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-Catenin1
Department of Medicine, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin, resulting in nuclear
accumulation and transcriptional activity of -catenin. Consistent
with this, we found that LPS caused an increase in the amounts of PI
3-kinase-dependent nuclear -catenin in human alveolar macrophages
and expression of genes that require nuclear -catenin for their
activation. This is the first demonstration that LPS exposure activates
AKT, inactivates GSK-3, and causes accumulation and transcriptional
activity of -catenin in the nucleus of any cell, including alveolar
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, I
B 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 -catenin. In the
resting cell, GSK-3 is in the cytoplasm in a complex with axin,
adenomatous polyposis coli (APC), and -catenin (37).
GSK-3 phosphorylates -catenin, which targets it for ubiquitination
and degradation, inhibiting the transcriptional activity of
-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 -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 -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, 
) and inactivated by Akt downstream of PI 3-kinase.
However, this has never been shown to correlate with increased
-catenin. Furthermore, no studies have shown that LPS inactivates
GSK-3 or increases -catenin. In this study we show that exposure to
LPS results in activation of Akt, inactivation of GSK-3, and
accumulation of -catenin in the nucleus. All of these events occur
downstream of PI 3-kinase, demonstrating a novel means of increasing
amounts of -catenin. These studies are the first to show that LPS
activates this pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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 x 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 x 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 x 30) on ice, and then spun at 100,000 x 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 x g for 10 min), and the supernatant was saved. Western analysis was performed as described below.
Immunoprecipitation
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 2x sample buffer for Western analysis or used in a kinase activity assay.
Western analysis
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 2x 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
-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 
ATP (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 2x 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
Statistical analysis of the densitometric data was performed by determining the fold increase of all the samples as they relate to the control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
).
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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.
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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.
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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).
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GSK-3 is one of the major downstream substrates of Akt.
Phosphorylation of serine 21 in the isoform and serine 9 in the
isoform results in an inactive enzyme that uncouples from its
constitutive complex (GSK-3, axin, APC, and -catenin)
(46). We found that LPS treatment resulted in the
phosphorylation of GSK-3 on these sites. Shown in Fig. 5A 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-3 is
preferentially phosphorylated. There are equal amounts of
unphosphorylated and , but the majority of the LPS-induced
phosphorylation is on the 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. 5B demonstrates that LPS causes a time-dependent
inactivation of GSK-3, consistent with the phosphorylation data shown
in Fig. 5A.
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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.
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-catenin in the
nucleus of alveolar macrophages
In the Wnt pathway, phosphorylation and inactivation of GSK-3
results in the accumulation of -catenin due to decreased
degradation. This increase in -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 -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
-catenin. We next wanted to determine whether the -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 -catenin accumulation. We found that inhibiting
PI 3-kinase blocked the LPS-induced increases in nuclear -catenin
(Fig. 8). This is the first description
of -catenin increases in the nucleus linked to the PI 3-kinase
pathway rather than the Wnt pathway.
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Finally, we wanted to determine whether there was physiological
relevance to our finding of increased -catenin. We evaluated the
amounts of two proteins whose transcription is linked to
-catenin/TCF/Lef-1 signaling (32, 47). It is not in the
scope of this paper to evaluate all the possible events downstream of
-catenin signaling. The two proteins we did evaluate have been
strongly linked to -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 -catenin is accompanied by specific increases in
gene transcription.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin. In alveolar macrophages, LPS
caused the nuclear accumulation of -catenin, and this was linked to
the PI 3-kinase pathway. LPS also activated genes that are dependent on
nuclear -catenin activity. These observations suggest a particular
sequence of events in LPS-treated alveolar macrophages that is shown in
Fig. 10.
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(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 , 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 
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 IB 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 -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 2 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 -catenin. When GSK-3 is removed from this complex, it can
no longer phosphorylate -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 -catenin accumulation and transcriptional
activity is inactivation of GSK-3 by the Wnt pathway (58).
