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Published online before print March 30, 2005
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/NF-
B pathway
Immunopathology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
1 Correspondence: Immunopathology Section, 30 Convent Drive, Building 30, Room 3A-300, NIDCR/NIH, Bethesda, MD 20892-4352. E-mail: lwahl{at}dir.nidcr.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and granulocyte macrophage-colony stimulating factor, leads to the production of significant amounts of MMP-9. Here, we show that LPS stimulation of monocytes results in MMP-9 production through a phosphatidylinositol-3 kinase (PI-3K)/Akt/inhibitor of 
B (IB) kinase- (IKK)/nuclear factor (NF)-B pathway. This new role for Akt in signaling leading to MMP-9 production was demonstrated by inhibitor and immunoprecipitation studies. LY294002 or wortmannin, inhibitors of PI-3K, suppressed LPS-induced Akt activity and MMP-9 production. Evidence for the participation of Akt in monocyte MMP-9 synthesis was demonstrated by the inhibition of MMP-9 by SH-5, a specific inhibitor of Akt. The mechanism by which Akt regulates MMP-9 is through the activation of NF-B, as shown by coimmunoprecipitation of the phosphorylated form of IKK and Akt as well as the SH-5 suppression of the dissociation of IB from NF-B and the activation of NF-B p65. The role of NF-B in regulation of MMP-9 was demonstrated further by the inhibition of MMP-9 production by proteasome inhibitors, lactacystin and MG-132, which prevented the ubiquitination and dissociation of IB from NF-B. This is the first demonstration that Akt is involved in the signaling pathway leading to the production of monocyte MMP-9 and provides an additional approach in the regulation of this enzyme in human primary monocytes.
Key Words: lipopolysaccharide • pleckstrin homology • PIP3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) induces a number of MMPs, including MMP-9 (gelatinase B), which degrades collagen denatured (gelatin) by MMP-1, -8, and -13 cleavage of fibrillar collagens, such as types I, II, and III. Additionally, MMP-9 degrades laminin and type IV collagen, components of the basement membrane. Thus, these enzymes are involved in the connective tissue loss associated with chronic inflammatory diseases as well as the migration of cells out of the bloodstream and through the ECM.
Production of MMPs by human monocytes/macrophages following stimulation with agents such as concanavalin A, LPS, or ECM components occurs, in part, through a prostaglandin E2 (PGE2)-cyclic adenosine monophosphate-dependent pathway [4
5
6
7
8
]. In contrast, stimulation of monocytes with GM-CSF and/or TNF- induces MMP-9 through a PG-independent pathway [9
]. However, cytokine-induced MMP-9 can be enhanced by the addition of exogenous PGE2 [10
]. These findings indicate that multiple pathways are involved in the induction of MMPs.
We have previously shown that LPS induces production of MMP-9 by monocytes through the nuclear factor (NF)-B pathway [10
]. However, the upstream components involved in the activation of NF-B that lead to MMP-9 production by monocytes still remain to be elucidated. Several studies using cell lines have demonstrated that NF-B can be regulated by Akt/protein kinase B (PKB) through inhibitor of B (IB) kinase (IKK)/IB in a cell- and stimulus-specific manner [11
12
13
14
]. Recent evidence has demonstrated that the cell specificity of NF-B activation by Akt is dependent on a high proportion of IKK to IKKß in cells [15
]. Therefore, our objective was to examine the potential role of Akt in the signaling pathway leading to the production of MMP-9 in monocytes stimulated with LPS. Here, we report that LPS induces the production of MMP-9 by human primary monocytes through a pathway involving Akt, IKK, and NF-B.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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agarose beads, anti-IKK, anti-IKKß, and protein A/G agarose beads (Santa Cruz Biotechnology, CA); LPS (Difco, Detroit, MI); wortmannin, LY294002, lactacystin, and MG-132 (Z-Leu-Leu-Leu-Chinese hamster ovary, Biomol, Plymouth Meeting, PA); SH-5 (Alexis Biochemicals, San Diego, CA); phospho-glycogen synthase kinase (GSK)-3/ß (Ser21/9) antibody, GSK-3 fusion protein, kinase buffer, and adenosine 5'-triphosphate (ATP; Cell Signaling Technology, Beverly, MA); elution buffer (Pierce Biotechnology, Rockford, IL); and nuclear extract kit, TransAMTM NF-B transcription factor assay kit (Active Motif, Carlsbad, CA).
