Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1003498 on August 17, 2004

Published online before print August 17, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1003498v1
76/5/1075    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boudreau, R. T. M.
Right arrow Articles by Lin, T.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boudreau, R. T. M.
Right arrow Articles by Lin, T.-J.
(Journal of Leukocyte Biology. 2004;76:1075-1081.)
© 2004 by Society for Leukocyte Biology

Phosphatase inhibition potentiates IL-6 production by mast cells in response to Fc{varepsilon}RI-mediated activation: involvement of p38 MAPK

Robert T. M. Boudreau*, David W. Hoskin*,{dagger} and Tong-Jun Lin*,{ddagger},1

* Departments of Microbiology & Immunology,
{dagger} Pathology, and
{ddagger} Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada

1 Correspondence: Isaac Walton Killam Health Centre, Department of Pediatrics, 8th Floor Immunology Research Laboratories, 5850 University Avenue, Halifax, Nova Scotia, Canada B3J 3G9. E-mail: tong-jun.lin{at}dal.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells are crucial effector cells in the immune response through mediator secretion and release of cytokines. A coordinated balance between protein kinases and phosphatases plays an essential role in the regulation of mast cell mediator secretion. We have previously shown that treatment of mast cells with okadaic acid (OA), a protein phosphatase 2A (PP2A) inhibitor, results in a dose-dependent increase in interleukin (IL)-6 production. We show here for the first time a synergism between OA and immunoglobulin E (IgE)-mediated IL-6 secretion by murine bone marrow-derived mast cells (BMMC). Selective p38 mitogen-activated protein kinase (p38 MAPK) inhibition reduces OA and IgE-mediated IL-6 production. Regulation of p38 MAPK by PP2A was demonstrated, as OA treatment caused a dose-dependent increase in p38 MAPK phosphorylation. Antigen-mediated activation of murine mast cells also resulted in an increase in p38 MAPK phosphorylation, which was potentiated by cotreatment of the cells with OA. Lastly, in two mast cell lines (human mast cell-1 5C6 and murine MC/9) and primary-cultured murine BMMC, we show by coimmunoprecipitation an interaction between p38 MAPK and PP2A. These data support a role for PP2A through interaction with p38 MAPK in the regulation of IgE-dependent mast cell activation.

Key Words: mast cell • protein phosphatase 2A (PP2A) • p38 mitogen-activated protein kinase • Fc{varepsilon} receptor • cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Upon binding immunoglobulin E (IgE) to high-affinity Fc{varepsilon} surface receptors (Fc{varepsilon}RI) and subsequent cross-linking with multivalent antigen, several critical phosphorylation-dependent signaling events are rapidly initiated. Immunoreceptor tyrosine activation motifs on the intracellular segments of the ß and {gamma} chains of the receptor become phosphorylated by src-kinase family members (e.g., lyn) upon receptor clustering, allowing for the recruitment of src homology 2-containing enzymes, for example, syk [1 ]. These kinases themselves become phosphorylated and are activated for the purposes of initiating various downstream intracellular signaling cascades responsible for initiating gene transcription.

The mitogen-activated protein kinases (MAPKs) are a highly conserved group of enzymes vital to the cellular signal transduction machinery [2 , 3 ]. Three principal members comprise this superfamily; the extracellular-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 MAPKs. Each becomes activated by dual phosphorylation on a key tri-peptide motif containing an intervening amino acid flanked by adjacent threonine and tyrosine residues [4 ]. They are crucial signaling molecules whose substrates are directly (as transcription factors) or indirectly involved in regulating gene expression, and they are implicated in the modulation of cytokine production in various cell types [5 6 7 8 ]. Inflammatory stimuli are known to induce p38 activation in several immune cell subsets, and this activation is necessary for proinflammatory phenotype expression [4 , 9 10 11 ]. Indeed, mast cell activation by stem cell factor, as well as antigen-induced Fc{varepsilon}RI aggregation in primary cultures and cell lines of murine mast cell systems, leads to the activation of p38 MAPK and subsequent release of proinflammatory cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) [6 , 12 , 13 ]. p38{alpha} MAPK is the best-studied isoform and along with p38ß MAPK, is selectively inhibited by the pyridinylimidazole inhibitor SB 203580, one of a class of agents originally prepared as cytokine synthesis inhibitors and found subsequently to act through selective inhibition of members of the MAPK enzyme family [14 ]. Consequently, insights into the regulation of p38 MAPK phosphorylation status and thus activity are crucial to our understanding of the mast cell’s involvement in inflammatory disease processes.

