Exposure of human alveolar macrophages to bacterial LPS results in activation of a number of signal transduction pathways. An early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation. We observed that LPS exposure results in phosphorylation of Akt (serine 473). We found this using both phosphorylation-specific Abs and also by in vivo phosphorylation with 32P-loaded cells. AKT activation resulted in the phosphorylation-dependent inactivation of glycogen synthase kinase (GSK-3) (serine 21/9). We found that both of these events were linked to PI 3-kinase because the PI 3-kinase inhibitors, wortmannin and LY294002, inhibited LPS-induced phosphorylation of both AKT and GSK-3. Inactivation of GSK-3 has been shown to reduce the ubiquitination of β-catenin, resulting in nuclear accumulation and transcriptional activity of β-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear β-catenin in human alveolar macrophages and expression of genes that require nuclear β-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of β-catenin in the nucleus of any cell, including alveolar macrophages.
Lipopolysaccharide (LPS) is an essential component of the cell wall of Gram-negative bacteria. It can interact with LPS-binding protein (LBP),3 allowing binding to CD14 and association with at least one other cell membrane receptor that contains an intracellular signaling domain (the most likely candidate being one of the Toll receptors) (1, 2, 3, 4, 5, 6). LPS signaling has been specifically linked to the Toll-like receptor 4 (TLR4) by a number of investigators (2, 4, 5, 7). The proximal signaling pathways activated by LPS are similar to those used by IL-1 or IL-18 (myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), TNFR-associated factor 6 (TRAF 6) (8, 9). More distally, LPS exposure has been linked to activation of a number of signaling cascades, ultimately resulting in the production of numerous inflammatory mediators (10, 11, 12).
Alveolar macrophages play a central role in the response of the lung to bacterial infection. They are specialized cells, which respond to infection by phagocytosis and killing of bacteria (13, 14, 15). They also respond to LPS by releasing large amounts of inflammatory mediators (12, 16, 17). We and others have shown that activation of various mitogen-activated protein (MAP) kinase pathways is necessary for expression of cytokine genes in alveolar macrophages in response to LPS (18, 19). More recently, we have shown that in human alveolar macrophages, LPS activates phosphatidylinositol 3 kinase (PI 3-kinase) (20).
PI 3-kinase is an enzyme that participates in multiple cell processes. It has been linked to cell growth, transformation, differentiation, insulin signaling, and cell survival (21, 22, 23, 24). It consists of a regulatory (p85) and a catalytic (p110) subunit. PI 3-kinase is recruited to the inner surface of the plasma membrane by Src homology 2 (SH2) domains in the regulatory unit. Once there, it catalyzes the transfer of ATP to the D-3 position of the inositol ring of membrane-localized phosphoinositides (22, 23, 25). Depending on the lipid substrate, this can generate three possible species. PI3P is constitutively present in all cells and its levels do not change after agonist stimulation. However, PI3,4P and PI3,4,5P are nominally absent in most cells, and amounts increase dramatically with stimulation of PI 3-kinase (26). The production of PI3,4,5P, especially, results in the recruitment to the membrane of a number of signaling molecules. The first kinase recruited and activated after PI 3-kinase activation is 3-phosphoinositide-dependent kinase (PDK-1), a kinase with multiple downstream substrates (27, 28). PDK-1 is recruited to PI 3-kinase-phosphorylated lipids (PI3,4,5P) by its pleckstrin homology (PH) domain. It then interacts with and phosphorylates various substrates of the AGC class of protein kinases (27, 28). Akt (protein kinase B), which is phosphorylated and activated by PDK-1 (on threonine 308 in the activation loop), is an important effector of the PI 3-kinase pathway (29, 30, 31). Activation of Akt results in the phosphorylation of a number of substrates that have potential importance in LPS signaling (glycogen synthase kinase (GSK-3), Bad, caspase 9, Forkhead transcription factors, Raf, IκB kinase, phosphodiesterase 3B, and endothelial NO synthase). Phosphorylation of these substrates by Akt results in either activation or inactivation depending on the substrate (32, 33, 34, 35).
