Skip Navigation


International Immunology Advance Access originally published online on March 28, 2006
International Immunology 2006 18(5):767-773; doi:10.1093/intimm/dxl012
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/5/767    most recent
dxl012v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lessmann, E.
Right arrow Articles by Huber, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lessmann, E.
Right arrow Articles by Huber, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A redundant role for PKC-{varepsilon} in mast cell signaling and effector function

Eva Lessmann1, Michael Leitges2,3, and Michael Huber1

1 Department of Molecular Immunology, Institute for Biology III, University of Freiburg and Max Planck Institute for Immunobiology, Stübeweg 51, 79108 Freiburg, Germany
2 Department of Medicine, Medical School Hannover, 30625 Hannover, Germany
3 Max Planck Institute for Experimental Endocrinology, 30625 Hannover, Germany

Correspondence to: Michael Huber; E-mail: huberm{at}immunbio.mpg.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and Discussion
 References
 
Protein kinase (PK) C-{varepsilon} is strongly expressed in mast cells (MCs) and activated in response to antigen-mediated high-affinity receptor for IgE (Fc{varepsilon}R1) engagement. A critical role of PKC-{varepsilon} in antigen-triggered activation of various signaling pathways was observed in basophilic leukemia cells. To study the function of PKC-{varepsilon} in MCs differentiated in vitro from murine bone marrow, we used our established PKC-{varepsilon} null mice. Unexpectedly, we did not reveal any difference in antigen-induced activation of many central signaling molecules (PKB, mitogen-activated protein kinase, p38, Jun-N-terminal kinase, phospholipase C-{gamma}1, Bruton's tyrosine kinase, PKD, Fos and PKC-{delta}) in time-course as well as dose-response studies between PKC-{varepsilon}-deficient and wild-type MCs. In correlation, antigen-triggered degranulation, release of arachidonic acid and secretion of IL-6 were unaltered by the loss of PKC-{varepsilon}. Furthermore, stimulation of MCs via different receptor systems [Steel factor receptor (c-kit) and toll-like receptor 4] did not lead to differences in the measured responses between both cell types. These results strongly suggest that PKC-{varepsilon} plays a redundant role in MCs stimulated by antigen as well as other well-known MC stimuli.

Keywords: degranulation, Fc{varepsilon}R1, IL-6, signal transduction, TLR4


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and Discussion
 References
 
The protein kinase (PK) C family of Ser-/Thr-kinases can be subdivided into three groups based on activation requirements. ‘Conventional’ PKCs ({alpha}, ßI, ßII, {gamma}) require both diacylglycerol (DAG) and calcium ions, ‘novel’ PKCs ({delta}, {varepsilon}, {eta}, {theta}) only depend on DAG and ‘atypical’ PKCs ({zeta}, {iota}, {lambda}) seem to be completely independent of both factors for their activation (1). Cells in general express more than one isoform of PKCs and thus, different PKC isoforms are thought to fulfill differential tasks in cells. However, there also might exist certain functional redundancies.

Mast cells (MCs) express several PKC isozymes and PKC in general has been demonstrated to be a promoter of ionophore-induced degranulation of RBL-2H3 MCs (2). Co-stimulation of MCs with ionophore and the phorbolester phorbol 12-myristate 13-acetate (PMA), which structurally mimicks DAG and constitutively activates conventional as well as novel PKCs, results in a synergistic degranulation response, whereas PMA alone does not induce degranulation at all (2). However, in the context of antigen stimulation of RBL-2H3 cells, PMA has also been demonstrated to be an attenuator of degranulation (2), suggesting that PKCs are involved in both the activation and termination of the secretory process. Most of attenuation of degranulation seems to be due to PMA-activated PKCs decreasing the opening probability of the store-operated calcium channels (3), thus reducing calcium influx into MCs, which is known to be important for the secretory response.

