| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cardiovascular Biology Research Group, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
1Correspondence: Cardiovascular Biology Research Programme, Department of Pharmacology, Centre for Life Sciences, National University of Singapore, 28 Medical Dr., Singapore 117456. E-mail: mbhatia{at}nus.edu.sg
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and novel PKC
and 
were selectively activated by SP via its primary neurokinin-1 receptor (NK-1R) on the cells. Activation of these PKC isoforms mediated the activation of downstream extracellular signal-regulated kinase-1/2 (ERK1/2) and the transcription factor NF-
B, which drove the transcription of inducible chemokines in macrophages. Additionally, phosphoinositide 3-kinase (PI3K)-Akt was also activated by SP/NK-1R in macrophages. Inhibition of PI3K-Akt pathway attenuated ERK1/2 and NF-B activation, suggesting it also played a part in SP-induced cellular inflammatory response. Kinetic analysis indicated that PKC isoforms induced early ERK1/2 activation, while PI3K-Akt contributed to the pathway at later time points. It was further demonstrated that PKC and PI3K-Akt were activated independent of each other. Collectively, our results suggest that SP/NK-1R activates two convergent proinflammatory signaling pathways, PKCs and PI3K-Akt, resulting in ERK1/2 and NF-B activation and chemokine production in mouse macrophages.—Sun, J., Ramnath, R. D., Tamizhselvi, R., Bhatia, M. Role of protein kinase C and phosphoinositide 3-kinase-Akt in substance P-induced proinflammatory pathways in mouse macrophages.
Key Words: neuroimmunoregulation • neuropeptides • leukocytes • inflammation • signaling kinases
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3
4
5
6
7
8)
. SP belongs to the tachykinin neuropeptide family. Its physiological and pathological activities are primarily mediated by the G protein-coupled receptor, neurokinin-1 receptor (NK-1R) on the effector cells (9)
. The modulatory influence of SP on inflammatory conditions is exerted largely by affecting multiple aspects of immune cell function (10
, 11)
.
In macrophages, SP is known to induce the production of inflammatory and cytotoxic mediators, including cytokines, chemokines, and free radicals, such as nitric oxide and reactive oxygen species and proteases, thereby contributing to macrophage-mediated inflammatory responses (10
, 12
13
14)
. It also enhances antigen presentation and phagocytotic activities of these cells during cellular immune responses (10
, 15)
. However, the detailed cellular signaling mechanisms of SP-induced cellular inflammation in macrophages still remain elusive.
In the present study, we have targeted two signaling pathways—protein kinase C (PKC) isoforms and phosphoinositide 3-kinase (PI3K)-Akt—for investigation in SP-evoked inflammatory pathways in macrophages.
The PKC protein family comprises phospholipid-dependent serine/threonine kinases (16)
subdivided into conventional (, βI, βII, and 
), novel (, , 
, and 
), and atypical (
, 
/
, and µ) subfamilies, based on their molecular structure and mode of activation (17
, 18)
. PKC activation is involved in regulation of cellular processes, including growth, migration, and inflammatory responses (19)
and has been shown to induce downstream release of inflammatory mediators, including lipid mediators, adhesion molecules, and chemokines in several in vitro models (20
21
22)
. However, the role of PKCs has not been investigated in neuropeptide-induced inflammatory responses in macrophages.
PI3K is a conserved family of signaling molecules involved in the regulation of cellular proliferation and survival. Akt is a direct downstream effector kinase (serine/threonine) of PI3K. PI3K-Akt has a documented role in regulating cellular growth, differentiation, adhesion, and inflammatory responses (23)
, although whether it is pro- or anti-inflammatory has been controversial, dependent on the cellular models and experimental conditions (24
25
26
27
28
29
30)
. The role of PI3K-Akt in neurokinin SP-induced macrophage cellular signaling is also unclear.