Purification of human monocytes and culture conditions
Human peripheral blood monocytes were obtained by leukapheresis of normal volunteers at the Department of Transfusion Medicine at the National Institutes of Health (Bethesda, MD). The monocytes were purified by counter-flow centrifugal elutriation as described previously [10
]. Monocytes were enriched to >90%, as determined by morphology, nonspecific esterase staining, and flow cytometry. Purified monocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker, Walkersville, MD), supplemented with 2 mM L-glutamine (Mediatech, Herndon, VA) and 10 µg/ml gentamicin sulfate (BioWhittaker) at 37°C in a humidified atmosphere containing 5% CO2. LPS, wortmannin, LY294002, lactacystin, and SH-5 were added to some of the cultures. Dimethyl sulfoxide (Me2SO) was used to dissolve some of the reagents, and the highest final concentration added to the culture was 0.1%. Unless otherwise stated, monocytes were adhered for 30 min before the addition of reagents. Each experiment was repeated a minimum of three times with different donors.
Detection of MMP-9 by Western blot analysis
For determination of the protein levels of MMP-9 produced by monocytes, proteins in the supernatants of 48 h-conditioned medium were precipitated with cold ethanol (final concentration of 60%) at –70°C as described previously [10
]. The proteins from equal portions of the conditioned medium were separated on a Novex 8–16% Tris-glycine polyacrylamide gel (Invitrogen, Carlsbad, CA) and then transferred onto nitrocellulose membranes, which were blocked with 5% nonfat dry milk in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCI, and 0.1% Tween 20. The membranes were then incubated overnight with primary antibodies. Western blots were analyzed by the addition of Alexa Fluor 680 secondary antibody (Molecular Probes, Eugene, OR), and the infrared fluorescence was detected with the Odyssey infrared imaging system (LI-COR, Lincoln, NE). The fluorescence intensity of each blot was measured with the densitometric program in the Odyssey infrared imaging system.
Immunopreciptation and Western blot assay
Monocytes were harvested at specific times after treatment with reagents and lysed in lysis buffer [20 mM, pH 7.5, Tris, 150 mM NaC1, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, and a cocktail of proteinase inhibitors (Roche Molecular Biochemicals, Indianapolis, IN)]. After equalizing the protein to 1.5 mg/300 µl in a 1.5-ml Eppendorf tube, the appropriate antibody bound to agarose beads was added, and the samples were rotated overnight at 4°C. After the beads were washed three times with lysis buffer, the samples were subjected to Western blot analysis as described above. The membranes were then stripped with elution buffer and reprobed with antibodies against the nonphosphorylated protein as a measure of equal loading. Controls for the immunoprecipitation used the same procedure, except agarose beads contained only mouse IgG.
Akt kinase assay
Monocytes were harvested at specific times after treatment with reagents and lysed in lysis buffer. After equalizing the protein to 1.0 mg/200 µl in a 1.5-ml Eppendorf tube, 10 µl Aktl/2 antibody was added, and the samples were rotated overnight at 4°C. Then 20 µl protein A/G agarose bead slurry was added, and the samples were rotated at room temperature for 2 h. The beads were washed twice with lysis buffer and kinase buffer, and the samples were suspended in 50 µl kinase buffer supplemented with 1 µl 10 mM ATP and 1 µg GSK-3 fusion protein and incubated at 30°C for 30 min. The phosphorylation of GSK-3 fusion protein was analyzed by Western blot with phosphor-GSK-3/ß (Ser2119) antibodies.