To shed more light on the differential functions of PKC isozymes, RBL-2H3 cells were reported to be permeabilized and washed to remove PKC molecules. Subsequently, recombinant PKC isozymes were re-added to the washed cells and MC responses measured (4, 5). Interestingly, different PKC isozymes were shown to be responsible for the regulation of different effector functions in these cells (4, 5). However, the loss of further regulatory proteins by the permeabilization procedure could not be ruled out using this approach. The pro-secretory function of PKC-ß, which was identified using this technique, was later verified with the help of PKC-ß-deficient bone marrow-derived mast cells (BMMCs) (4, 6). In contrast, PKC-{delta} was suggested to be of pro-secretory function (4); however, analysis of PKC-{delta}-deficient BMMCs revealed that PKC-{delta} is a negative regulator of MC degranulation (7). These examples clearly demonstrate the need for verification of differential PKC functions by alternative approaches, for instance the use of BMMCs from mice deficient for different PKC isozymes.

Using the technique of protein over-expression (about 100-fold) in RBL-2H3 cells, PKC-{varepsilon} has been suggested to be implicated in the suppression of phospholipase (PL) A2 activation in MCs (8), indicating its involvement in negative regulation of calcium mobilization and/or mitogen-activated protein kinase (MAPK) activation. Both signaling events are known to be crucial for the activation of PLA2 (9). Furthermore, using the aforementioned permeabilization approach, PKC-{varepsilon} has been shown to positively affect transcription of Fos/Jun transcription factors (10), indicating a positive effect of PKC-{varepsilon} on activation of MAPK pathways. Moreover, involvement of PKC-{varepsilon} in the regulation of MAPK pathways has been suggested in various cellular systems on the basis of different techniques (antisense oligonucleotides, pharmacological inhibitors, over-expression and heterologous expression of constitutively active as well as dominant-negative mutants) (11–14). Using PKC-{varepsilon}-deficient BMMCs, the role of PKC-{varepsilon} for the activation of MAPK as well as additional pathways in response to antigen and further relevant stimuli was re-investigated. In addition, since the negative regulatory effect of PKC-{delta} on MC degranulation was only of mild nature, PKC-{varepsilon}-deficient BMMCs were analyzed in this respect to test whether PKC-{varepsilon} also exerts some function for the process of mediator secretion.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and Discussion
 References
 
Mice and cell culture
PKC-{varepsilon} knockout mice generation has been described previously (15). In brief, E14 ES cells (129/Ola) were used for the targeting experiment following the standard procedures of the gene-targeting approach in mouse. Homologously recombined ES cell clones were then introduced by injection into C57Bl/6 blastocystes. Germline transmission of the injected ES cells was identified by crossing chimeric males to C57Bl/6 females and subsequently the presence of agouti coat color in the F1 progeny. F1 heterozygote breedings gave rise to homozygous animals, which correspond to the hybrid (129/Ola x C57Bl/6). Subsequently F1 heterozygote animals were backcrossed on the 129/Sv wild-type background for at least five generations. Bone marrow cells (1 x 106 ml–1) from 6- to 8-week-old PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– mice (129/Sv and 129/Ola x C57BL/6) were cultured (37°C, 5% CO2) in a single-cell suspension in RPMI 1640 medium containing 20% FCS, 1% X63Ag8-653-conditioned medium as a source of IL-3 (16), 2 mM L-glutamine, 1 x 10–5 M 2-mercaptoethanol, 50 units ml–1 penicillin and 50 mg ml–1 streptomycin. In weekly intervals, the non-adherent cells were reseeded at 1 x 106 cells ml–1 in fresh medium. By 4–6 weeks in culture, more than 99% of the cells were Steel factor receptor (c-kit) and high-affinity receptor for IgE (Fc{varepsilon}R1) positive as assessed by FACS using phycoerythrin-labeled anti-c-kit antibodies (Pharmingen, Mississauga, Canada) and FITC-labeled IgE (anti-erythropoietin 26), respectively.