Of particular note is GSK-3, a previously described member of the Wnt signaling pathway (for reviews of this pathway, see papers by Behrins and Morin; Refs. 36 and 37). In the Wnt pathway, GSK-3 phosphorylation and inactivation leads to the accumulation and nuclear translocation of β-catenin. In the resting cell, GSK-3 is in the cytoplasm in a complex with axin, adenomatous polyposis coli (APC), and β-catenin (37). GSK-3 phosphorylates β-catenin, which targets it for ubiquitination and degradation, inhibiting the transcriptional activity of β-catenin. Inhibition of GSK-3 happens after Wnt binds to its receptor (Frizzled), which activates Dishevelled, resulting in phosphorylation of GSK-3 and accumulation of β-catenin (36, 37, 38, 39). Mutations in this pathway have been commonly observed in some cancers, including colorectal carcinomas (36, 39). This is thought to be due to the effect of β-catenin on T cell factor (TCF)/Lef-1-driven transcription of pro-growth genes. Some of the genes known to be transcriptionally activated by TCF/Lef-1 signaling include c-jun, matrilysin, c-myc, fibronectin, cyclin D1, fra-1, e cadherin, and fibronectin (32, 40, 41, 42). Besides the Wnt pathway, GSK-3 is also phosphorylated (serine 21/9, αβ) and inactivated by Akt downstream of PI 3-kinase. However, this has never been shown to correlate with increased β-catenin. Furthermore, no studies have shown that LPS inactivates GSK-3 or increases β-catenin. In this study we show that exposure to LPS results in activation of Akt, inactivation of GSK-3, and accumulation of β-catenin in the nucleus. All of these events occur downstream of PI 3-kinase, demonstrating a novel means of increasing amounts of β-catenin. These studies are the first to show that LPS activates this pathway.
Materials and Methods
Isolation of human alveolar macrophages
Alveolar macrophages were obtained from bronchoalveolar lavage as previously described (43). Briefly, normal volunteers with a lifetime nonsmoking history, no acute or chronic illness, and no current medications underwent bronchoalveolar lavage. The lavage procedure used five 25-ml aliquots of sterile, warmed saline in each of three segments of the lung. The lavage fluid was filtered through two layers of gauze and centrifuged at 1500 × g for 5 min. The cell pellet was washed twice in HBSS without Ca2+ and Mg2+ and suspended in complete medium, Roswell Park Memorial Institute (RPMI) 1640 tissue culture medium (Life Technologies/BRL, Gaithersburg, MD) with 100 ng/ml LBP (a gift from Peter Tobias, Scripps Research Institute, La Jolla, CA) and gentamicin (80 μg/ml). Differential cell counts were determined using a Wright-Giemsa-stained cytocentrifuge preparation. All cell preparations had between 90 and 100% alveolar macrophages. This study was approved by the Committee for Investigations Involving Human Subjects at the University of Iowa.
Isolation of whole cell and nuclear extracts
Alveolar macrophages were cultured for various times with or without 1 μg/ml LPS. Whole cell protein was obtained by lysing the cells on ice for 20 min in 500 μl of lysis buffer (0.05 M Tris-HCl, pH 7.4, 0.15 M NaCl, 1% Nonidet P-40, 0.5 M PMSF, 50 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μg/ml pepstatin, 0.4 mM sodium orthovanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate, all obtained from Roche, Indianapolis, IN). The lysates were then sonicated for 20 s and spun at 15,000 × g for 10 min, and the supernatant was saved. Protein determinations were made using the method of Bradford (44).
The nuclear pellets were prepared by resuspending cells in 400 μl of lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA), placing them on ice for 15 min, and then vigorously mixing after the addition of 25 μl of 10% Nonidet P-40. After a 30-s centrifugation (16,000 × g, 4°C), the pelleted nuclei were resuspended in 50 μl of extraction buffer (50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol) and incubated on ice for 20 min. Nuclear extracts were stored at −70°C.
Isolation of cytoplasmic and membrane proteins
Alveolar macrophages were cultured for various times with and without LPS at 1 μg/ml. Cell pellets were suspended in 200 μl lysis buffer (see whole cell protocol) without Tween 20 detergent, pulse sonicated (1 s × 30) on ice, and then spun at 100,000 × g for 1 h. The supernatant (cytoplasmic fraction) was saved at −70°. The membrane pellet was resuspended in 100 μl lysis buffer with 1% Tween 20 and sonicated for 5 s on ice. After 20 min, cell debris was removed (15,000 × g for 10 min), and the supernatant was saved. Western analysis was performed as described below.