To this end, we investigated the participation of PKC isoforms and PI3K-Akt and the possible crosstalk between them in SP-induced inflammatory response in macrophages. A mouse monocyte/macrophage cell line RAW 264.7 and primary peritoneal macrophages were used as our cellular models. Elucidation of key signaling molecules involved contributes to understanding of the interaction between the neuronal and immune systems and provides the basis for therapeutic intervention in neurogenic inflammatory diseases.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
translocation inhibitor peptide (PKC TIP) and ERK1/2 inhibitor PD98059 were obtained from Calbiochem (San Diego, CA, USA). PKC TIP was synthesized by Sigma Genosys (Japan). Dulbecco modified Eagle medium (DMEM), heat-inactivated FBS, Novex 10% tris-glycine polyacrylamide gels, and Novex transfer buffer were obtained from Invitrogen (Gaithersburg, MD, USA). PKC, PKC, PKC, phospho-PKC (Thr638), phospho-PKC (Thr505), phospho-Akt (Ser-473), Akt, IB, phospho-NF-B p65 (Ser-276), phospho-p44/42 MAPK (phospho-ERK1/2), and p44/42 MAPK (ERK1/2) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-PKC (Ser-729), hypoxanthine-guanine phosphoribosyltransferase antibody (HPRT), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SuperSignal West Pico chemiluminescent substrate and X-ray films were obtained from Pierce (Rockford, IL, USA). Bradford protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Nuclear extract kit and TransAM NF-B p65 transcription factor assay kit were obtained from Active Motif (Carlsbad, CA, USA). Murine MIP-2 and MCP-1 Duoset ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA).
Cell culture and treatment
RAW 264.7 cells were grown in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained at 37°C in a humidified atmosphere containing 5% CO2. For treatments, cells were seeded onto 6-well plates and allowed to grow overnight to 70–80% confluence. Cells were deprived of serum for at least 2 h before experiments. SP and CP96,345 were dissolved in sterile saline (0.9%). PKC TIP and PKC TIP were dissolved in sterile water. All the other inhibitors were dissolved in dimethyl sulfoxide (DMSO) and diluted in serum-free DMEM medium to the desired concentrations, such that the final DMSO concentration was <0.1%. SP stimulation of cells was carried out at cell culture conditions. In some experiments, cells were pretreated with CP96,345 (1 µM) for 10 min, or PKC TIP, PKC TIP for 2 h, or chelerythrine chloride, Gö6976, LY294002, or triciribine for 1 h before addition of SP.
Isolation of peritoneal macrophages
Resident peritoneal macrophages were isolated as described previously, with modifications (31)
. Briefly, closed peritoneal lavage was performed on anesthetized mice using 10 ml ice-cold PBS. Peritoneal exudate cells were collected, washed once with PBS, and resuspended in supplemented DMEM. Peritoneal macrophages were allowed to adhere in 6-well plates at 37°C in a humidified 5% CO2 incubator for 2 h, after which nonadherent contaminating cells were removed, and the primary macrophage cultures were maintained for an additional 2 h before they were subjected to SP treatment and subsequent experiments. The preparations routinely contain >95% macrophages, as verified by microscopic examination with Turk’s staining.
ELISA
Chemokine concentration in the medium of cultured RAW 264.7 cells or primary peritoneal macrophages was determined using the murine MIP-2 and MCP-1 Duoset ELISA kits, according to the instructions of the manufacturer. Samples were run in triplicate for each condition in 3 independent experiments. Absorbance was measured at 450 nm by a microplate reader (Tecan Systems, San Jose, CA, USA). Results were expressed as picograms per milliliter of each chemokine.
Whole-cell lysate preparation and Western blot analysis
At the end of designated treatment, cells were lysed with chilled radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktails. Protein concentrations were determined by the Bradford protein assay. Protein samples (50–100 µg) were separated on Novex 10% Tris-glycine polyacrylamide gels and transferred onto polyvinylidene difluoride membranes by electroblotting in Novex transfer buffer containing 20% (v/v) methanol. Membranes were then washed, blocked, and probed overnight with rabbit anti-mouse PKC, PKC, PKC, phospho-PKC (Thr638), phospho-PKC (Thr505), phospho-PKC (Ser-729), phospho-IB, IB, phospho-NF-B p65, phospho-p44/42 MAPK (phospho-ERK1/2), p44/42 MAPK (ERK1/2), phospho-Akt (Ser-473), Akt (1:1000 dilution), or HPRT (1:2000) antibody, followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (1:2000) for 2 h. Membranes were washed and then incubated in SuperSignal West Pico chemiluminescent substrate before exposure to X-ray films. The intensity of bands was quantified using LabWorks Image Analysis software (UVP). HPRT was used as the housekeeping protein.