Reagents
Polyclonal anti-P-p38 (T180/Y182), anti-P-PKB (S473), anti-P-Bruton's tyrosine kinase (Btk) (Y223), anti-P-Jun-N-terminal kinase (JNK) (T183/Y185), anti-inhibitor of NF-{kappa}B (I{kappa}B{alpha}), anti-P-I{kappa}B{alpha} (S32) and anti-P-PKD (S744/S748) antibodies were purchased from Cell Signaling Technology (Frankfurt a. M., Germany). Polyclonal anti-P-phospholipase (PL) C-{gamma}1 (Y783) (sc-12943-R), anti-PKC-{alpha}, anti-PKC-ß, anti-PKC-{delta} (C-17), anti-PKC-{varepsilon} (C-15), anti-panFos (K-25) and anti-actin (I-19) antibodies were obtained from Santa Cruz (Heidelberg, Germany). Anti-P-PKC-{delta} (Y311) was from Stressgen (Victoria, BC, Canada). Polyclonal anti-p85 (#06-195) was purchased from Upstate/Biomol (Hamburg, Germany) and monoclonal anti-P-MAPK antibodies (T202/Y204) (12D4) were from Nanotools (Teningen, Germany). Monoclonal antibody against the ß-subunit of the Fc{varepsilon}R1 was a generous gift from Dr. R. Siraganian (Bethesda, MD, USA). DNP-HSA containing 30–40 moles DNP per mole albumin, monoclonal IgE with specificity for DNP (clone SPE-7) and PMA were purchased from SIGMA (Deisenhofen, Germany). Thapsigargin was obtained from Calbiochem (Schwalbach, Germany). Recombinant human IGF-1 was purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany) and recombinant murine Steel factor (SF) from Biosource (Nivelles, Belgium). Lipid A (liquid form) from Salmonella minnesota LPS (R595) was from Alexis (Gruenberg, Germany).

Toluidine blue staining of BMMCs
Cytospin slides were prepared by centrifugation of 5 x 104 cells per 200 µl RPMI/0.1% BSA at 700 r.p.m. for 10 min in a cytofuge. Cells were air dried for 5 min. Then, cells were stained with toluidine blue staining solution [1.92 g anhydrous citric acid (Roth, Karlsruhe, Germany), 0.2 g toluidine blue O (SIGMA) in 100 ml 50% EtOH] for 15 min and subsequently destained by gently flushing with dH2O for 10 s. The slides were allowed to dry overnight before cover slips were fixed with Permount (Fisher Scientific, NJ, USA).

Degranulation assay
For degranulation studies, cells were pre-loaded with 0.2 µg ml–1 IgE anti-DNP overnight. The cells were then washed and re-suspended in Tyrode's buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose and 0.1% BSA in 10 mM Hepes, pH 7.4). The cells were adapted to 37°C for 20 min and treated for 30 min at 37°C as mentioned. The degree of degranulation was determined by measuring release of ß-hexosaminidase (17).

Measurement of arachidonic acid release
For measuring the activity of cPLA2 or rather the conversion of incorporated radioactive-labeled arachidonic acid and the release of lipid mediators, 0.5 x 106 cells per 500 µl RPMI 1640 supplemented with 10% FCS without IL-3 were incubated with 0.1 µCi ml–1 [14C]-arachidonic acid (Amersham Biosciences, Braunschweig, Germany) for 24 h and 0.2 µg ml–1 IgE overnight. After washing four times with Tyrode's buffer, the cells were adjusted to a concentration of 0.5 x 106 cells per 200 µl Tyrode's buffer and allowed to adapt to 37°C for 15 min. The cells were then stimulated for 30 min at 37°C. The reaction was stopped by incubating the samples for 10 min in icewater and centrifuging for 5 min at 600 x g. The supernatant was collected to measure the released amount of radioactivity and the pellets were lysed in 200 µl Tyrode's buffer containing 0.5% NP40 for 30 min on ice. The lysates were centrifuged at 15 700 x g for 10 min to remove the insoluble material. Fifty microliters of the supernatant or accordingly 50 µl of the lysate were then mixed with 3 ml Ecoscint A (DROL, Reichertshausen, Germany) and measured in a [14C] routine program in a Beckmann LS 1801 scintillation counter. The percentage of release was calculated from the counts per minute counted from the supernatant as well as the lysate.

BMMC stimulation and western blotting
IgE-pre-loaded cells (0.2 µg ml–1 IgE; overnight) were washed twice in PBS and re-suspended (2 x 107 ml–1) in RPMI 1640 containing 0.1% BSA. Cells were adapted to 37°C for 30 min and stimulated with the indicated concentrations of DNP-HSA or lipid A. After stimulation, cells were pelleted and solubilized with 0.5% Nonidet P-40/0.5% deoxycholate in 4°C phosphorylation solubilization buffer (18). After normalizing for protein content, the post-nuclear supernatants were separated by SDS–PAGE and subjected to western blot analysis as described previously (18).