Protein phosphatase 2A (PP2A) is the major family of serine/threonine phosphatases in mammalian cells, containing a highly conserved 36-kDa catalytic ("c") subunit regulated by a 65-kDa regulatory ("a") subunit (together constituting the core "ac" holoenzyme) and an additional associated regulatory ("b") subunit [15 ]. PP2A is involved in various aspects of cell biology including cell-cycle regulation, carcinogenesis and transformation, translation, apoptosis, and not surprisingly, signal transduction [16 ]. This phosphatase has been implicated in the regulation of stress-activated protein kinase (SAPK) pathways [17 , 18 ], and the regulatory "a" subunit coprecipitates with JNK [19 ]. Furthermore, PP2A inhibition with the dinoflagellate polyether metabolite okadaic acid (OA) has resulted in increased JNK and activated protein-1 activity, as well as interleukin (IL)-1ß production [19 ]. Although circumstantial evidence in vitro has long suggested the mutual regulation of PP2A and p38 MAPK, only recently have the two enzymes been shown to coprecipitate in platelets [20 ] and endothelial cells [21 ]. Furthermore, the involvement of p38 MAPK in inflammatory cytokine production by mast cells is well-documented, but with the exception of one previous study by our laboratory, the role of PP2A in mast cell cytokine release has not been investigated. Originally thought of as having solely a housekeeping role in maintaining phosphorylation homeostasis, PP2A has, at least in mast cells, become well-appreciated for taking a proactive and necessary role in the secretory response [22 , 23 ]. We have previously shown that mast cell treatment with OA resulted in significant IL-6 production [24 ] and that this effect is a result of, at least in part, a functional association between PP2A and protein kinase C (PKC){alpha}. However, the additional involvement of PP2A in mast cell signaling has not been further investigated.

In the present study, we provide evidence that the catalytic subunit of the serine/threonine phosphatase PP2A (PP2Ac) and p38{alpha} MAPK is associated in mast cells and that this association mediates the direct regulation of p38{alpha} MAPK phosphorylation and thus activation by PP2A. Furthermore, we demonstrate that this functional association is involved in Fc{varepsilon}RI-mediated signaling as determined by IL-6 production in response to immunological activation. This work clarifies the contribution made by PP2A in mast cell cytokine release and highlights a possible target for therapeutic intervention in the search for methods of controlling aberrant inflammatory responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Rabbit antiphospho-p38{alpha} MAPK (Thr180/Tyr182) and anti-total p38{alpha} MAPK antibody (Ab) for use in immunoblot analyses were from Cell Signaling Technology of New England Biolabs (Beverly, MA). Mouse anti-PP2A catalytic subunit (PP2Ac) Ab were from Transduction Laboratories of BD Biosciences (Mississauga, ON). Protein A/G PLUS-agarose immunoprecipitation beads, secondary Ab for immunoblot analyses [donkey anti-rabbit IgG–horseradish peroxidase (HRP) and donkey anti-mouse IgG–HRP], anti-total p38{alpha} MAPK for use in coimmunoprecipitation experiments, and antifocal adhesion kinase (anti-FAK) were from Santa Cruz Biotechnology (Santa Cruz, CA). RPMI 1640, OA, aprotinin, pepstatin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), prostaglandin E2, sodium deoxycholate, Nonidet P-40 (NP-40), Triton X-100, sodium orthovanadate, sodium fluoride, EDTA, and EGTA were from Sigma/Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was from Medicorp (Montreal, PQ). Iscove’s modified Dulbecco’s medium (IMDM), Dulbecco’s modified Eagle’s medium, and cell culture supplementary components [modified Eagle’s medium (MEM) nonessential amino acid solution, penicillin, streptomycin, and HEPES buffer solution] were from Gibco/BRL of Invitrogen-Life Technologies (Burlington, ON). SB 203580 was from Calbiochem-Novabiochem (San Diego, CA). Trinitrophenol (TNP)–bovine serum albumin (BSA) was from Biosearch Technologies, Inc. (Novato, CA). All other reagents and chemicals were of the highest analytical grade available.

Cell culture
Cell lines and primary cultures were maintained at 37°C in a sterilized, humidified atmosphere containing 5% CO2. The human mast cell line-1 (HMC-1) 5C6 was maintained in IMDM supplemented with 10% FBS and 50 units/ml each of penicillin and streptomycin. The MC/9 murine mast cell line (American Type Culture Collection, Manassas, VA, CRL 8306) was maintained in RPMI-1640 medium supplemented with 20% WEHI-3B-conditioned medium, 15% FBS, 1% MEM nonessential amino acid solution, 50 units/ml each penicillin and streptomycin, and 50 µM ß-mercaptoethanol. Murine primary-cultured bone marrow-derived mast cells (BMMC) were harvested from the femurs and tibias of C57BL/6 mice from Charles River Laboratories (Montreal, PQ) and maintained as described previously [24 ]. Following 4–5 weeks of culture, mast cell purity of >98% was achieved as assessed by toluidine blue staining (pH=1.0) of fixed cytocentrifuged preparations. Mature mast cells were identified by their morphological features and granule prevalence.

Murine mast cell sensitization
One day (20–24 h) prior to experimental immunological activation, MC/9 or BMMC were passively sensitized. Briefly, cells were resuspended in fresh complete medium, supplemented with TIB141-conditioned medium, enriched in IgE directed against TNP, at a ratio of 3:1. MC/9 were typically sensitized at 0.2 million/ml final density; BMMC at 0.5 million/ml. Following sensitization, experimental groups were washed extensively with RPMI 1640, supplemented with 10% FBS alone, resuspended at a higher density (1–5 million/ml) in wash medium, and activated by the addition of 10 ng/ml TNP–BSA.

