V5, and the total amount of plasmid was kept constant by supplementing it with appropriate amounts of Empty:V5 and 2 µg of pGL3NF-kappa B response-element reporter construct. Cells were incubated with 500 ng/ml LPS and assayed for luciferase activity 24 h later. All samples were normalized with pRenilla. Data are the mean ± standard deviation of four independent trials. RLU, relative light units. B, KP-392 decreases NF-kappa B activity in a dose-dependent manner. Cells were transfected with equivalent amounts of pGL3NF-kappa B and pRenilla. After transfection, cells were incubated with 500 ng/ml LPS and increasing amounts of KP-392, and the total amount of drug vehicle was kept constant by the relevant supplementary addition of Me2SO for 24 h, diluted in Dulbecco's modified Eagle's medium with 5% FBS. Cells were harvested and assessed for luciferase activity. Data are the mean ± standard deviation of six independent trials. C, ILK regulates iNOS transcription in a NF-kappa B-dependent manner. J774 cells were transfected with the indicated plasmids (full-length wild-type murine iNOS promoter or double mutant NF-kappa B binding sites murine iNOS promoter). Cells were then incubated with 500 ng/ml LPS and increasing concentrations of KP-392. After 24 h, cells were harvested and assessed for luciferase activity. Data are the mean ± standard deviation of four independent trials.

Inhibition of ILK Suppresses Ikappa B Ser-32 Phosphorylation and Prevents Its Degradation-- To gain further insight into the mechanism of the ILK-induced regulation of NF-kappa B, we examined the effects of inhibition of ILK activity on Ikappa B phosphorylation and degradation. As shown in Fig. 5, LPS treatment of J774 cells results in a stimulation of phosphorylation of Ikappa B on Ser-32, the site for ubiquitin-mediated degradation of Ikappa B (10). As can be seen in Fig. 5, LPS treatment also leads to degradation of Ikappa B, which correlates with its phosphorylation on Ser-32. Exposure of cells to 50 µM of KP-392 ILK inhibitor for 1 h prior to LPS stimulation markedly inhibits Ikappa B phosphorylation, thus stabilizing and preventing its degradation (Fig. 5), and subsequent NF-kappa B activation by retaining NF-kappa B in the cytoplasm.


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  		Fig. 5.   Inhibition of ILK suppresses Ikappa B Ser-32 phosphorylation and prevents its degradation. Ikappa B and Ikappa B Ser-32 phosphorylation and protein levels were determined by Western blot analysis in J774 cells exposed to KP-392 (50 µM) for 1 h prior to exposure to LPS (1 µg/ml) and KP-392 (50 µM) for the indicated times.

Inhibition of ILK Suppresses LPS-stimulated COX-2 Expression in J774 Cells and Human Macrophages-- The regulation of iNOS production differs between human and mouse macrophages, in large part because of the differences between the human and murine iNOS promoters. It is known that the induction of another pro-inflammatory protein, COX-2, by LPS stimulation in mouse macrophages also involves the activation of NF-kappa B (53-56). Recent publications have also identified NF-kappa B as a major regulator of COX-2 expression in humans (29, 57, 58). Therefore, to investigate whether the ILK inhibitor is effective in decreasing inflammatory responses and regulating NF-kappa B activity in humans, we analyzed LPS-stimulated cyclooxygenase-2 expression.

We first determined whether ILK was present in human peripheral macrophages. As can be observed in Fig. 6, ILK expression is readily detectable in human alveolar macrophages. We next incubated J774 cells and human peripheral monocyte-derived macrophages with LPS and increasing amounts of KP-392. As shown in Fig. 7, A and B, LPS stimulated COX-2 is inhibited in a dose-dependent manner with increasing amounts of ILK inhibitor, in both mouse and human macrophages.


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  		Fig. 6.   Expression of integrin-linked kinase in human alveolar macrophages. Human lung biopsy specimens were fixed with paraformaldehyde and imbedded in paraffin. A, rabbit IgG control staining on lung biopsy. The paraffin blocks were sliced into 5-µm-thick sections and stained with hematoxylin, and incubated with rabbit IgG antibody, at the same concentration as the ILK antibody. Staining was carried out as indicated under "Experimental Procedures." This is a representation of 36 different fields and 6 different cases. B, ILK staining on lung biopsy. A section from the same block of tissue was stained with anti-ILK (Upstate Biotechnology Inc.) antibody. This is a representation of 36 different fields and 6 different cases.


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  		Fig. 7.   A, inhibition of ILK suppresses COX-2 expression in J774 cells. Cells were incubated with LPS (500 ng/ml), and the indicated amounts of KP-392 for 24 h. COX-2 expression were measured by Western blot analysis. B, inhibition of ILK suppresses COX-2 expression in human peripheral macrophages. Cells were incubated with LPS (500 ng/ml), and the indicated amounts of KP-392 for 24 h. COX-2 expression were measured by Western blot analysis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The expression of the inducible form of nitric oxide synthase, which catalyzes the production of nitric oxide from L-arginine, is regulated by the transcription factor NF-kappa B in murine macrophages in response to LPS and cytokines (1). Macrophage-derived NO is an important host defense and microbial and tumor cell killing agent (1), as well as a regulator of proinflammatory genes in vivo. The ability to modulate iNOS expression could potentially control chronic and acute inflammatory diseases; therefore, it is important to understand the regulation of iNOS.