Alveolar macrophages were cultured in complete medium with or without LPS (1 μg/ml; Sigma, St. Louis, MO). After isolating protein, 200–600 μg from each sample was removed for immunoprecipitation. The samples were cleared by incubating for 2 h with 1 μg/sample of rabbit IgG and 10 μl/sample of GammaBind Sepharose (Pharmacia, Piscataway, NJ). After centrifuging, the supernatants were transferred to a tube containing 3 μg/sample of Ab bound to GammaBind Sepharose, then rotated at 4°C overnight. The beads were subsequently washed three times with high salt buffer (1 M Tris-HCl pH 7.4, 0.50 M NaCl, and 1% Nonidet P-40) and three times with lysis buffer without protease inhibitors. The immunoprecipitated complexes were released with 2× sample buffer for Western analysis or used in a kinase activity assay.
Western analysis for the presence of particular proteins or for phosphorylated forms of proteins was performed on whole cell, cytosol/membrane, or nuclear proteins from alveolar macrophage experiments. Protein (50–100 μg) was mixed 1:1 with 2× sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05% bromophenol blue, and 1.25 M Tris-HCl pH 6.8; all obtained from Sigma), loaded onto a 10% SDS-PAGE gel, and run at 40 V for 2 h. Cell proteins were transferred to nitrocellulose (ECL; Amersham, Arlington Heights, IL) overnight at 30 V. Equal loading of the protein groups on the blots was evaluated using Ponceau S, a staining solution designed for detecting proteins on nitrocellulose membranes (Sigma). Images of the Ponceau S stain are included in some of the figures to demonstrate equal loading of the samples. The nitrocellulose was then blocked with 5% milk in TBST for 1 h, washed, and then incubated with the primary Ab (anti-phosphorylated extracellular signal-related kinase, Sigma; all other phospho-specific Abs, Cell Signaling, Beverly, MA; anti-PDK-1, Upstate Biotechnology, Waltham, MA; other nonphospho-specific Abs, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. The blots were washed four times with TBST and incubated for 1 h with HRP-conjugated anti-IgG Ab (Jackson ImmunoResearch, West Grove, PA). Immunoreactive bands were developed using a chemiluminescent substrate (ECL Plus, Amersham, Arlington Heights, IL or SuperSignal West Femto, Pierce, Rockford, IL). An autoradiograph was obtained, with exposure times of 10 s to 2 min.
In vivo phosphorylation of Akt
Alveolar macrophages were labeled with 1.25 mCi of 32Pi/group (NEN Life Science Products, Boston, MA) in phosphate-free RPMI 1640 without serum for 3 h at 37°C. The cells were harvested and placed in RPMI 1640 with 100 ng/ml LBP and treated with LPS for various times at 37°C. The cells were harvested, resuspended in lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M Na3PO4, pH 7.2, 2 mM Na3VO4, 1 μM okadaic acid, 100 μg/ml PMSF, 50 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μg/ml pepstatin, all obtained from Roche), and sonicated. Akt was immunoprecipitated from the lysate, and the sample was separated on a 10% SDS-PAGE discontinuous gel as described above. The gel was dried, and an autoradiograph was obtained.
GSK-3 activity assay
After immunoprecipitating GSK-3 from LPS-treated alveolar macrophages, the protein-containing pellet was washed twice with kinase buffer (20 mM MgCl2, 25 mM HEPES, 20 mM β-glycerophosphate, 20 mM p-nitrophenylphosphate, 20 mM sodium orthovanadate, and 2 mM DTT). The pellet was then suspended in 20 μl kinase buffer and the following were added: 20 μM ATP, 5 μCi γATP (32) (BLU 002Z; DuPont-NEN, Boston, MA), and 10 μg myelin basic protein (MBP; Sigma). The total volume of sample plus additions at this point was 25 μl. The reaction was continued for 30 min at 25°C and then stopped by the addition of 25 μl/sample of 2× sample buffer. The samples were boiled for 5 min, then run on a 12% SDS-PAGE gel. The gel was dried, and autoradiography performed to visualize the 32P-labeled MBP. Densitometry was performed on films, and fold difference was calculated as experimental sample/control sample.
PI 3-kinase activity assay
After culture, whole cell lysates were obtained and PI 3-kinase was immunoprecipitated using an Ab to the p85 regulatory subunit of PI 3-kinase (528107; Calbiochem, San Diego, CA). Activity was assayed by measuring the amount of tyrosine phosphorylation of the immunoprecipitated protein (45). The immunoprecipitation product was divided into two portions, and two separate gels were run (20% for equal loading analysis and the remainder for analyzing tyrosine phosphorylation). The gels were transferred to nitrocellulose, and Western blot analysis was performed. The activity blot was stained with an Ab to phosphorylated tyrosines (PY20; Santa Cruz Biotechnology) and visualized with chemiluminescence (ECL Plus; Amersham). Equal loading was determined by staining the remaining blot for p85.