Subcellular fractionation
Cytosol–membrane fractionation of BMMCs was performed as previously described (19). As a change to the published protocol, the membrane pellet obtained after the first round of ultracentrifugation was solubilized in hypotonic lysis buffer containing 0.5% Nonidet P-40 and 0.5% of deoxycholate.

IL-6 measurement
Mouse IL-6 ELISA (BD Biosciences, Heidelberg, Germany) was performed according to the manufacturer's instructions.


   Results and Discussion
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
References
 
So far, PKC-ß and PKC-{delta} have been demonstrated to be of pro- and anti-secretory nature in BMMCs, respectively (6, 7). Beside these two isoforms, PKC-{varepsilon} also is prominently expressed in BMMCs and becomes activated after antigen stimulation as indicated by its translocation to the membrane fraction (Fig. 1A). To study the role of PKC-{varepsilon} in BMMC activation in response to antigen, BMMCs were differentiated in vitro from the bone marrow of our established PKC-{varepsilon} null mice, where the PKC-{varepsilon} gene is disrupted by insertion of a LacZ/neo resistance cassette into exon 1 [first described in (15)]. PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs differentiated comparably well, showed equal expression of the surface markers Fc{varepsilon}R1 and c-kit (Fig. 1B), had comparable appearance when stained with toluidine blue (Fig. 1C) and demonstrated no alteration in the extent of proliferation in response to IL-3 (data not shown) or in their survival capacity after IL-3 withdrawal (data not shown). Furthermore, PKC-{varepsilon}–/– BMMCs did not show de-regulated expression and activation (membrane translocation upon antigen stimulation) of PKC-{alpha}, PKC-ß and PKC-{delta} (Fig. 1D) compared with PKC-{varepsilon}+/+ BMMCs. This was also true for phosphorylation of PKC-{delta} at Y311 (Fig. 1D) and T505 (data not shown). PKC-{varepsilon}, as expected, was absent from preparations of PKC-{varepsilon}–/– BMMCs (Fig. 4B).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1. Activation of PKC-{varepsilon} after antigen stimulation of BMMCs. (A) BMMCs were pre-loaded with IgE (0.2 µg ml–1), stimulated for 1 min with the indicated concentrations of antigen (DNP-HSA) and subcellular fractionation was performed. Based on cell equivalents, comparable amounts of cytosol and membrane fractions were analyzed by western blotting against PKC-{varepsilon} (top panel), I{kappa}B{alpha} (middle panel; control for cytosol fraction) and Fc{varepsilon}R1ß (bottom panel; control for membrane fraction). Comparable results were obtained in at least two independent experiments with different BMMC clones. (B) PKC-{varepsilon}+/+ (upper panels) and PKC-{varepsilon}–/– BMMCs (lower panels) were analyzed by FACS with respect to Fc{varepsilon}R1 (left panels) and c-kit expression (right panels). Comparable results were obtained with 15 different PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMC clones. (C) PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs were stained according to Methods. Comparable results were obtained using two different BMMC clones each. (D) IgE-loaded PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs were stimulated for the indicated times with 20 ng ml–1 antigen (DNP-HSA) and subcellular fractionation was performed. Based on cell equivalents, comparable amounts of cytosol and membrane fractions were analyzed by immunoblotting against PKC-{alpha} (top panel), PKC-ß (second panel from top), PKC-{delta} (third panel from top) and P-PKC-{delta} (Y311) (bottom panel). Comparable results were obtained in four independent experiments with different BMMC clones.