In this paper we have provided novel data indicating that the integrin-linked kinase, ILK, which couples integrins and growth factors to downstream signaling pathways (11), can regulate iNOS expression and NO production in murine macrophages and regulate the expression of a pro-inflammatory protein, COX-2, in both murine and human macrophages. ILK is a phosphatidylinositol 3-kinase-dependent kinase (17-19, 59) capable of regulating the phosphorylation and activation of PKB/Akt, which has recently been shown to regulate NF-kappa B activation by activating Ikappa B kinase (8, 9). Here we have shown that ILK can also regulate NF-kappa B activation in a physiologically relevant system. Our data indicates that ILK activity is rapidly stimulated in response to LPS in murine macrophages, and this results in the phosphorylation and degradation of Theta kappa beta . Importantly, we have shown here that inhibition of ILK using a specific small molecule inhibitor results in the inhibition of Theta kappa beta phosphorylation on Ser-32 and the prevention of its degradation. In addition, ILK inhibition suppresses LPS-stimulated NF-kappa B promoter activity as well as iNOS promoter activity and NO production. The inhibition of ILK also inhibits iNOS protein expression. Furthermore, the stable expression of dominant-negative ILK decreased the iNOS expression and NO production in LPS-stimulated J774 cells, and the over-expression of wild-type ILK enhanced iNOS expression and NO production in LPS-stimulated J774 cells.

The precise mechanism involved in the regulation of Ikappa B phosphorylation by ILK is not yet clear and is under investigation. A possible mechanism is that ILK is a critical upstream mediator of NF-kappa B activation through its capacity to regulate PKB/Akt kinase activity by phosphorylating Ser-473 (18). In this paper we have demonstrated that ILK-stimulated NF-kappa B activity is inhibited by dominant-negative PKB/Akt. This implicates PKB/Akt in the ILK regulation of NF-kappa B.

Our data further suggest that ILK may play a pivotal role in the regulation of NO production by coupling integrin and LPS signaling. It has been shown that NO production is significantly stimulated in the presence of integrin activation (61, 62). Furthermore, ligation of alpha 5beta 1 integrin with a specific antibody stimulates NO production (50, 61, 62). Because ILK can interact directly with the cytoplasmic domains of integrin beta 1 and beta 3 subunits and can couple integrins to the actin cytoskeleton and downstream signaling components such as PKB/Akt (11), it is likely that the integrin-mediated stimulation of NO involves ILK. Thus ILK appears to be an important mediator of NO production by iNOS in macrophages and may play a role in other cell types such as endothelial cells, chondrocytes, and osteoblasts.

Because human and mouse iNOS promoters are very different, we have also demonstrated the role of ILK on inflammatory molecule expression in human macrophages by assessing the expression of COX-2. The LPS-inducible expression of COX-2, also a proinflammatory enzyme, is regulated by NF-kappa B in both mice and human promoters (28, 34, 60, 61). As shown by our results, ILK plays a role in regulating the expression of COX-2 in human macrophages. Because ILK has been shown to regulate transcription factors such as AP-1 and CREB (15, 16), it is probable that ILK could regulate the transcription of COX-2 through an NF-kappa B-, AP-1-, and/or CREB-dependent manner. Investigations are currently under way to determine the identity of the transcription factors involved in ILK-regulated transcription activity of the COX-2 promoter.

Macrophages can produce NO and prostaglandins, which are regulated by the expression of iNOS and COX-2 upon stimulation with LPS. Our results show that inhibiting ILK function using either a small molecule (KP-392) or dominant-negative ILK cDNA can result in the inhibition of NF-kappa B activity as well as NF-kappa B-dependent expression of both iNOS and COX-2. These results implicate ILK as a novel player in the regulation of NO and prostaglandin production and suggest that the inhibition of ILK may be of therapeutic benefit in controlling destructive inflammatory processes triggered by NO and COX-2.

    ACKNOWLEDGEMENT

We thank Louise Clarke for help in preparing this manuscript.

    FOOTNOTES

* This work was supported by grants from the Canadian Institute of Health Research (CIHR) (to S. D. and A. M.), the National Cancer Institute of Canada (to S. D.), and the CIHR M.D./Ph.D. program (studentship to C. T.).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: Jack Bell Research Center, 2660 Oak St., Vancouver, British Columbia V6H 3Z6, Canada. Tel.: 604-875-5655; Fax: 604-875-5452; E-mail: Sdedhar@interchange.ubc.ca.

Published, JBC Papers in Press, November 27, 2001, DOI 10.1074/jbc.M108673200

    ABBREVIATIONS

The abbreviations used are: iNOS, inducible nitric oxide; ILK, integrin-linked kinase; PKB/Akt, protein kinase B; NO, nitric oxide; COX-1 and -2, cyclooxygenase-1 and -2; LPS, lipopolysaccharide; AP-1, activator protein-1; CREB, cAMP-responsive element-binding protein; FBS, fetal bovine serum; TBS, Tris-buffered saline.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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