Preparation of total cellular lysate
Experimental treatments leading to the acquisition of total cellular lysates for immunoblot analysis were typically carried out at densities of 1–5 million cells/ml. At the appropriate times, cells were harvested by centrifugation at 500 gfor 5 min at 4°C. Cell pellets were immediately resuspended in ice-cold lysis buffer [25 mM Tris-HCl, pH=7.5, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] containing freshly added 5 µg/ml leupeptin and pepstatin, 1 mM PMSF, 1 mM dithiothreitol, 100 µM sodium orthovanadate, 10 mM sodium fluoride, 10 µM phenylarsine oxide, and 10 µg/ml aprotinin. Lysates were left on ice for at least 30 min and transferred to Eppendorf tubes for clarification at 15,000 g for 10 min at 4°C to remove cellular debris.

Coimmunoprecipitation
For coimmunoprecipitation studies, samples typically consisted of 10–20 million cells that were harvested by centrifugation at 500 g for 5 min at 4°C. Cell pellets were immediately resuspended in 1 ml ice-cold radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH=7.5, 150 mM sodium chloride, 50 mM sodium phosphate, 0.25% sodium deoxycholate, 0.1% NP-40, 1 mM sodium orthovanadate, and 1 mM sodium fluoride) containing freshly added 5 mM EDTA and EGTA, 10 µg/ml leupeptin and pepstatin, 1 mM PMSF, and 10 µg/ml aprotinin. Lysates were left on ice for at least 30 min and transferred to Eppendorf tubes for clarification at 15,000 g for 10 min at 4°C to remove cellular debris. To 500 µl clarified RIPA lysate, 1 µg primary Ab was added, and the sample was incubated for 1 h at 4°C with end-over-end mixing. Immunoreactive proteins and protein complexes were then precipitated by the addition of 20 µl of a protein A/G PLUS-agarose bead slurry and incubated at 4°C overnight with end-over-end mixing. The beads were pelleted by centrifugation at 1200 g for 5 min at 4°C, and the supernatant was discarded. The bead pellet was washed at least four times with ice-cold phosphate-buffered saline (sodium chloride concentration adjusted to 1 M) before the addition of 40 µl 3x SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and storage at –20°C until further SDS-PAGE and immunoblot analysis.

Cytokine release experiments
Following overnight sensitization and extensive washing, BMMC were resuspended in RPMI 1640 supplemented with 10% FBS at a density of 0.5 million cells/ml, and a total volume of 500 µl per test was aliquotted to Eppendorf tubes. Activators and/or inhibitors were added, and samples were incubated for further 20–24 h at 37°C in a sterilized, humidified atmosphere containing 5% CO2. Supernatants were then harvested and frozen for the subsequent determination of IL-6 concentration by enzyme-linked immunosorbent assay (ELISA) as described previously [24 ].

Data analysis
Comparisons between experimental treatments were analyzed by the one-sided Wilcoxon scores nonparametric procedure. A statistical value of P < 0.05 was established as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Synergistic effects of OA on Fc{varepsilon}RI-mediated IL-6 production
We have previously shown that inhibition of PP2A by OA treatment increases IL-6 production by primary-cultured murine BMMC in a dose-dependent manner [24 ]. To further identify relevant downstream effects of direct modulation of cytokine production by PP2A, we decided to explore the effects of OA and antigen treatment on mast cell IL-6 secretion. Consistent with previous findings from our laboratory, Figure 1 shows a significant increase in IL-6 production by BMMC upon overnight treatment with a suboptimal dose (100 nM) of OA alone (275.3±128.7 pg/ml) compared with control levels (1.6±1.2 pg/ml). Our system also displays a robust IL-6 response in cells treated with TNP–BSA antigen (1518.8±360.0 pg/ml). It is remarkable that when BMMC were treated with OA, followed 15 min later by 10 ng/ml antigen for an additional 20–24 h incubation, a strong, synergistic effect on IL-6 production was seen (3646.0±894.1 pg/ml). These data support a role for PP2A in the regulation of mast cell signaling and IL-6 production in response to immunological activation through Fc{varepsilon}RI aggregation.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. OA and Fc{varepsilon}RI-mediated activation synergistically induce IL-6 production in mast cells. BMMC were sensitized with IgE directed against TNP for 20–24 h and activated by the addition of 10 ng/ml TNP–BSA for an additional 20–24 h in the absence or presence of a 15-min pretreatment with 100 nM OA. Supernatants were analyzed by ELISA for IL-6 production. OA and TNP–BSA each caused a significant induction of cytokine release compared with control (NT), and coincubation with both agents resulted in a synergistic IL-6 response. Results are the means ± SE of eight experiments, with each condition assayed in duplicate (*, P<0.05, compared with NT; **, P<0.05, compared with OA or TNP–BSA alone).