Subcellular fractionation
After treatment, cells grown on 6- or 12-well plates were washed with ice-cold PBS and lysed in Buffer B (20 mM Tris, 50 mM NaCl, 2 mM EDTA, 10 mM β-mercaptoethanol, 1% Nonidet P-40). Cell lysates were collected and sheared in 1-ml syringes by being passed 10 times through a 25-gauge needle. Lysates were centrifuged at 18,000 g for 30 min, and the supernatant was collected as cytosolic fraction. The pellet was resuspended in Buffer B containing 1% Triton X-100, shaken on ice for 60 min, and centrifuged at 18,000 g for 30 min. The supernatant was collected as membrane fraction.
Nuclear extract preparation
Cell nuclear fractions were extracted using a nuclear extract kit. Briefly, cells were washed, collected in ice-cold PBS in the presence of phosphatase inhibitors, and then centrifuged at 300 g for 5 min. Cell pellets were resuspended in a hypotonic buffer, treated with detergent, and centrifuged at 14,000 g for 30 s. After collection of the cytoplasmic fraction, the nuclear pellet was lysed and nuclear proteins were solubilized in lysis buffer containing protease inhibitors. Protein concentrations were determined by the Bradford protein assay.
Statistical analysis
Data are expressed as means ± SD. Statistical analyses were performed by independent t test or, when multiple comparisons were made, by one-way ANOVA with post hoc Tukey’s test using SPSS ver. 13.0 (SPSS, Chicago, IL, USA). A value of P < 0.05 was considered a statistically significant difference.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, , and activation in mouse macrophages via the NK-1R in a time-dependent manner, , and was detected in unstimulated RAW 264.7 cells, with a large percentage of each isoform residing in the cytosolic fraction. Incubation of cells with 10 nM SP provoked the cytosol-to-membrane translocation and thus activation of PKC, , and , with maximal activation observed at 3 min of stimulation for PKC, and 5 min for PKC and PKC (Fig. 1A-D
). Consistent with PKC translocation results, SP also induced phosphorylation of PKC (Thr638), (Thr505), and (Ser-729) in the cells as demonstrated using specific phospho-PKC antibodies, which detect PKC isoforms only when phosphorylated at indicated specific sites (Fig. 1E-H
). No change in the subcellular localization of other PKC isoforms was observed in the cells after SP treatment (data not shown).
|
The differential kinetics of PKC activation in the cells prompted us to investigate whether PKC activation occurred in a hierarchical manner. We analyzed PKC activation in the cells following SP stimulation in the presence of isoform-specific PKC inhibitors: Gö6976 (PKC inhibitor), PKC TIP (specific PKC inhibitor), and PKC TIP (specific PKC inhibitor). Pretreatment of cells with Gö6976, PKC TIP, or PKC TIP blocked the translocation and activation of PKC, , and , respectively, without affecting other PKC isoforms (data not shown), showing that these inhibitors are specific and the conventional PKC is activated in parallel with novel PKC and . Furthermore, SP-induced PKC isoform activation was found to be NK-1R-mediated. Pretreatment of cells with a selective NK-1R antagonist CP96,345 abolished SP-induced translocation and thus activation of PKC, , and (Fig. 2A-D
). These results suggest that SP activates PKC, , and in mouse macrophages via the NK-1R.