 

Figure 4
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 4. Comparable responses to lipid A and SF in PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs. (A) PKC-{varepsilon}+/+ (black bars) and PKC-{varepsilon}–/– BMMCs (white bars) were stimulated with 10 µg ml–1 lipid A for 3 h. IL-6 content of the cellular supernatants was determined by ELISA. Each bar is the mean of triplicates ± SD. Similar results were obtained in independent experiments using four different BMMC clones. n.d., not detected. (B) PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs were stimulated with 10 µg ml–1 lipid A for the indicated times. Post-nuclear supernatants were analyzed by immunoblotting against P-I{kappa}B{alpha} (top panel), I{kappa}B{alpha} (second panel from top), PKC-{varepsilon} (third panel from top) and actin (bottom panel). Data are representative of two independent experiments using different BMMC clones. (C) PKC-{varepsilon}+/+ (black bars) and PKC-{varepsilon}–/– BMMCs (white bars) were stimulated with 100 ng ml–1 SF for 3 h. IL-6 content of the cellular supernatants was determined by ELISA. Each bar is the mean of triplicates ± SD. Similar results were obtained in independent experiments using two different BMMC clones. n.d., not detected.

 
Engagement of the IgE-bound Fc{varepsilon}R1 by multivalent antigens results in the fast release (within minutes) of pre-stored mediators from secretory granules (degranulation) as well as the slower secretion (within hours) of newly synthesized cytokines. Measurement of degranulation revealed no significant difference between PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs, irrespective of whether degranulation was triggered by different concentrations of antigen or by the tumor promoter thapsigargin (Fig. 2A). Moreover, production and secretion of IL-6 and tumor necrosis factor-{alpha} (TNF-{alpha}) in response to antigen was indistinguishable between the two cell types studied (Fig. 2B; data not shown). Furthermore, though PKC-{varepsilon} has been implicated in the suppression of PLA2 activation in RBL-2H3 cells (8), no significant difference in the release of arachidonic acid in response to different concentrations of antigen was observed (Fig. 2C).


Figure 2
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Comparable antigen-triggered degranulation, arachidonic acid release and IL-6 secretion in PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs. (A) IgE-loaded PKC-{varepsilon}+/+ (black bars) and PKC-{varepsilon}–/– BMMCs (white bars) were stimulated with the indicated concentrations of antigen (DNP) or thapsigargin (thaps) for 30 min. Subsequently, degranulation was assessed by ß-hexosaminidase assays. Each bar is the mean of duplicates ± SD. Comparable results with comparable SD were obtained in independent experiments using four different PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMC clones. (B) IgE-loaded PKC-{varepsilon}+/+ (black bars) and PKC-{varepsilon}–/– BMMCs (white bars) were stimulated with the indicated concentrations of antigen for 3 h. IL-6 content of the cellular supernatants was determined by means of ELISA. Each bar is the mean of triplicates ± SD. Similar results were obtained in four independent experiments using different BMMC clones. n.d., not detected. (C) IgE-loaded PKC-{varepsilon}+/+ (black bars) and PKC-{varepsilon}–/– BMMCs (white bars) were stimulated with the indicated concentrations of antigen for 30 min. Arachidonic acid release was measured as described in Methods. Each bar is the mean of triplicates ± SD. Similar results were obtained in two independent experiments using different BMMC clones.

 
Degranulation has been demonstrated to be dependent on activation of the phosphatidylinositol 3-kinase (PI3K) pathway as well as calcium mobilization (20). Correspondingly, antigen-triggered activation of PKB downstream of PI3K and PLC-{gamma}1 upstream of calcium mobilization were not different between PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs in dose-response and time-course studies (Fig. 3A and B). Furthermore, no differences in activation of the Ser-/Thr-kinases, MAPK, p38 and JNK were observed (Fig. 3A and B), which have been shown to be necessary for optimal IL-6 secretion in response to antigen (21).


Figure 3
View larger version (46K):
[in this window]
[in a new window]
  		Fig. 3. Antigen-triggered activation of signaling pathways in PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs. (A) IgE-pre-loaded PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs were stimulated with the indicated concentrations of antigen (DNP) for 5 min. Post-nuclear supernatants were analyzed by western blotting against P-PKB (top panel), P-MAPK (second panel from top), P-p38 (third panel from top), P-PLC-{gamma}1 (fourth panel from top) and p85 (bottom panel). (B) IgE-loaded PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs were stimulated with 20 ng ml–1 antigen for the indicated time points. Post-nuclear supernatants were analyzed by immunoblotting against P-PKB (top panel), P-MAPK (second panel from top), P-p38 (third panel from top), P-JNK (fourth panel from top) and PLC-{gamma}1 (bottom panel). (C) PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs were treated as described in (B). Post-nuclear supernatants were analyzed by western blotting against P-Btk (top panel), P-PKD (middle panel) and PLC-{gamma}1 (bottom panel). In (B) and (C), two different clones of PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs are shown. (D) IgE-pre-loaded PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs were stimulated with 20 ng ml–1 antigen (DNP) for the indicated time points. Post-nuclear supernatants were analyzed by western blotting with anti-panFos (upper panel) and anti-PLC-{gamma}1 antibodies (lower panel). Data are representative of at least two independent experiments using different BMMC clones [P-PKB (n = 4), P-MAPK (n = 5), P-p38 (n = 6), P-JNK (n = 2), P-Btk (n = 3), P-PKD (n = 2), Fos (n = 3)].