 
To examine whether PP2A has a role in the regulation of additional cytokine production such as TNF, BMMC were treated with various concentrations of OA (10, 50, 100 nM) for 18 h. Cell-free supernatants were used to determine TNF production by ELISA. OA induced a concentration-dependent TNF production (untreated mast cells: undetectable; OA 10 nM: 15.2 pg/ml; OA 50 nM: 151.9 pg/ml; OA 100 nM: 230.6 pg/ml), suggesting a potential role for PP2A in the regulation of TNF production by mast cells.

p38{alpha} MAPK inhibition blocks OA- and Fc{varepsilon}RI-induced IL-6 production
We next carried out experiments in which the specific p38{alpha} MAPK inhibitor SB 203580 was used in an attempt to block IL-6 production in response to OA alone, Fc{varepsilon}RI aggregation, or both methods of stimulation. SB 203580 dose-dependently inhibited OA (Fig. 2a )- and antigen-induced (Fig. 2b) IL-6 production, as well as cytokine release in response to mast cell activation by both methods of stimulation (Fig. 2c) . A suboptimal dose of the compound (1 µM) elicited significant inhibition in all three contexts. It is interesting that treatment with the highest dose of SB 203580 (10 µM) led to the inhibition of IL-6 to varying degrees, providing further evidence for a complex regulatory role for PP2A in p38 MAPK-mediated cytokine production in this cell type.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. IL-6 production induced by OA and/or Fc{varepsilon}RI-mediated activation is differentially regulated by p38{alpha} MAPK. BMMC were sensitized with IgE directed against TNP for 20–24 h and activated by the addition of (a) 100 nM OA, (b) 10 ng/ml TNP–BSA, or (c) both for an additional 20–24 h in the presence of increasing concentrations of the p38 MAPK inhibitor SB 203580 (SB). Supernatants were analyzed by ELISA for IL-6 production. A differential dose-dependent decrease in IL-6 release was seen in response to each induction context. Results are the mean ± SE of eight experiments, with each condition assayed in duplicate (*, P<0.05, compared with 0 µM SB 203580 control).

 
Treatment with OA enhances p38{alpha} MAPK phosphorylation
To examine if inhibition of PP2A by OA modulates p38{alpha} MAPK phosphorylation in mast cells, HMC-1, murine MC/9, and BMMC were treated with various doses of the inhibitor (0.1, 0.5, and 1 µM) for 1 h. Total cellular lysates were obtained and subjected to SDS-PAGE and immunoblot analysis with Ab to p38{alpha} MAPK, which is dually phosphorylated on Thr180/Tyr182 (Fig. 3 , upper panels), or total p38{alpha} MAPK (Fig. 3 , lower panels). The phosphorylation of p38{alpha} MAPK was shown to be enhanced following OA treatment in HMC-1 (Fig. 3a) , as well as murine MC/9 (Fig. 3b) , and BMMC (Fig. 3c) . The increases in phosphorylated p38{alpha} MAPK were largely dose-dependent and were not a result of changes in total p38{alpha} MAPK levels, as lysates, when immunoblotted with a phosphorylation-independent Ab to total p38{alpha} MAPK protein, showed similar levels, even in OA-treated samples.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. Treatment of mast cells with OA results in enhanced phosphorylation of p38{alpha} MAPK. HMC-1 (a), MC/9 (b), and BMMC (c) were treated with increasing concentrations of OA at various concentrations for 1 h, and a total cellular lysate was obtained. Samples were subjected to SDS-PAGE and immunoblot analysis with Ab, recognizing p38{alpha} MAPK, which is dually phosphorylated on Thr180/Tyr182 (upper panels). Membranes were also immunoblotted with a nonphospho-specific Ab to confirm equal protein levels in each lane by revealing total p38{alpha} MAPK.

 
Effect of OA on Fc{varepsilon}RI-mediated phosphorylation of p38{alpha} MAPK
To investigate if the inhibition of PP2A and resulting p38{alpha} MAPK phosphorylation is involved in Fc{varepsilon}RI-mediated mast cell activation, we treated murine MC/9 mast cells and BMMC with OA, antigen, or both and subjected the lysates to SDS-PAGE and immunoblot analysis with total and phospho-specific p38{alpha} MAPK Ab. Cells were sensitized with IgE directed against a TNP antigen before activation. As shown in Figure 4 , p38{alpha} MAPK phosphorylation was increased in response to the addition of 10 ng/ml TNP antigen in MC/9 and BMMC. These sensitized cells, when treated with OA for 10 or 60 min, showed increased levels of phosphorylated p38{alpha} MAPK. However, when sensitized cells were pretreated with OA for the indicated times prior to addition of the TNP antigen, p38{alpha} MAPK phosphorylation was robustly enhanced in MC/9 cells (Fig. 4a) and BMMC (Fig. 4b) . Indeed, observation of band intensity clearly shows a greater-than-additive effect when compared with treatment with OA or antigen alone. The lower panels show samples immunoblotted with Ab to total p38 MAPK.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 4. OA treatment potentiates Fc{varepsilon}RI-mediated phosphorylation of p38{alpha} MAPK. SDS-PAGE and Western analysis with phospho-specific and total p38{alpha} MAPK Ab were used to determine the effect of OA on p38{alpha} MAPK phosphorylation in response to immunological activation. MC/9 mast cells (a) and mouse BMMC (b) were sensitized with IgE directed against TNP (IgE). Cells were activated with 10 ng/ml TNP–BSA for 20 min (IgE. + TNP), 500 nM OA for the indicated times (IgE. + OA), or both (IgE. + OA + TNP). OA and TNP–BSA each induced moderate increases in p38{alpha} MAPK phosphorylation, and OA pretreatment followed by TNP–BSA activation resulted in a greater-than-additive effect on p38{alpha} MAPK phosphorylation at both time-points assayed. The lower panels confirm equal, total p38{alpha} MAPK protein levels in response to all experimental treatments, indicating the increase in phosphorylation status is not a result of variations in total protein concentration within the lysate.