|
PKC, , and are involved in SP-induced chemokine production in mouse macrophages
We have previously examined the time course of SP-induced chemokine production by mouse macrophages (12)
. A significant increase in chemokine secretion by SP-stimulated macrophages was observed after 4 h treatment with SP. To examine whether PKC isoforms are involved in SP-induced chemokine production in mouse macrophages, RAW 264.7 cells were treated with the broad-spectrum PKC inhibitor Che (0.1 and 1 µM) or Gö6976 (0.1 and 1 µM) or the chemical PKC inhibitor rottlerin (1 µM) for 1 h or PKC TIP (1, 5, and 10 µM) or PKC TIP (1, 10, and 30 µM) for 2 h before SP stimulation for 4 h. Subsequently, the cell supernatant was obtained for ELISA determination of MIP-2 and MCP-1 secretion levels. None of these inhibitors affected the basal levels of chemokine production by mouse macrophages (data not shown). The inhibitors dose dependently inhibited SP-induced MIP-2 and MCP-1 production (Fig. 3
). Only partial inhibition of the chemokine secretion was observed with PKC inhibitors. The results suggest that activation of PKC, , contributes to SP-induced chemokine expression in mouse macrophages.
|
PKC, , and are involved in SP-induced ERK1/2 activation in mouse macrophages
To investigate how PKCs are involved in SP-induced chemokine production, we studied whether PKC isoforms mediate SP-induced ERK1/2 activation. The time course of SP-induced ERK1/2 activation has been studied previously, and the maximal ERK1/2 activity was observed 15–30 min after SP stimulation of cells. On the basis of this result, RAW 264.7 cells were pretreated with Gö6976 (0.1 µM) for 1 h or PKC TIP (5 µM) and PKC TIP (10 µM) for 2 h before they were treated with 10 nM of SP for 15 min. Subsequently, activation of ERK1/2 was determined by Western blot analysis of cellular phospho-ERK1/2 levels. The results showed that blockade of the activation of PKC, , or was associated with markedly lower ERK1/2 expression levels, although it did not completely inhibit the activation of ERK1/2 (Fig. 4A-C
). These results suggest that these PKC isoforms are important for SP-induced ERK1/2 phosphorylation and activation.
|
Inhibition of PKC, , and attenuated SP-induced NF-B activation in mouse macrophages
We then investigated whether PKC isoforms mediate SP-induced NF-B activation. NF-B activation was assessed by phosphorylation and degradation of its inhibitory protein IB and the nuclear translocation of NF-B p65. On the basis of our earlier study, induction of NF-B activation by SP was most significant 30 min after treatment. Therefore, cells were pretreated with various inhibitors for 1 or 2 h before SP stimulation for 30 min. Subsequently, whole-cell lysates were prepared for Western blot analysis of phospho-IB and total IB levels. In separate experiments, after the same treatment, cytosolic and nuclear fractions of cells were prepared to analyze nuclear translocation of phospho-NF-B p65. The results demonstrated that Gö6976, PKC TIP, or PKC TIP pretreatment was associated with markedly less IB phosphorylation and degradation (Fig. 5A-C
) and significantly attenuated the nuclear translocation of phospho-p65 (Fig. 5D, E
).
|
SP induces Akt phosphorylation in mouse macrophages via the NK-1R in a time-dependent manner
The activity of PI3K was determined by measuring the phosphorylation of its direct downstream effector Akt. To examine whether SP induces Akt phosphorylation in the cells, cells were treated with 10 nM of SP for 0–60 min before whole-cell lysates were prepared and probed with specific phospho-Akt antibodies. Significant increase in Akt phosphorylation was observed 15 min after SP stimulation. Increased phospho-Akt levels persisted until 30 min after treatment and declined thereafter (Fig. 6A, B
). Moreover, incubation of cells with the NK-1R antagonist CP96,345 (1 µM) almost completely blocked SP-evoked Akt phosphorylation (Fig. 6C, D
), showing that the effect of SP on PI3K-Akt activation was mediated via the NK-1R.