 
Btk, via its PH domain, is capable of interacting with various PKC isozymes, e.g. PKC-{varepsilon}, and the phosphorylation of Btk by PKCs results in the down-regulation of its enzymatic activity (22). However, no difference was observed between PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs with respect to Btk autophosphorylation at Y223 (Fig. 3C), suggesting no functional interaction between PKC-{varepsilon} and Btk in response to antigen. Moreover, PKC-{varepsilon} has been implicated in the control of activation of PKD and expression of the Fos transcription factor in RBL-2H3 cells (10, 23). However, no alterations in antigen-triggered activation of PKD or expression of Fos family transcription factors were measured between PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs (Fig. 3C and D).

Studies in a macrophage cell line have demonstrated that PKC-{varepsilon} was critical for LPS-induced nuclear factor-{kappa}B (NF-{kappa}B) activation and TNF-{alpha} and IL-12 production (24, 25). Along this line, macrophages from PKC-{varepsilon} knockout mice have severe deficiencies and host defense against bacterial infection is severely compromised resulting in increased mortality (25). Since MCs are located at the interfaces between host and environment and are capable of participating in innate immunity to pathogens (26), e.g. gram-negative bacteria, we also analyzed the role of PKC-{varepsilon} within this response. However, not only was there no significant difference between PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs concerning lipid A-induced IL-6 secretion (Fig. 4A) but also did we not observe alterations in the phosphorylation and degradation of IkB{alpha}, the inhibitor of NF-{kappa}B (Fig. 4B).

PKC-{varepsilon} has been shown to play differential roles for the proper integration of signals downstream of receptor tyrosine kinases (RTKs) (27, 28). The dominant RTK on the surface of MCs is c-kit, the receptor for SF (26). When analyzing this signaling system, however, we did not find differences between PKC-{varepsilon}+/+ and PKC-{varepsilon}–/– BMMCs with respect to SF-induced IL-6 secretion (Fig. 4C), enhancement of antigen-triggered degranulation (data not shown) and activation of several signaling pathways (PKB, MAPK and p38; data not shown). Furthermore, stimulation with insulin-like growth factor-I, a ligand of a further RTK expressed on MCs (29), did not reveal any detectable difference between the analyzed clones (data not shown). These data strongly suggest that in MCs, PKC-{varepsilon} is not involved in the regulation of signal transduction downstream of RTKs.

The experiments described here did not reveal any defect in the development and function of BMMCs derived from PKC-{varepsilon}-deficient mice. This is in strict contrast to the established role of PKC-{varepsilon} in macrophages and thus in an effective innate immune response (25). Although PKC-{varepsilon} has been implicated to play a role in Fc{varepsilon}R1 signaling in the MC line RBL-2H3 (8, 10), our data suggest that PKC-{varepsilon} deficiency has no specific effect on antigen-induced signal transduction in BMMCs, though PKC-{varepsilon} is strongly expressed in these cells and activated after antigen stimulation (Fig. 1A). PKC-{varepsilon} rather seems to play a redundant role in MC signaling and its absence might be compensated by the recruitment of other BMMC-expressed conventional and/or novel PKC isotypes. Interestingly, in a comparable study addressing the regulatory role of PKC-{varepsilon} in T cell proliferation, PKC-{varepsilon}-deficient T cells were shown to have similar physiological thresholds for activation in vitro as compared with wild-type T cells (30).


   Acknowledgements
 
We thank Kerstin Fehrenbach for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft through grant LE1207/3-1 to M.L. and HU794/2-1 to M.H.