NF-B p65 activiation assay
Monocytes were harvested at specific times after treatment with reagents, and the nuclear extracts and the activation assay of p65 were performed according to the instructions of the manufacturer of the TransAMTM/NF-B kit. Briefly, 2 µg/well nuclear extract protein was added to a microtiter plate coated with a specific oligonucleotide of p65. The coated plate was then incubated for 1 h at room temperature with mild agitation, after which a primary antibody recognizing the p65 was added, and the plate was incubated for an additional 1 h at room temperature. Anti-IgG horseradish peroxidase-conjugated secondary antibody was then added, and the plate was incubated for 1 h at room temperature. At the end of the hour, the developing and stop solution were added, and an optical density of 450 nm (OD450) was read on a Wallac Victor 1420 multilabel counter (Perkin Elmer Life Sciences, Shelton, CT). Three duplicates were done for each sample.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (MG-132) was added to monocyte cultures. As shown in Figure 2C
, Akt1/2 activity induced by LPS was inhibited by wortmannin and SH-5, whereas the NF-B inhibitor MG-132 had no effect. These findings demonstrate that PI-3K regulates phosphorylation of Akt, whereas NF-B is downstream and does not influence Akt in monocytes.
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B is involved in the regulation of monocyte MMP-9 expressionB. To determine the role of NF-B in the regulation of MMP-9, proteasome inhibitors (MG-132 and lactacystin), which suppress NF-B activation by blocking IB degradation, were added to monocytes. Both proteasome inhibitors were effective in the suppression of MMP-9 production, and 10 µM MG-132 or 2.5 µM lactacystin caused complete inhibition of MMP-9 (Fig. 3A
and 3B
).
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B activation in LPS-stimulated monocytesB activation through its interaction with IKK but not IKKß, which is involved in the phosphorylation of IB bound to NF-B [13
14
15
]. Once IB is phosphorylated, it is ubiquitinated and degraded by the proteasome complex, allowing NF-B to translocate to the nucleus. To determine if Akt regulated NF-B activation in the monocyte in this manner, we first examined the IKK/KKß proportion in human monocytes because of the variation in the IKK/KKß ratio found in various cell lines [15
]. As shown in Figure 4A
, IKK is predominant, whereas IKKß is expressed at low levels in monocytes. This is in contract to the monocyte cell line THP-1, in which there is a substantial level of IKKß. Second, we analyzed the interaction between IKK and Akt. As shown in Figure 4B
, using primary human monocytes, there is an association of Akt1/2 with IKK that is independent of monocyte activation. Use of mouse IgG as a control in the immunoprecipitation procedure did not pull down Akt1/2 or IKK (Fig. 4C)
.
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in the Akt/IKK complex. As shown in Figure 5A
, the phosphorylation of Akt (Ser473) and IKK (Thr23) in the complex of IKK/Akt corresponded to the dose of LPS (Fig. 5A) . Additionally, the phosphorylation of Akt and IKK in the complex was inhibited by wortmannin and SH-5 but not by MG-132 (Fig. 5B)
. Finally, we analyzed the degradation of IB by Western blot (Fig. 6A
and 6C
) and the activation of p65 by enzyme-linked immunosorbent assay (ELISA; Fig.6B
and 6D
) after the treatment of LPS in the absence or presence of specific inhibitors. Activation of monocytes with LPS resulted in degradation of IB and activation of p65, which were blocked by inhibitors of PI-3K (wortmannin), Akt (SH-5), and proteasome (MG-132; Fig. 6
). These findings demonstrate that Akt is a central component in the pathway leading to the downstream activation of NF-B in monocytes and the subsequent production of MMP-9. The LPS-induced PI-3K/Akt/IKK/NF-B pathway leading to monocyte MMP-9 production and the specific inhibitors of this pathway are summarized in Figure 7
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three members of the Akt family have been isolated, and these are now referred to as Akt1/PKB, Akt2/PKBß, and Akt3/PKB