|
PI3K-Akt proceeds through ERK1/2 and NF-B activation to mediate chemokine production induced by SP
We further determined the role of PI3K-Akt in SP-induced signaling pathways leading to chemokine expression in macrophages. A specific inhibitor of PI3K-Akt LY294002 (1, 5, and 10 µM) and a specific inhibitor of Akt triciribine (1, 5, and 10 µM) were added to the cells 1 h prior to SP stimulation, and the inhibitor effects of SP-induced chemokine secretion were examined. As shown in Fig. 7
, both inhibitors dose-dependently inhibited SP-provoked MIP-2 and MCP-1 production by macrophages. LY294002 and triciribine were both effective at a low dose of 5 µM. Western blot analysis further demonstrated that inhibition of PI3K-Akt pathway significantly blocked SP-induced ERK1/2 activation (Fig. 8A-C
). Both LY294002 and triciribine markedly attenuated phosphorylation and degradation of IB (Fig. 9A-C
) and diminished nuclear translocation of phospho-NF-B p65 (Fig. 9D, E
). These results suggest that PI3K-Akt is involved in SP-elicited signaling pathway leading to the chemokine expression.
|
|
|
PKCs and PI3K-Akt are two independent, convergent pathways mediating SP-induced ERK1/2-NF-B activation and chemokine production in mouse macrophages
To determine the hierarchy order of PKC isoforms and PI3K-Akt in SP-induced signal transduction cascade in mouse macrophages, we examined PKC isoform and PI3K-Akt activation in the presence of PI3K-Akt and PKC inhibitors. The results demonstrate that pretreatment of cells with Gö6976, PKC TIP, or PKC TIP had no effect on SP-induced PI3K-Akt activation (Fig. 10A, B
). Neither did the specific PI3K-Akt pathway inhibitor LY294002 or Akt inhibitor triciribine affect PKC isoform activation (Fig. 10C
). These data suggest that PKCs and PI3K-Akt are two independent pathways induced by SP in mouse macrophages.
|
SP induces PKC and PI3K-Akt pathway in mouse primary peritoneal macrophages
SP-induced PKC and PI3K-Akt activation was also demonstrated in mouse primary peritoneal macrophages. SP dose of 100 nM optimized earlier to induce significant chemokine response in primary peritoneal macrophages was used to treat isolated macrophages. Subsequently, membrane and cytosolic fractions of cells were prepared for Western blot analysis of PKC, , and . In separate experiments, whole-cell lysates were prepared to detect phospho-Akt expression levels. The results clearly demonstrated that SP induced the translocation of all these PKC isoforms from the cytosol to the membrane. An induction of Akt phosphorylation was also observed in SP-treated peritoneal macrophages (Fig. 11A, B
). This indicates that SP-induced PKC and PI3K-Akt activation is not a cell line-specific effect.
|
We further verified the roles of these two signaling pathways in SP-induced chemokine production. On the basis of earlier data showing a significant increase in chemokine production 24 h after SP treatment of peritoneal macrophages, isolated macrophages were pretreated with various pathway inhibitors for 1 or 2 h before they were subjected to SP stimulation for 24 h. Subsequently, secretion levels of MIP-2 and MCP-1 in the cell medium were analyzed by ELISA. The results demonstrated that inhibitors of both signaling pathways significantly attenuated SP-induced MIP-2 and MCP-1 production by primary peritoneal macrophages (Fig. 11C, D
).
Signal transduction pathways elicited by SP in mouse macrophages
SP stimulation of mouse macrophages via its preferential NK-1R induces two convergent signaling pathways: PKCs and PI3K-Akt. Both pathways activate the ERK1/2 MAPK, which, in turn, induces the classical activation pathway of NF-B involving phosphorylation and degradation of its inhibitory protein IB and NF-B p65 nuclear translocation. NF-B activation promotes the expression of its responsive proinflammatory chemokines, MIP-2 and MCP-1 (Fig. 12
).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B activation and chemokine expression.