   Abbreviations
 
BMMCs, bone marrow-derived mast cells
Btk, Bruton's tyrosine kinase
c-kit, Steel factor receptor
DAG, diacylglycerol
Fc{varepsilon}R1, high-affinity receptor for IgE
I{kappa}B{alpha}, inhibitor of NF-{kappa}B
JNK, Jun-N-terminal kinase
MAPK, mitogen-activated protein kinase
MCs, mast cells
NF-{kappa}B, nuclear factor-{kappa}B
PI3K, phosphatidylinositol-3-kinase
PK, protein kinase
PL, phospholipase
PMA, phorbol 12-myristate 13-acetate
RTK, receptor tyrosine kinase
SF, Steel factor
TNF-{alpha}, tumor necrosis factor-{alpha}

   Notes
 
Transmitting editor: I. Pecht

Received 29 March 2005, accepted 17 February 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 

  1. Tan S. L. and Parker P. J. 2003. Emerging and diverse roles of protein kinase C in immune cell signalling. Biochem. J. 376: 545.[CrossRef][ISI][Medline]
  2. Sagi-Eisenberg R., Lieman H. and Pecht I. 1985. Protein kinase C regulation of the receptor-coupled calcium signal in histamine-secreting rat basophilic leukaemia cells. Nature 313: 59.[CrossRef][Medline]
  3. Parekh A. B. and Penner R. 1995. Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells. Proc. Natl. Acad. Sci. USA 92: 7907.[Abstract/Free Full Text]
  4. Ozawa K., Szallasi Z., Kazanietz M. G., Blumberg P. M., Mischak H., Mushinski J. F. and Beaven M. A. 1993. Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells. J. Biol. Chem. 268: 1749.[Abstract/Free Full Text]
  5. Ozawa K., Yamada K., Kazanietz M. G., Blumberg P. M. and Beaven M. A. 1993. Different isozymes of protein kinase C mediate feedback inhibition of phospholipase C and stimulatory signals for exocytosis in rat RBL-2H3 cells. J. Biol. Chem. 268: 2280.[Abstract/Free Full Text]
  6. Nechushtan H., Leitges M., Cohen C., Kay G. and Razin E. 2000. Inhibition of degranulation and interleukin-6 production in mast cells derived from mice deficient in protein kinase Cbeta. Blood 95: 1752.[Abstract/Free Full Text]
  7. Leitges M., Gimborn K., Elis W., Kalesnikoff J., Hughes M. R., Krystal G. and Huber M. 2002. Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation. Mol. Cell. Biol. 22: 3970.[Abstract/Free Full Text]
  8. Chang E. Y., Szallasi Z., Acs P., Raizada V., Wolfe P. C., Fewtrell C., Blumberg P. M. and Rivera J. 1997. Functional effects of overexpression of protein kinase C-alpha, -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3. J. Immunol. 159: 2624.[Abstract]
  9. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A. and Davis R. J. 1993. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269.[CrossRef][ISI][Medline]
  10. Razin E., Szallasi Z., Kazanietz M. G., Blumberg P. M. and Rivera J. 1994. Protein kinases C-beta and C-epsilon link the mast cell high-affinity receptor for IgE to the expression of c-fos and c-jun. Proc. Natl Acad. Sci. USA 91: 7722.[Abstract/Free Full Text]
  11. Paruchuri S., Hallberg B., Juhas M., Larsson C. and Sjolander A. 2002. Leukotriene D(4) activates MAPK through a Ras-independent but PKCepsilon-dependent pathway in intestinal epithelial cells. J. Cell. Sci. 115: 1883.[Abstract/Free Full Text]
  12. Maulon L., Mari B., Bertolotto C., Ricci J. E., Luciano F., Belhacene N., Deckert M., Baier G. and Auberger P. 2001. Differential requirements for ERK1/2 and P38 MAPK activation by thrombin in T cells. Role of P59Fyn and PKCepsilon. Oncogene 20: 1964.[CrossRef][ISI][Medline]
  13. Hamilton M., Liao J., Cathcart M. K. and Wolfman A. 2001. Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules. J. Biol. Chem. 276: 29079.[Abstract/Free Full Text]
  14. Ohashi K., Kanazawa A., Tsukada S. and Maeda S. 2005. PKCepsilon induces interleukin-6 expression through the MAPK pathway in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. 327: 707.[CrossRef][ISI][Medline]