In alveolar macrophages, the result of LPS-induced -catenin
accumulation is unknown. In other systems, -catenin stabilizes a
transcription complex containing -catenin and the transcription
factor TCF/Lef-1. The -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 -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-B in LPS signaling. LPS is a
strong inducer of NF-B activity, and many of the LPS-induced
inflammatory mediators have important NF-B sites (IL-6, IL-8,
TNF-) (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 IB kinase (IKK) phosphorylation at threonine
23, leading to IB degradation and NF-B translocation to the
nucleus (65, 66). Secondly, Madrid et al. have shown a
further function for Akt in NF-B 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-B translocation and necessary for
the transcriptional activity of NF-B. One more study suggests a
possible pathway by which the findings in this study could relate to
LPS-induced NF-B activation in alveolar macrophages. A study by
Hoeflich using GSK-3 knockout mice has the very interesting finding
that GSK-3 is required for the NF-B-mediated survival response. Mice
lacking GSK-3 resemble mice lacking the components of the NF-B
pathway. Fibroblasts from the GSK-3-/- mice had
a decreased NF-B reporter gene response after TNF- or IL-1
stimulation (68). It is interesting to speculate that the
inactivation of GSK-3 has a role to play in NF-B 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 -catenin. This results in increases in at least two
of the proteins known to be linked to -catenin, cyclin D1, and
connexin 43. The possible importance of this pathway has yet to be
discovered but could include -catenin-driven gene expression and
possible effects on NF-B activation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martha M. Monick, Division of Pulmonary, Critical Care, and Occupational Medicine Room 100, Ekstein Medical Research Building, University of Iowa Hospitals and Clinic, Iowa City, IA 52242.
3 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.
Received for publication October 24, 2000. Accepted for publication January 30, 2001.
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 C. L. Avvisato, X. Yang, S. Shah, B. Hoxter, W. Li, R. Gaynor, R. Pestell, A. Tozeren, and S. W. Byers Mechanical force modulates global gene expression and beta-catenin signaling in colon cancer cells J. Cell Sci., August 1, 2007; 120(15): 2672 - 2682. [Abstract] [Full Text] [PDF]
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A. Moeenrezakhanlou, D. Nandan, L. Shephard, and N. E. Reiner 1{alpha},25-Dihydroxycholecalciferol activates binding of CREB to a CRE site in the CD14 promoter and drives promoter activity in a phosphatidylinositol-3 kinase-dependent manner J. Leukoc. Biol., May 1, 2007; 81(5): 1311 - 1321. [Abstract] [Full Text] [PDF]
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 C.-H. Liu, F. S. Machado, R. Guo, K. E. Nichols, A. W. Burks, J. C. Aliberti, and X.-P. Zhong Diacylglycerol kinase {zeta} regulates microbial recognition and host resistance to Toxoplasma gondii J. Exp. Med., April 16, 2007; 204(4): 781 - 792. [Abstract] [Full Text] [PDF]
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 C. C. McGown and Z. L. S. Brookes Beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide Br. J. Anaesth., February 1, 2007; 98(2): 163 - 175. [Abstract] [Full Text] [PDF]
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 N. Matsuda, Y. Hayashi, Y. Takahashi, and Y. Hattori Phosphorylation of Endothelial Nitric-Oxide Synthase Is Diminished in Mesenteric Arteries from Septic Rabbits Depending on the Altered Phosphatidylinositol 3-Kinase/Akt Pathway: Reversal Effect of Fluvastatin Therapy J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1348 - 1354. [Abstract] [Full Text] [PDF]