Our findings identified that the conventional PKC isoform and the novel PKC isoforms and were selectively activated by SP in mouse macrophages. Presence of a small fraction of membrane-bound active PKC forms in the unstimulated control cells suggests some activation of PKCs in resting-state cells. This may explain a basal level of chemokine production by mouse macrophages. Similar activation of PKC in unstimulated acinar cells was reported by Thrower et al. (32)
, causing baseline zymogen activation. SP had previously been shown to activate PKC isoforms for various cellular responses in vitro. SP induces PKC phosphorylation and activation to mediate interleukin-8 (IL-8) expression in human colonic epithelial cells (33)
. PKC is also the isoform involved in SP-evoked chemokine secretion by mouse pancreatic acini (20)
. Furthermore, we have noted that the conventional PKC was activated by SP in our cellular models in parallel with the novel PKC and , as the isozyme-specific inhibitors did not cross-inhibit activation of other isoforms. Activation of conventional and novel PKCs may occur in a hierarchical order, depending on the cellular models. Trushin et al. (34)
observed that PKC acts upstream of novel PKC to activate NF-B in T cells. It is suggested that PKC may be involved directly or indirectly in the induction of PKC phosphorylation at the plasma membrane (34)
.
Isozyme-specific PKC inhibitors are necessary to define the involvement of different PKC isoforms in SP-induced cellular responses (35)
. Gö6976, a widely used Ca2+-dependent, conventional PKC inhibitor (36
37
38)
, was used in our investigation for selective blockade of the actions of PKC but not for those of PKC and . Rottlerin is used by many studies as a selective PKC inhibitor (33
, 39
40
41)
. However, the broad action of this inhibitor on other PKC isoforms and other kinases has raised considerable concern (42)
. To address this concern, in our study, we used a specific translocation inhibitor peptide PKC TIP or PKC TIP, which is designed to competitively inhibit the binding of PKC or to specific anchoring proteins and hence inhibit their translocation to subcellular sites (43
44
45)
. The specificity of the two inhibitors was tested and confirmed in our results. To our knowledge, these translocation inhibitor peptides are employed for the first time in mouse macrophages for studying the roles of different PKC isoforms in SP-induced cellular signaling.
Using these isoform-specific PKC inhibitors, we further demonstrated that PKC, , and mediated an SP-induced inflammatory pathway leading to ERK1/ 2-NF-B activation and chemokine expression. Our report is the first to demonstrate that differential PKC isoforms are involved in SP-induced ERK1/2 activation and inflammatory responses in immune cells. Few previous in vitro studies have investigated the role of PKCs and MAPKs in SP-induced release of inflammatory mediators (20
, 21)
. SP is known to enhance soluble ICAM-1 production by cardiac fibroblasts via an ERK1/2- and PKC-mediated mechanism, suggesting a role for SP in modulation of inflammatory process in the heart (21)
. Our group recently demonstrated that SP stimulates production of MCP-1, MIP-1, and MIP-2 by pancreatic acinar cells via a PKC-dependent mechanism. The present study further extends earlier findings that PKC isoforms are involved in SP-induced cellular proinflammatory responses to the immune cell macrophages.
It was noted that inhibitors of PKC isoforms did not completely block chemokine production. This suggests that there may be a second PKC-independent signaling mechanism contributing to the process, which prompted us to study the involvement of another candidate, the PI3K-Akt pathway, in this response. Our observation indeed confirmed the activation of PI3K-Akt pathway induced in the cells after 15 min of SP stimulation and sustained for up to 30 min. For further investigation, we employed a specific inhibitor of PI3K-Akt pathway LY294002 and an Akt inhibitor triciribine. LY294002 has been extensively used to study the role of PI3K in the regulation of different intracellular pathways (29
, 46
, 47)
. Triciribine is a synthetic tricyclic nucleoside, which acts as a potent, specific inhibitor of the Akt signaling pathway. It selectively inhibits phosphorylation and activation of Akt but does not inhibit Akt kinase activity or known upstream Akt activators including PI3K. It also exhibits little effect toward cellular signaling pathways mediated by PKC, PKA, SGK, Stat3, p38, ERK1/2, or JNK. Pretreatment with either LY294002 or triciribine attenuated SP-induced phosphorylation and activation of ERK1/2.
PI3K-Akt was activated later (15 min) than PKC isoforms (3–5 min) in the cells. The differential kinetics of their activation suggest that PKCs may be the primary mechanism to mediate an early activation of ERK1/2, and PI3K-Akt serves as a second mechanism, setting in at later time points to sustain ERK1/2 activation. This is further supported by the observation that PKC inhibitors attenuated the early activation of ERK1/2 induced by SP, while the effects of PI3K-Akt inhibitors are only effective at later time points.