  15. Wheeler D. L., Ness K. J., Oberley T. D. and Verma A. K. 2003. Protein kinase Cepsilon is linked to 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor-alpha ectodomain shedding and the development of metastatic squamous cell carcinoma in protein kinase Cepsilon transgenic mice. Cancer Res. 63: 6547.[Abstract/Free Full Text]
  16. Karasuyama H. and Melchers F. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18: 97.[ISI][Medline]
  17. Huber M., Helgason C. D., Scheid M. P., Duronio V., Humphries R. K. and Krystal G. 1998. Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells. EMBO J. 17: 7311.[CrossRef][ISI][Medline]
  18. Liu L., Damen J. E., Cutler R. L. and Krystal G. 1994. Multiple cytokines stimulate the binding of a common 145-kilodalton protein to Shc at the Grb2 recognition site of Shc. Mol. Cell. Biol. 14: 6926.[Abstract/Free Full Text]
  19. Huber M., Hughes M. R. and Krystal G. 2000. Thapsigargin-induced degranulation of mast cells is dependent on transient activation of phosphatidylinositol-3 kinase. J. Immunol. 165: 124.[Abstract/Free Full Text]
  20. Huber M., Helgason C. D., Damen J. E., Liu L., Humphries R. K. and Krystal G. 1998. The Src homology 2-containing inositol phosphatase (Ship) is the gatekeeper of mast cell degranulation. Proc. Natl Acad. Sci. USA 95: 11330.[Abstract/Free Full Text]
  21. Kalesnikoff J., Baur N., Leitges M., Hughes M. R., Damen J. E., Huber M. and 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.[Abstract/Free Full Text]
  22. Yao L., Kawakami Y. and Kawakami T. 1994. The pleckstrin homology domain of Bruton tyrosine kinase interacts with protein kinase C. Proc. Natl Acad. Sci. USA 91: 9175.[Abstract/Free Full Text]
  23. Matthews S. A., Rozengurt E. and Cantrell D. 2000. Protein kinase D. A selective target for antigen receptors and a downstream target for protein kinase C in lymphocytes. J. Exp. Med. 191: 2075.[Abstract/Free Full Text]
  24. Aksoy E., Amraoui Z., Goriely S., Goldman M. and Willems F. 2002. Critical role of protein kinase C epsilon for lipopolysaccharide-induced IL-12 synthesis in monocyte-derived dendritic cells. Eur. J. Immunol. 32: 3040.[CrossRef][ISI][Medline]
  25. Castrillo A., Pennington D. J., Otto F., Parker P. J., Owen M. J. and Bosca L. 2001. Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection. J. Exp. Med. 194: 1231.[Abstract/Free Full Text]
  26. Galli S. J., Maurer M. and Lantz C. S. 1999. Mast cells as sentinels of innate immunity. Curr. Opin. Immunol. 11: 53.[CrossRef][ISI][Medline]
  27. Brodie C., Bogi K., Acs P., Lazarovici P., Petrovics G., Anderson W. B. and Blumberg P. M. 1999. Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells. Cell Growth Differ. 10: 183.[Abstract/Free Full Text]
  28. Saito Y., Hojo Y., Tanimoto T., Abe J. and Berk B. C. 2002. Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor. J. Biol. Chem. 277: 23216.[Abstract/Free Full Text]
  29. Rodriguez-Tarduchy G., Collins M. K., Garcia I. and Lopez-Rivas A. 1992. Insulin-like growth factor-I inhibits apoptosis in IL-3-dependent hemopoietic cells. J. Immunol. 149: 535.[Abstract]
  30. Gruber T., Thuille N., Hermann-Kleiter N., Leitges M. and Baier G. 2005. Protein kinase Cepsilon is dispensable for TCR/CD3-signaling. Mol. Immunol. 42: 305.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/5/767    most recent
dxl012v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (5)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lessmann, E.
Right arrow Articles by Huber, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lessmann, E.
Right arrow Articles by Huber, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?