. They are products of distinct genes but are highly related, exhibiting greater than 80% homology at the amino acid level [21
]. The three genes are expressed differentially: Akt1/PKB and Akt2/PKBß are widely displayed, and Akt3/PKB has a restricted tissue distribution. Of interest in our studies is that Akt1 and Akt2 are expressed and associated with IKK in control or activated monocytes (Fig. 4B)
. This indicates that Akt associates with IKK constitutively, at least in the monocyte. This is consistent with the finding in other cell types stimulated with platelet-derived growth factor [14
], TNF [13
], and LPS [22
]. IKK is a complex composed of three subunits: IKK (IKK1), IKKß (IKK2), and IKK {NF-B essential modulator (NEMO), IKß kinase associated protein-1 (IKKAP-1) [23
]}. IKK and IKKß are the catalytic subunits of the complex, sharing 52% overall sequence identity and 65% identity in their catalytic domains. The third subunit, IKK/NEMO, is the regulatory subunit and is not related to the catalytic subunits [24
]. Expression and the ratio of IKK to IKKß, which homo- and heterodimerize, vary among cell types. NF-B, in the cells with a high proportion of IKK to IKKß, is sensitive to Akt activity [15
]. Our data show that IKK is predominant in human primary monocyte, whereas IKKß is barely detectable. This is in contrast to a monocyte cell line THP-1, which has a significant amount of IKKß (Fig. 4A)
. These data indicate that there may be considerable variation in the ratio of IKK to IKKß between primary monocytes and monocyte cell lines, such as THP-1. Gel-filtration analysis indicates that IKK is a large complex, 700–900 kDa in size, suggesting the presence of additional components. Recently, Cdc 37 and Hsp 90 were suggested to serve as components of the IKK complex [25
]. Based on our data (Fig. 4B)
, we hypothesize that Aktl and Akt2 may also serve as additional components of the IKK homodimmer complex in the primary monocyte. Cell stimulation with a variety of agonists triggers signal transduction pathways that ultimately result in activation of a specific IKK [23
]. Our data show that Akt binds to IKK constitutively, and their phosphorylation occurs following with LPS stimulation (Fig. 5A) ; moreover, the phosphorylation of Akt is regulated by PI-3K, and Akt subsequently phosphorylates IKK, as demonstrated with inhibitors (Fig. 5B)
.
Phosphorylation of IBs by phosphorylated IKK tags them for polyubiquitination by a specific ubiquitin ligase belonging to the Skp-l/Cul/F box family [26
]. The actual recognition of N-terminally phosphorylated IBs is carried out by a WD repeat- and F box-containing protein, ß-TrCP [26
]. Upon ubiquitination, the IB proteins are degraded rapidly by the proteasome complex, thereby freeing NF-B, which then enters the nucleus, binds to DNA, and activates transcription. It has been shown that Akt regulates NF-B activation directly through activation of IKK or phosphorylation of RelA [27
, 28
]. Our findings demonstrated that the PI-3K/Akt pathway also regulates IB degradation and activation of NF-B in human primary monocytes stimulated by LPS (Fig. 6)
.
In summary, the findings presented here demonstrate that activation of monocytes by LPS induces MMP-9 through a PI-3K/Akt/IKK/NF-B pathway, as outlined in Figure 7
. This pathway can be interrupted by inhibition of PI-3K, Akt, or the proteasome complex. These data further our understanding of the pathways included in the regulation of MMP-9 and suggest a possible avenue for intervention in connective tissue destruction.
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ACKNOWLEDGEMENTS |
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Received September 7, 2004; revised February 1, 2005; accepted March 7, 2005.
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D. H. Li, J. W. Tung, I. H. Tarner, A. L. Snow, T. Yukinari, R. Ngernmaneepothong, O. M. Martinez, and J. R. Parnes CD72 Down-Modulates BCR-Induced Signal Transduction and Diminishes Survival in Primary Mature B Lymphocytes J. Immunol., May 1, 2006; 176(9): 5321 - 5328. [Abstract] [Full Text] [PDF]
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