 
PP2Ac and p38{alpha} MAPK are physically associated in human and murine mast cells
To determine if PP2A and p38 MAPK are associated in this cell type, primary-cultured murine BMMC as well as the murine MC/9 and HMC-1 mast cell lines were used in our investigation. Immunoprecipitation and subsequent immunoblot analysis revealed the constitutive expression of p38{alpha} MAPK and the PP2Ac in all three populations of unstimulated mast cells (Fig. 5b and 5d , respectively). As seen in Figure 5a , immunoprecipitates of p38{alpha} MAPK that were immunoblotted with Ab directed against PP2Ac revealed the presence of this enzyme. Reciprocally, immunoprecipitates of PP2Ac were immunoblotted with an Ab directed against p38{alpha} MAPK, and in Figure 5c , it can be seen that p38{alpha} MAPK is present in PP2A immunoprecipitates. Therefore, PP2Ac and p38{alpha} MAPK are physically associated in mast cells. To exclude the possibility of the nonspecific binding of PP2Ac with p38{alpha} MAPK, PP2Ac immunoprecipitates of HMC-1, MC/9, and BMMC were immunoblotted with anti-FAK Ab. Although mast cells express FAK, no FAK protein was detected in PP2Ac immunoprecipitates. Reciprocally, there was no PP2Ac protein found in FAK immunoprecipitates (data not shown). Further, mast cell (HMC-1) lysates were immunoprecipitated with anti-FAK Ab or anti-p38{alpha} MAPK Ab and probed with anti-FAK Ab, and no p38{alpha} MAPK was seen in FAK immunoprecipitates (Fig. 5e) . This serves as an additional negative control for our immunoprecipitation experiment.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 5. PP2Ac and p38{alpha} MAPK are physically associated in mast cells. HMC-1, MC/9, and BMMC were grown to subconfluence and harvested for lysis in RIPA buffer. (a) Lysates were immunoprecipitated (IP) with Ab to p38{alpha} MAPK and subjected to immunoblot (WB) analysis with Ab to PP2Ac. (b) Lysates were immunoprecipitated with Ab to p38{alpha} MAPK and subjected to immunoblot analysis with Ab to p38{alpha} MAPK. (c) Lysates were immunoprecipitated with Ab to PP2Ac and subjected to immunoblot analysis with Ab to p38{alpha} MAPK. (d) Lysates were immunoprecipitated with Ab to PP2Ac and subjected to immunoblot analysis with Ab to PP2Ac. Mast cells constitutively express p38{alpha} MAPK (b) and PP2Ac (d). The presence of PP2Ac in p38{alpha} MAPK immunoprecipitates (a) and p38{alpha} MAPK in PP2Ac immunoprecipitates (c) demonstrates the physical association between these two enzymes in mast cells. (e) HMC-1 lysates were immunoprecipitated with Ab to FAK or p38{alpha} MAPK and were immunoblotted with anti-FAK Ab.

 
To examine if phosphorylated p38{alpha} MAPK following TNP stimulation is associated with PP2Ac, mast cell lysates from TNP-treated or untreated BMMC were immunoprecipitated with anti-PP2Ac Ab and then probed with antiphospho-p38{alpha} MAPK. There was little change of phospho-p38{alpha} MAPK in the PP2Ac immunoprecipitates from TNP-treated samples compared with that from untreated mast cells, suggesting that TNP-induced, phophorylated p38{alpha} MAPK was likely released from the p38{alpha} MAPK–PP2Ac complex (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A classical method of mast cell activation, in vivo and in vitro, involves the clustering Fc{varepsilon}RI by the addition of antigen to cells that have been previously sensitized with IgE directed against the antigen [1 ]. The role of p38 MAPK in Fc{varepsilon}RI-mediated mast cell activation and subsequent cytokine production has been relatively well-characterized. This enzyme is activated in response to receptor aggregation in primary-cultured murine BMMC as well as the MC/9 mast cell line [12 , 13 ]. Furthermore, p38 MAPK has been implicated positively in the production of a number of mast cell-derived cytokines such as TNF [13 ], IL-4 [25 ], IL-8 [26 ], and it is interesting, negatively in human mast cell-derived granulocyte/macrophage-colony stimulating factor production [27 ].

Complete activation of the main isoforms of p38 MAPK requires dual phosphorylation on a specific activation motif consisting of two adjacent threonine and tyrosine residues separated by a single intervening residue. In p38{alpha} MAPK, threonine 180 and tyrosine 182 are the relevant residues [28 ]. Thus, it was appropriate to investigate the possible regulation of threonine phosphorylation, specifically, by the most common mammalian phosphatase, PP2A.