The role of PI3K-Akt in inflammation remains disputable in the literature (24)
. Some researchers have shown that PI3K-Akt pathway positively regulates proinflammatory signaling in monocytes/macrophages. It is required for LPS induction of gene expression, including cytokines, chemokines, reactive oxygen species, nitric oxide, and other proinflammatory mediators triggered by a range of stimuli in monocytes/macrophages (48
49
50
51
52)
. In contrast, other studies show that the PI3K-Akt pathway negatively regulates LPS signaling and expression of proinflammatory genes, including cytokines, inducible NO synthase, and nitrite in monocytes/macrophages (28
29
30)
. This pathway also induces expression of antiinflammatory heme oxygenase-1 and IL-10 (24)
. Our study, for the first time, suggests that PI3K-Akt has a proinflammatory role in SP-induced ERK1/2-NF-B activation and chemokine expression.
Activation of PI3K-Akt pathway is found to be independent of PKC isoforms in our cellular system, although some earlier reports have indicated that PKC isoforms may be closely linked to PI3K-Akt stimulation (50)
. Distinct stimuli applied to the cells triggering unique receptor engagement pattern may account for the differential interaction of the intracellular pathways. PKC and PI3K-Akt pathways likely converge and activate ERK1/2-NF-B, leading to chemokine production.
In summary, in the present study, we identified the involvement of PKC isoforms , , and PI3K-Akt pathway upstream of ERK1/2 and NF-B to mediate SP/NK-1R-induced proinflammatory chemokine production in mouse macrophages. PKCs and PI3K-Akt lead two independent convergent pathways resulting in the response. Our study is the first in mouse macrophages to elucidate key intracellular signaling molecules involved in SP/NK-1R-induced macrophage inflammatory responses. It contributes to the current understanding of the interplay between immune and nervous systems, and the key signaling molecules uncovered may be the potential therapeutic target for macrophage-mediated neurogenic inflammatory conditions.
|
ACKNOWLEDGMENTS |
|---|
Received for publication October 2, 2008. Accepted for publication October 30, 2008.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B transactivation and chemokine response in murine macrophages via ERK1/2 and p38 MAPK signaling pathways. Am. J. Physiol. Cell Physiol. 294,C1586-C1596B pathway. Br. J. Pharmacol. 145,385-396[CrossRef][Medline] and - modulate caerulein-induced zymogen activation in pancreatic acinar cells. Am. J. Physiol. Gastrointest. Liver Physiol. 294,G1344-G1353B kinase and NF-B in T lymphocytes. Mol. Cell. Biol. 23,7068-7081B-dependent transcription: Roles for isoforms of novel protein kinase C. Cell. Signal. 20,1338-1348[CrossRef][Medline] activation in pancreatic cancer cells. Am. J. Physiol. Gastrointest. Liver Physiol. 294,G899-G905 and PI3K in human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 38,483-490 and -
regulate NF-B activation induced by cholecystokinin and TNF- in pancreatic acinar cells. Am. J. Physiol. Gastrointest. Liver Physiol. 287,G582-G591 PKC and epsilon PKC. Proc. Natl. Acad. Sci. U. S. A. 98,11114-11119This article has been cited by other articles:
|
|
 |
|
  W. Wang, Q. Li, J. Zhang, H. Wu, Y. Yin, Q. Ge, and Y. Zhang Hemokinin-1 Activates the MAPK Pathway and Enhances B Cell Proliferation and Antibody Production J. Immunol., April 1, 2010; 184(7): 3590 - 3597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chandras, Y. Koutmani, E. Kokkotou, C. Pothoulakis, and K. P. Karalis Activation of Phosphatidylinositol 3-Kinase/Protein Kinase B by Corticotropin-Releasing Factor in Human Monocytes Endocrinology, October 1, 2009; 150(10): 4606 - 4614. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
P < 0.05 vs. SP.