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D. P. Sester, K. Brion, A. Trieu, H. S. Goodridge, T. L. Roberts, J. Dunn, D. A. Hume, K. J. Stacey, and M. J. Sweet CpG DNA Activates Survival in Murine Macrophages through TLR9 and the Phosphatidylinositol 3-Kinase-Akt Pathway J. Immunol., October 1, 2006; 177(7): 4473 - 4480. [Abstract] [Full Text] [PDF]
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K. Sakai, H. Suzuki, H. Oda, T. Akaike, Y. Azuma, T. Murakami, K. Sugi, T. Ito, H. Ichinose, S. Koyasu, et al. Phosphoinositide 3-Kinase in Nitric Oxide Synthesis in Macrophage: CRITICAL DIMERIZATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE J. Biol. Chem., June 30, 2006; 281(26): 17736 - 17742. [Abstract] [Full Text] [PDF]
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 X. Cai, M. Li, J. Vrana, and M. D. Schaller Glycogen synthase kinase 3- and extracellular signal-regulated kinase-dependent phosphorylation of paxillin regulates cytoskeletal rearrangement. Mol. Cell. Biol., April 1, 2006; 26(7): 2857 - 2868. [Abstract] [Full Text] [PDF]
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 A. T. Franco, D. A. Israel, M. K. Washington, U. Krishna, J. G. Fox, A. B. Rogers, A. S. Neish, L. Collier-Hyams, G. I. Perez-Perez, M. Hatakeyama, et al. Activation of {beta}-catenin by carcinogenic Helicobacter pylori PNAS, July 26, 2005; 102(30): 10646 - 10651. [Abstract] [Full Text] [PDF]
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 D. N. Everly Jr., S. Kusano, and N. Raab-Traub Accumulation of Cytoplasmic {beta}-Catenin and Nuclear Glycogen Synthase Kinase 3{beta} in Epstein-Barr Virus-Infected Cells J. Virol., November 1, 2004; 78(21): 11648 - 11655. [Abstract] [Full Text] [PDF]
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M. M. Monick, R. K. Mallampalli, M. Bradford, D. McCoy, T. J. Gross, D. M. Flaherty, L. S. Powers, K. Cameron, S. Kelly, A. H. Merrill Jr., et al. Cooperative Prosurvival Activity by ERK and Akt in Human Alveolar Macrophages is Dependent on High Levels of Acid Ceramidase Activity J. Immunol., July 1, 2004; 173(1): 123 - 135. [Abstract] [Full Text] [PDF]
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H. Fang, R. A. Pengal, X. Cao, L. P. Ganesan, M. D. Wewers, C. B. Marsh, and S. Tridandapani Lipopolysaccharide-Induced Macrophage Inflammatory Response Is Regulated by SHIP J. Immunol., July 1, 2004; 173(1): 360 - 366. [Abstract] [Full Text] [PDF]
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 J. Sun, M. E. Hobert, A. S. Rao, A. S. Neish, and J. L. Madara Bacterial activation of {beta}-catenin signaling in human epithelia Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G220 - G227. [Abstract] [Full Text] [PDF]
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 M. M. Monick, K. Cameron, L. S. Powers, N. S. Butler, D. McCoy, R. K. Mallampalli, and G. W. Hunninghake Sphingosine Kinase Mediates Activation of Extracellular Signal-Related Kinase and Akt by Respiratory Syncytial Virus Am. J. Respir. Cell Mol. Biol., June 1, 2004; 30(6): 844 - 852. [Abstract] [Full Text] [PDF]
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X. Cao, G. Wei, H. Fang, J. Guo, M. Weinstein, C. B. Marsh, M. C. Ostrowski, and S. Tridandapani The Inositol 3-Phosphatase PTEN Negatively Regulates Fc{gamma} Receptor Signaling, but Supports Toll-Like Receptor 4 Signaling in Murine Peritoneal Macrophages J. Immunol., April 15, 2004; 172(8): 4851 - 4857. [Abstract] [Full Text] [PDF]