Studies aimed at elucidating the function of PP2A have been carried out almost exclusively with the use of the selective PP2A inhibitor OA, which at concentrations of less than 1 µM, has no detectable inhibitory effect on PP1, PP2B, or PP2C [29 ]. Indeed, OA has been instrumental in virtually every initial mast cell examination of PP2A function and has led to more sophisticated transient translocation studies [22 ] and the finding that PP2A is actually required for the mast cell secretory response [30 ]. Recent, well-designed studies have defined a temporal role for PP2A in the mast cell secretory response [30 ], as well as implicating the serine/threonine phosphatase in calcium ionophore and antigen-induced mast cell degranulation [31 ]. It is surprising, however, that there has been no work to date regarding the involvement of this crucial enzyme in signaling events downstream of receptor-mediated activation leading to cytokine production.

Earlier studies have also provided evidence describing the mutual regulation of PP2A and p38 MAPK, two of the most significant molecules involved in cellular signal transduction. Recent immunoprecipitation data have confirmed the physical association between the two enzymes in two mammalian systems [20 , 21 ]. Through pharmacological studies with OA, PP2A has been shown to be involved in the control of phosphorylation status of various MAPK family members, including p38 MAPK [32 ], although it has been more strongly implicated in the direct regulation of the SAPK signaling cascade [18 ]. It is interesting that a recent study has indicated that p38 MAPK might, through regulating the activity of PP2A, indirectly inhibit upstream components of the ERK–MAPK cascade responsible for collagenase 1 production in human and mouse fibroblasts [33 ]. Clearly, there is a relationship between these two enzymes in a number of types of cellular activation. In the present study, we provide the first evidence in mast cells that the catalytic subunit of PP2A is physically associated with p38{alpha} MAPK and that this interaction is responsible for allowing possible direct control of p38{alpha} MAPK phosphorylation status by PP2A.

Previous investigations in our laboratory have shown that PP2Ac is also associated with a partnered serine/threonine kinase–PKC{alpha} [24 ]. One can thus easily envision a method of regulation of p38 MAPK signaling, where a balance is maintained between PP2A and PKC enzymatic activity acting to directly influence p38{alpha} MAPK phosphorylation at threonine 180. In the nonstimulated condition, threonine phosphorylation is maintained at a very low basal level by PP2A, and when the mast cell becomes activated by antigen-induced Fc{varepsilon}RI aggregation, PKC activity can override this phosphatase activity to enhance p38 MAPK activation and affect downstream transcriptional activation.

It has been shown that mast cell IL-6 production is regulated by the transcription factor nuclear factor (NF)-{kappa}B binding to specific {kappa}B elements in the IL-6 promoter [34 , 35 ]. p38 MAPK [36 ] and various PKC isoforms, some through the direct activation of p38 MAPK [37 ], have been shown to augment NF-{kappa}B transactivation [38 ] and thus, might contribute to control of IL-6 gene expression at this level of transcriptional regulation. Alternatively, there is recent evidence that in epithelial cells, activated via Toll-like receptor 5 to produce IL-8, p38 MAPK inhibition by SB 203580 had no effect on NF-{kappa}B activity but instead, reduced protein expression by 75% by influencing translation [39 ]. PP2A could consequently contribute a major influence on p38 MAPK, PKC, and other kinase activities at a number of signaling levels involved in gene expression, ensuring that physiological responses proceed at an appropriate intensity upon cellular activation.

We thus propose a working model of PP2A-mediated regulation of p38 MAPK-related signaling pathways involved in the immunological activation of mast cells (Fig. 6 ). Upon binding of antigen-specific IgE to the high-affinity Fc{varepsilon}RI on the mast cell surface, antigen-mediated receptor aggregation leads to the activation of numerous upstream receptor-associated as well as nonreceptor-associated kinase cascades. These ultimately lead to the activation of p38 MAPK through enhancement of its phosphorylation on key threonine and/or tyrosine residues. This might occur, in the case of threonine phosphorylation, through the action of specific kinases (such as PKC) or through the alleviation of serine/threonine phosphatase (PP2A) pressure, which normally maintains p38 MAPK in a hypo-phosphorylated state under basal conditions. Activated p38 MAPK is then free to induce the transactivation and binding of NF-{kappa}B to IL-6 promoter elements. Consequently, p38 MAPK and its regulatory partner PP2A are critically involved in mediating extracellular activation signals that ultimately result in the induction of mast cell inflammatory cytokines, namely IL-6.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 6. Proposed model of the PP2A regulatory role in p38{alpha} MAPK signaling in the mast cell.

 


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the Nova Scotia Health Research Foundation, and the Isaac Walton Killam (IWK) Health Centre to T-J. L. T-J. L. is the recipient of a CIHR New Investigator award and an Investigatorship award from IWK Health Centre. R. T. M. B. is the recipient of a Cancer Research and Education (CaRE)–Nova Scotia Trainee award, with funding from Cancer Care Nova Scotia and the Canadian Cancer Society. The authors acknowledge the excellent technical assistance of Fang Liu for generation and long-term maintenance of murine BMMC cultures.