Statistical analysis of the densitometric data was performed by determining the fold increase of all the samples as they relate to the control.
LPS activates PI 3-kinase in alveolar macrophages
We initially wanted to confirm that LPS activated PI 3-kinase in alveolar macrophages. We did this by immunoprecipitating the p85 regulatory unit from PI 3-kinase and evaluating tyrosine phosphorylation using a phosphorylated tyrosine-specific Ab (45). We found that LPS treatment resulted in the tyrosine phosphorylation of the p85 subunit of PI 3-kinase and that this activation was inhibited by LY 294002, a PI 3-kinase inhibitor (Fig. 1).
LPS causes membrane translocation of PDK-1
PDK-1 is a constitutively active kinase, the activity of which is regulated by binding to PI3,4P and PI3,4,5P on the inner plasma membrane (22, 30). To evaluate the effect of LPS on this kinase, we harvested both cytosolic and membrane proteins and evaluated PDK-1 translocation. We found that LPS caused a time-dependent increase in the amount of PDK-1 found in the membrane of alveolar macrophages (Fig. 2). These two observations, PI 3-kinase activation and membrane translocation of PDK-1, suggested our next experiments: to examine possible LPS activation of kinases known to be downstream of PDK-1.
LPS causes phosphorylation and activation of Akt
Akt is a major substrate of PDK-1. Toker and Newton have shown that PDK-1 phosphorylation of threonine 308 in the activation loop of the catalytic domain of Akt allows autophosphorylation of serine 473 (a hydrophobic phosphorylation site) in the carboxyl-terminal (30). These two events result in generation of a catalytically competent enzyme. We evaluated Akt activation using phosphorylation-specific Abs for both Thr308 and Ser473. Our findings are shown in Fig. 3. We found that LPS caused a sustained increase in amounts of phosphorylated Akt, on both Thr308 and Ser473. Equal loading of the proteins is demonstrated with an Ab specific for total Akt. Shown also is densitometry from data obtained from the phosphorylated Ser473 Ab. LPS caused a rapid activation of Akt, peaking at 30 min, but remaining above baseline up to 6 h posttreatment.
LPS exposure results in Akt phosphorylation in vivo
To confirm the data found with the phosphorylation-specific Abs, we performed an in vivo phosphorylation assay. Alveolar macrophages were loaded with 32P and then treated with LPS. Whole cell protein was isolated, and then Akt was immunoprecipitated and an SDS-PAGE gel was run. This experiment demonstrates ‘in cell’ phosphorylation of Akt after LPS exposure in alveolar macrophages (Fig. 4).
LPS causes phosphorylation and inactivation of GSK-3
GSK-3 is one of the major downstream substrates of Akt. Phosphorylation of serine 21 in the α isoform and serine 9 in the β isoform results in an inactive enzyme that uncouples from its constitutive complex (GSK-3, axin, APC, and β-catenin) (46). We found that LPS treatment resulted in the phosphorylation of GSK-3 on these sites. Shown in Fig. 5,A is an LPS time course demonstrating GSK-3 phosphorylation. Like Akt, GSK-3 demonstrated peak phosphorylation at 15–30 min, with some increases in phosphorylation seen out to 6 h. Of note is the fact that GSK-3β is preferentially phosphorylated. There are equal amounts of unphosphorylated α and β, but the majority of the LPS-induced phosphorylation is on the β isoform. The significance of this is yet to be determined. To confirm that phosphorylation at this site does indeed result in GSK-3 inactivation, we performed a kinase activity assay using immunoprecipitated GSK-3. Alveolar macrophages were treated with LPS, whole cell lysates were obtained, and GSK-3 was immunoprecipitated. GSK-3 activity was determined by evaluating the in vitro ability of GSK-3 to phosphorylate the generic substrate MBP. Fig. 5,B demonstrates that LPS causes a time-dependent inactivation of GSK-3, consistent with the phosphorylation data shown in Fig. 5 A.
LPS-induced phosphorylation of Akt and GSK-3 is linked to PI 3-kinase
To demonstrate that both of these events were tightly linked to LPS-induced PI 3-kinase activation, we treated alveolar macrophages with PI 3-kinase inhibitors (wortmannin and LY294002) and evaluated the appearance of phosphorylated forms. Fig. 6 demonstrates that both Akt and GSK-3 phosphorylation were downstream of PI 3-kinase.
LPS exposure results in increased amounts of β-catenin in the nucleus of alveolar macrophages
In the Wnt pathway, phosphorylation and inactivation of GSK-3 results in the accumulation of β-catenin due to decreased degradation. This increase in β-catenin has been linked to transcriptional activation of genes containing TCF/Lef-1 sites (32, 40, 41, 42). We wanted to determine whether the phosphorylation and inactivation of GSK-3 by LPS could mimic the effects of activation of the Wnt pathway. Alveolar macrophages were cultured with LPS for various time points, nuclear protein was isolated, and Western analysis was performed for β-catenin. Fig. 7 demonstrates that in alveolar macrophages, LPS-induced activation of PI 3-kinase and Akt and inactivation of GSK-3 resulted in the nuclear accumulation of β-catenin. We next wanted to determine whether the β-catenin accumulation was downstream of PI 3-kinase activation. We treated alveolar macrophages with LPS with and without LY294002, a specific PI 3-kinase inhibitor (see Fig. 1). We then evaluated the effect of LY294002 on nuclear β-catenin accumulation. We found that inhibiting PI 3-kinase blocked the LPS-induced increases in nuclear β-catenin (Fig. 8). This is the first description of β-catenin increases in the nucleus linked to the PI 3-kinase pathway rather than the Wnt pathway.
LPS increases amounts of cyclin D1 and connexin 43
Finally, we wanted to determine whether there was physiological relevance to our finding of increased β-catenin. We evaluated the amounts of two proteins whose transcription is linked to β-catenin/TCF/Lef-1 signaling (32, 47). It is not in the scope of this paper to evaluate all the possible events downstream of β-catenin signaling. The two proteins we did evaluate have been strongly linked to β-catenin signaling and will serve as an example of possible downstream PI 3-kinase sequelae to LPS signaling. In Fig. 9, we show that LPS caused increases in the amounts of both cyclin D1 and connexin 43 protein in whole cell protein samples from treated alveolar macrophages. These data suggest that the activation of signaling modules that result in the accumulation of β-catenin is accompanied by specific increases in gene transcription.
This study evaluates the role of LPS in the activation of components of the PI 3-kinase pathway. We have previously shown that in alveolar macrophages, LPS increased PI 3-kinase activity (20). Using this information as a starting point, we have found that downstream of PI 3-kinase, LPS activates PDK-1 and Akt and inactivates GSK-3. We evaluated the implications of this by looking at the nuclear accumulation of β-catenin. In alveolar macrophages, LPS caused the nuclear accumulation of β-catenin, and this was linked to the PI 3-kinase pathway. LPS also activated genes that are dependent on nuclear β-catenin activity. These observations suggest a particular sequence of events in LPS-treated alveolar macrophages that is shown in Fig. 10.
LPS has been shown to activate PI 3-kinase in alveolar macrophages by our laboratory and in other systems by other investigators (48, 49, 50). There has been little work performed on the possible downstream effects of that activation. LPS activation of PI 3-kinase has been linked to activation of the atypical PKC isoform, ζ (51). Venkataraman et al. have shown that in B cells, proliferation is dependent on PI 3-kinase. In macrophages, Procyk et al. have linked LPS-induced extracellular signal-related kinase activation to PI 3-kinase. Several papers by Herrera-Velit have linked LPS signaling (adherence, PKC ζ, lyn) to activation of PI 3-kinase (48, 51, 52). These studies are consistent with our previous study and suggest that LPS activation of PI 3-kinase plays an important role in the cellular response to LPS.
Downstream of PI 3-kinase, there are no reports of LPS-induced activation of Akt. Akt is a serine/threonine kinase that has significant homology to PKC ε and PKA (25). It has been described as a mediator of the effects of insulin, growth factors, cytokines, and various other factors. It is known to be activated by platelet-derived growth factor, insulin, and cell adhesion (53, 54, 55). Downstream affects of Akt include inhibition of a number of pro-apoptotic factors (GSK-3, caspase 9, Forkhead transcription factors, and Bad) and activation of IκB kinase, PDE-3B, eNOS, and p70S6 kinase (32, 33, 34, 35). A large number of studies have shown that constitutively active Akt blocks apoptosis induced by a diverse array of stimuli (UV light, matrix detachment, DNA damage, and anti-fas treatment) (25). Although evaluating the relationship between LPS and apoptosis is beyond the scope of this study, it is interesting to speculate that LPS activation of Akt in alveolar macrophages is related to maintaining cell viability during the release of inflammatory mediators.
We have shown here that LPS activation of Akt results in the inactivation of GSK-3 and increases in nuclear β-catenin. GSK-3 is a constitutively active enzyme that is negatively regulated by the Wnt pathway and by Akt-induced phosphorylation (25, 56, 57). The other previously reported regulator of GSK-3 is found in colon cancer where PKC β2 inactivates GSK-3, resulting in increased cell division and tumorigenesis (39). Phosphorylation of GSK-3 on serine 21/9 results in GSK-3 dissociating from the complex of APC, axin, and β-catenin. When GSK-3 is removed from this complex, it can no longer phosphorylate β-catenin and target it for ubiquitination and proteosome degradation (58). This negative regulation of GSK-3 has been linked to Wnt signaling, growth factors, insulin, and cell/fibronectin interactions (58, 59). The only signal that has been linked to β-catenin accumulation and transcriptional activity is inactivation of GSK-3 by the Wnt pathway (58). In alveolar macrophages, the result of LPS-induced β-catenin accumulation is unknown. In other systems, β-catenin stabilizes a transcription complex containing β-catenin and the transcription factor TCF/Lef-1. The β-catenin-TCF/Lef-1 transcription complex can activate or suppress gene transcription (34, 60, 61). Genes that have been shown to contain TCF/Lef-1 sites and to be positively regulated by β-catenin include c-myc, fibronectin, cyclin D1, c-jun, fra-1, e cadherin, matrilysin, and connexin 43 (32, 40, 41, 42). Our data show that LPS up-regulates transcription of the cyclin D1 and connexin 43 genes. Whether or not LPS results in the up-regulation of any of the other TCF/Lef-1-dependent genes is still to be determined.
One interesting area to be considered because of these findings is the relationship between PI 3-kinase and NF-κB in LPS signaling. LPS is a strong inducer of NF-κB activity, and many of the LPS-induced inflammatory mediators have important NF-κB sites (IL-6, IL-8, TNF-α) (62, 63, 64). Three observations relevant to this pathway are of interest. First, two studies (using platelet-derived growth factor and TNF as stimuli) have demonstrated that Akt mediates IκB kinase α (IKKα) phosphorylation at threonine 23, leading to IκB degradation and NF-κB translocation to the nucleus (65, 66). Secondly, Madrid et al. have shown a further function for Akt in NF-κB activation, up-regulation of the transactivation potential of p65/RelA by phosphorylation of domain 1 (67). In addition, Sizemore et al. have demonstrated that IL-1 signals through IL-1 receptor accessory protein to PI 3-kinase, resulting in phosphorylation of the p65/RelA subunit (24). This process is distinct from NF-κB translocation and necessary for the transcriptional activity of NF-κB. One more study suggests a possible pathway by which the findings in this study could relate to LPS-induced NF-κB activation in alveolar macrophages. A study by Hoeflich using GSK-3β knockout mice has the very interesting finding that GSK-3 is required for the NF-κB-mediated survival response. Mice lacking GSK-3 resemble mice lacking the components of the NF-κB pathway. Fibroblasts from the GSK-3−/− mice had a decreased NF-κB reporter gene response after TNF-α or IL-1 stimulation (68). It is interesting to speculate that the inactivation of GSK-3 has a role to play in NF-κB activation.
This study demonstrates the presence of an important new signaling pathway for LPS in alveolar macrophages. We found that LPS activates Akt, phosphorylates and inactivates GSK-3, and causes nuclear accumulation of β-catenin. This results in increases in at least two of the proteins known to be linked to β-catenin, cyclin D1, and connexin 43. The possible importance of this pathway has yet to be discovered but could include β-catenin-driven gene expression and possible effects on NF-κB activation.
This work was supported by a Department of Veterans Affairs Merit Review, National Institutes of Health Grants ES-09607 and HL-60316 (to G.W.H.), and National Institutes of Health Grant HL03860 (to A.B.C.).
Abbreviations used in this paper: LBP, LPS-binding protein; PI 3-kinase, phosphatidylinositol 3 kinase; GSK-3, glycogen synthase kinase; PDK-1, 3-phosphoinositide-dependent kinase; APC, adenomatous polyposis coli; MBP, myelin basic protein; TCF, T cell factor.