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 M. Aga, J. J. Watters, Z. A. Pfeiffer, G. J. Wiepz, J. A. Sommer, and P. J. Bertics Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-{kappa}B signaling pathways in murine RAW 264.7 macrophages Am J Physiol Cell Physiol, April 1, 2004; 286(4): C923 - C930. [Abstract] [Full Text] [PDF]
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D. Xie, D. Yin, X. Tong, J. O'Kelly, A. Mori, C. Miller, K. Black, D. Gui, J. W. Said, and H. P. Koeffler Cyr61 Is Overexpressed in Gliomas and Involved in Integrin-Linked Kinase-Mediated Akt and {beta}-Catenin-TCF/Lef Signaling Pathways Cancer Res., March 15, 2004; 64(6): 1987 - 1996. [Abstract] [Full Text] [PDF]
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 U. Uhlig, H. Fehrenbach, R. A. Lachmann, T. Goldmann, B. Lachmann, E. Vollmer, and S. Uhlig Phosphoinositide 3-OH Kinase Inhibition Prevents Ventilation-induced Lung Cell Activation Am. J. Respir. Crit. Care Med., January 15, 2004; 169(2): 201 - 208. [Abstract] [Full Text] [PDF]
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M. M. Monick, T. O. Yarovinsky, L. S. Powers, N. S. Butler, A. B. Carter, G. Gudmundsson, and G. W. Hunninghake Respiratory Syncytial Virus Up-regulates TLR4 and Sensitizes Airway Epithelial Cells to Endotoxin J. Biol. Chem., December 26, 2003; 278(52): 53035 - 53044. [Abstract] [Full Text] [PDF]
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J. A. Morrison, A. J. Klingelhutz, and N. Raab-Traub Epstein-Barr Virus Latent Membrane Protein 2A Activates {beta}-Catenin Signaling in Epithelial Cells J. Virol., November 15, 2003; 77(22): 12276 - 12284. [Abstract] [Full Text] [PDF]
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H.-K. Lim, Y.-A. Choi, W. Park, T. Lee, S. H. Ryu, S.-Y. Kim, J.-R. Kim, J.-H. Kim, and S.-H. Baek Phosphatidic Acid Regulates Systemic Inflammatory Responses by Modulating the Akt-Mammalian Target of Rapamycin-p70 S6 Kinase 1 Pathway J. Biol. Chem., November 14, 2003; 278(46): 45117 - 45127. [Abstract] [Full Text] [PDF]
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J. Y. Lee, J. Ye, Z. Gao, H. S. Youn, W. H. Lee, L. Zhao, N. Sizemore, and D. H. Hwang Reciprocal Modulation of Toll-like Receptor-4 Signaling Pathways Involving MyD88 and Phosphatidylinositol 3-Kinase/AKT by Saturated and Polyunsaturated Fatty Acids J. Biol. Chem., September 26, 2003; 278(39): 37041 - 37051. [Abstract] [Full Text] [PDF]
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M. M. Monick, L. S. Powers, N. S. Butler, and G. W. Hunninghake Inhibition of Rho Family GTPases Results in Increased TNF-{alpha} Production After Lipopolysaccharide Exposure J. Immunol., September 1, 2003; 171(5): 2625 - 2630. [Abstract] [Full Text] [PDF]
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 S. Dihlmann, S. Klein, and M. v. K. Doeberitz Reduction of {beta}-Catenin/T-Cell Transcription Factor Signaling by Aspirin and Indomethacin Is Caused by an Increased Stabilization of Phosphorylated {beta}-Catenin Mol. Cancer Ther., June 1, 2003; 2(6): 509 - 516. [Abstract] [Full Text] [PDF]
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D.-W. Park, J.-R. Kim, S.-Y. Kim, J.-K. Sonn, O.-S. Bang, S.-S. Kang, J.-H. Kim, and S.-H. Baek Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression J. Immunol., February 15, 2003; 170(4): 2093 - 2099. [Abstract] [Full Text] [PDF]
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S. Ahmad, A. Ahmad, E. Gerasimovskaya, K. R. Stenmark, C. B. Allen, and C. W. White Hypoxia Protects Human Lung Microvascular Endothelial and Epithelial-like Cells against Oxygen Toxicity: Role of Phosphatidylinositol 3-Kinase Am. J. Respir. Cell Mol. Biol., February 1, 2003; 28(2): 179 - 187. [Abstract] [Full Text] [PDF]
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S. Bozinovski, J. E. Jones, R. Vlahos, J. A. Hamilton, and G. P. Anderson Granulocyte/Macrophage-Colony-stimulating Factor (GM-CSF) Regulates Lung Innate Immunity to Lipopolysaccharide through Akt/Erk Activation of NFkappa B and AP-1 in Vivo J. Biol. Chem., November 1, 2002; 277(45): 42808 - 42814. [Abstract] [Full Text] [PDF]
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E. Oviedo-Orta and W. H. Evans Gap junctions and connexins: potential contributors to the immunological synapse J. Leukoc. Biol., October 1, 2002; 72(4): 636 - 642. [Abstract] [Full Text] [PDF]
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M. M. Monick, P. K. Robeff, N. S. Butler, D. M. Flaherty, A. B. Carter, M. W. Peterson, and G. W. Hunninghake Phosphatidylinositol 3-Kinase Activity Negatively Regulates Stability of Cyclooxygenase 2 mRNA J. Biol. Chem., August 30, 2002; 277(36): 32992 - 33000. [Abstract] [Full Text] [PDF]
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X. Ye and S. F. Liu Lipopolysaccharide Down-regulates Sp1 Binding Activity by Promoting Sp1 Protein Dephosphorylation and Degradation J. Biol. Chem., August 23, 2002; 277(35): 31863 - 31870. [Abstract] [Full Text] [PDF]
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S. L. Christian, P. V. Sims, and M. R. Gold The B Cell Antigen Receptor Regulates the Transcriptional Activator {beta}-Catenin Via Protein Kinase C-Mediated Inhibition of Glycogen Synthase Kinase-3 J. Immunol., July 15, 2002; 169(2): 758 - 769. [Abstract] [Full Text] [PDF]
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M.M. Monick and G.W. Hunninghake Activation of second messenger pathways in alveolar macrophages by endotoxin Eur. Respir. J., July 1, 2002; 20(1): 210 - 222. [Abstract] [Full Text] [PDF]
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L. Samavati, M. M. Monick, S. Sanlioglu, G. R. Buettner, L. W. Oberley, and G. W. Hunninghake Mitochondrial KATP channel openers activate the ERK kinase by an oxidant-dependent mechanism Am J Physiol Cell Physiol, July 1, 2002; 283(1): C273 - C281. [Abstract] [Full Text] [PDF]
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R. A. Zirngibl, Y. Senis, and P. A. Greer Enhanced Endotoxin Sensitivity in Fps/Fes-Null Mice with Minimal Defects in Hematopoietic Homeostasis Mol. Cell. Biol., April 15, 2002; 22(8): 2472 - 2486. [Abstract] [Full Text] [PDF]
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 E. Cario, D. Brown, M. McKee, K. Lynch-Devaney, G. Gerken, and D. K. Podolsky Commensal-Associated Molecular Patterns Induce Selective Toll-Like Receptor-Trafficking from Apical Membrane to Cytoplasmic Compartments in Polarized Intestinal Epithelium Am. J. Pathol., January 1, 2002; 160(1): 165 - 173. [Abstract] [Full Text] [PDF]
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A. Thiele, M. Wasner, C. Muller, K. Engeland, and S. Hauschildt Regulation and Possible Function of {beta}-Catenin in Human Monocytes J. Immunol., December 15, 2001; 167(12): 6786 - 6793. [Abstract] [Full Text] [PDF]
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M. M. Monick, R. K. Mallampalli, A. B. Carter, D. M. Flaherty, D. McCoy, P. K. Robeff, M. W. Peterson, and G. W. Hunninghake Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages J. Immunol., November 15, 2001; 167(10): 5977 - 5985. [Abstract] [Full Text] [PDF]
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