Received October 22, 2003; revised July 20, 2004; accepted July 21, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rivera, J. (2002) Molecular adapters in Fc {epsilon}RI signaling and the allergic response Curr. Opin. Immunol. 14,688-693[CrossRef][Medline]
  2. Robinson, M. J., Cobb, M. H. (1997) Mitogen-activated protein kinase pathways Curr. Opin. Cell Biol. 9,180-186[CrossRef][Medline]
  3. Weston, C. R., Lambright, D. G., Davis, R. J. (2002) Signal transduction. MAP kinase signaling specificity Science 296,2345-2347[Abstract/Free Full Text]
  4. Arbabi, S., Maier, R. V. (2002) Mitogen-activated protein kinases Crit. Care Med. 30(Suppl. 1),S74-S79[CrossRef][Medline]
  5. Arbabi, S., Garcia, I., Bauer, G. J., Maier, R. V. (1999) Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway J. Immunol. 162,7441-7445[Abstract/Free Full Text]
  6. Ishizuka, T., Terada, N., Gerwins, P., Hamelmann, E., Oshiba, A., Fanger, G. R., Johnson, G. L., Gelfand, E. W. (1997) Mast cell tumor necrosis factor {alpha} production is regulated by MEK kinases Proc. Natl. Acad. Sci. USA 94,6358-6363[Abstract/Free Full Text]
  7. Shapiro, L., Dinarello, C. A. (1995) Osmotic regulation of cytokine synthesis in vitro Proc. Natl. Acad. Sci. USA 92,12230-12234[Abstract/Free Full Text]
  8. Rosengart, M. R., Arbabi, S., Garcia, I., Maier, R. V. (2000) Interactions of calcium/calmodulin-dependent protein kinases (CaMK) and extracellular-regulated kinase (ERK) in monocyte adherence and TNF{alpha} production Shock 13,183-189[Medline]
  9. Nick, J. A., Avdi, N. J., Gerwins, P., Johnson, G. L., Worthen, G. S. (1996) Activation of a p38 mitogen-activated protein kinase in human neutrophils by lipopolysaccharide J. Immunol. 156,4867-4875[Abstract]
  10. Nick, J. A., Avdi, N. J., Young, S. K., Knall, C., Gerwins, P., Johnson, G. L., Worthen, G. S. (1997) Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP J. Clin. Invest. 99,975-986[Medline]
  11. Ridley, S. H., Sarsfield, S. J., Lee, J. C., Bigg, H. F., Cawston, T. E., Taylor, D. J., DeWitt, D. L., Saklatvala, J. (1997) Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels J. Immunol. 158,3165-3173[Abstract]
  12. Ishizuka, T., Kawasome, H., Terada, N., Takeda, K., Gerwins, P., Keller, G. M., Johnson, G. L., Gelfand, E. W. (1998) Stem cell factor augments Fc {varepsilon} RI-mediated TNF-{alpha} production and stimulates MAP kinases via a different pathway in MC/9 mast cells J. Immunol. 161,3624-3630[Abstract/Free Full Text]
  13. Ishizuka, T., Chayama, K., Takeda, K., Hamelmann, E., Terada, N., Keller, G. M., Johnson, G. L., Gelfand, E. W. (1999) Mitogen-activated protein kinase activation through Fc {varepsilon} receptor I and stem cell factor receptor is differentially regulated by phosphatidylinositol 3-kinase and calcineurin in mouse bone marrow-derived mast cells J. Immunol. 162,2087-2094[Abstract/Free Full Text]
  14. Lee, J. C., Kumar, S., Griswold, D. E., Underwood, D. C., Votta, B. J., Adams, J. L. (2000) Inhibition of p38 MAP kinase as a therapeutic strategy Immunopharmacology 47,185-201[CrossRef][Medline]
  15. Lechward, K., Awotunde, O. S., Swiatek, W., Muszynska, G. (2001) Protein phosphatase 2A: variety of forms and diversity of functions Acta Biochim. Pol. 48,921-933[Medline]
  16. Janssens, V., Goris, J. (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling Biochem. J. 353,417-439[CrossRef][Medline]
  17. Keyse, S. M. (2000) Protein phosphatases and the regulation of mitogen-activated protein kinase signalling Curr. Opin. Cell Biol. 12,186-192[CrossRef][Medline]
  18. Tamura, S., Hanada, M., Ohnishi, M., Katsura, K., Sasaki, M., Kobayashi, T. (2002) Regulation of stress-activated protein kinase signaling pathways by protein phosphatases Eur. J. Biochem. 269,1060-1066[Medline]
  19. Shanley, T. P., Vasi, N., Denenberg, A., Wong, H. R. (2001) The serine/threonine phosphatase, PP2A: endogenous regulator of inflammatory cell signaling J. Immunol. 166,966-972[Abstract/Free Full Text]
  20. Sundaresan, P., Farndale, R. W. (2002) p38 mitogen-activated protein kinase dephosphorylation is regulated by protein phosphatase 2A in human platelets activated by collagen FEBS Lett. 528,139-144[CrossRef][Medline]
  21. Lee, T., Kim, S. J., Sumpio, B. E. (2003) Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain J. Cell. Physiol. 194,349-355[CrossRef][Medline]
  22. Ludowyke, R. I., Holst, J., Mudge, L. M., Sim, A. T. (2000) Transient translocation and activation of protein phosphatase 2A during mast cell secretion J. Biol. Chem. 275,6144-6152[Abstract/Free Full Text]
  23. Peirce, M. J., Munday, M. R., Peachell, P. T. (1999) Role of protein phosphatases in the regulation of human mast cell and basophil function Am. J. Physiol. 277,C1021-C1028
  24. Boudreau, R. T., Garduno, R., Lin, T. J. (2002) Protein phosphatase 2A and protein kinase C{alpha} are physically associated and are involved in Pseudomonas aeruginosa-induced interleukin 6 production by mast cells J. Biol. Chem. 277,5322-5329[Abstract/Free Full Text]
  25. Hirasawa, N., Sato, Y., Fujita, Y., Ohuchi, K. (2000) Involvement of a phosphatidylinositol 3-kinase-p38 mitogen activated protein kinase pathway in antigen-induced IL-4 production in mast cells Biochim. Biophys. Acta. 1456,45-55[Medline]
  26. Feoktistov, I., Goldstein, A. E., Biaggioni, I. (1999) Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells Mol. Pharmacol. 55,726-734[Abstract/Free Full Text]
  27. Kimata, M., Inagaki, N., Kato, T., Miura, T., Serizawa, I., Nagai, H. (2000) Roles of mitogen-activated protein kinase pathways for mediator release from human cultured mast cells Biochem. Pharmacol. 60,589-594[CrossRef][Medline]
  28. Doza, Y. N., Cuenda, A., Thomas, G. M., Cohen, P., Nebreda, A. R. (1995) Activation of the MAP kinase homologue RK requires the phosphorylation of Thr-180 and Tyr-182, and both residues are phosphorylated in chemically stressed KB cells FEBS Lett. 364,223-228[CrossRef][Medline]
  29. Millward, T. A., Zolnierowicz, S., Hemmings, B. A. (1999) Regulation of protein kinase cascades by protein phosphatase 2A Trends Biochem. Sci. 24,186-191[CrossRef][Medline]
  30. Holst, J., Sim, A. T., Ludowyke, R. I. (2002) Protein phosphatases 1 and 2A transiently associate with myosin during the peak rate of secretion from mast cells Mol. Biol. Cell 13,1083-1098[Abstract/Free Full Text]
  31. Ludowyke, R. I., Warton, K., Scurr, L. L. (1998) Inhibition of antigen and calcium ionophore induced secretion from RBL-2H3 cells by phosphatase inhibitors Cell Biol. Int. 22,855-865[CrossRef][Medline]
  32. Chen, K. D., Lai, M. T., Cho, J. H., Chen, L. Y., Lai, Y. K. (2000) Activation of p38 mitogen-activated protein kinase and mitochondrial Ca(2+)-mediated oxidative stress are essential for the enhanced expression of grp78 induced by the protein phosphatase inhibitors okadaic acid and calyculin A J. Cell. Biochem. 76,585-595[CrossRef][Medline]
  33. Westermarck, J., Li, S. P., Kallunki, T., Han, J., Kahari, V. M. (2001) p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression Mol. Cell. Biol. 21,2373-2383[Abstract/Free Full Text]
  34. Marquardt, D. L., Walker, L. L. (2000) Dependence of mast cell IgE-mediated cytokine production on nuclear factor-{kappa}B activity J. Allergy Clin. Immunol. 105,500-505[CrossRef][Medline]
  35. Kalesnikoff, J., Baur, N., Leitges, M., Hughes, M. R., Damen, J. E., Huber, M., Krystal, G. (2002) SHIP negatively regulates IgE + antigen-induced IL-6 production in mast cells by inhibiting NF-{kappa} B activity J. Immunol. 168,4737-4746[Abstract/Free Full Text]
  36. Madrid, L. V., Mayo, M. W., Reuther, J. Y., Baldwin, A. S., Jr (2001) Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-{kappa} B through utilization of the I{kappa} B kinase and activation of the mitogen-activated protein kinase p38 J. Biol. Chem. 276,18934-18940[Abstract/Free Full Text]
  37. Rahman, A., Anwar, K. N., Uddin, S., Xu, N., Ye, R. D., Platanias, L. C., Malik, A. B. (2001) Protein kinase C-{delta} regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase Mol. Cell. Biol. 21,5554-5565[Abstract/Free Full Text]
  38. Lallena, M. J., Diaz-Meco, M. T., Bren, G., Paya, C. V., Moscat, J. (1999) Activation of I{kappa}B kinase ß by protein kinase C isoforms Mol. Cell. Biol. 19,2180-2188[Abstract/Free Full Text]
  39. Yu, Y., Zeng, H., Lyons, S., Carlson, A., Merlin, D., Neish, A. S., Gewirtz, A. T. (2003) TLR5-mediated activation of p38 MAPK regulates epithelial IL-8 expression via a post-transcriptional mechanism Am. J. Physiol. Gastrointest. Liver Physiol. 285,G282-G290[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Dent. Res.Home page
C.S. Patil and K.L. Kirkwood
p38 MAPK Signaling in Oral-related Diseases
J. Dent. Res., September 1, 2007; 86(9): 812 - 825.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
Y. Liu, M. Zhu, K. Nishida, T. Hirano, and W. Zhang
An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response
J. Exp. Med., January 22, 2007; 204(1): 93 - 103.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1003498v1
76/5/1075    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boudreau, R. T. M.
Right arrow Articles by Lin, T.-J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boudreau, R. T. M.
Right arrow Articles by Lin, T.-J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS