Activation of mast cells by incorporation of cholesterol into rafts
Thomas Baumruker1,
Robert Csonga1,
Edith Pursch1,
Andrea Pfeffer1,
Nicole Urtz1,
Sue Sutton2,
Elisa Bofill-Cardona1,
Michael Cooke2 and
Eva Prieschl1
1 Novartis Research Institute, Brunner Strasse 59, 1235 Vienna, Austria 2 Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
Correspondence to: T. Baumruker; E-mail: thomas.baumruker{at}pharma.novartis.com
Transmitting editor: S. J. Galli
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Abstract
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IgE plus antigen-stimulated mast cells degranulate, synthesize leukotrienes and secrete cytokines. According to the coalescence model this process is initiated in specific membrane compartments termed rafts. There, enhanced levels of glycosphingolipids and cholesterol stabilize the interaction of Fc
RI and Lyn, and thus facilitate the first steps of signal transduction. Enforced changes in raft architecture by cholesterol deprivation and exogenous application of glycosphingolipids influence these early events by loss of tyrosine kinase activity or receptor-independent signal initiation respectively. Here we show that exogenously added cholesterol accumulates in rafts and activates mast cells. An investigation of the signaling events reveals that in contrast to IgE plus antigen-mediated stimulation, cholesterol triggers p38 mitogen-activated protein kinase and preferentially induces expression of FosB. Consequently, a comparative large-scale microarray analysis demonstrates that a number of IgE plus antigen-induced immediate early genes (peak expression at 30 min after induction) are repressed by cholesterol. These changes further translate into altered expression levels and time kinetics of a number of early genes (peak expression at 2 h after stimulation). As the most prominent example for cholesterol-dependent genes, we identified PAI1 (plasminogen activator inhibitor 1), a protein regarded as a risk factor for atherosclerosis.
Keywords: basophil, gene regulation, lipid mediator, mast cell, signal transduction, transcription factor
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Introduction
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Endogenous or diet-related hyper-cholesteremia comprises a major risk factor for the development of atherosclerotic cardiovascular diseases. The first signs in the pathology of such diseases are deposited lipids, fatty streaks and the conversion of macrophages into foam cells. Interestingly, mast cells are located at such sites and exogenously added oxidized low-density lipoprotein (LDL) has been reported to promote mast cell degranulation (14). Conversely, depletion of cholesterol attenuates signaling events after Fc
RI triggering in this cell type by altering the raft architecture (5). According to the coalescence model, this specific membrane compartment stabilizes receptortyrosine kinase interactions, thereby facilitating the first steps of cell activation. The hypothesis is based on a significantly reduced lateral diffusion time of membrane (signaling) proteins in rafts, which is attributed to the elevated concentrations of glycosphingolipids and cholesterol. The sphingolipid hydrocarbon chains are rigid due to their gel and/or lamellar crystalline character. Cholesterol incorporation provides a domain environment that is tightly packed and in a lipid-ordered state. While the low in-plane elasticity is equal to or even exceeds that of the purely sphingolipid gel state, it maintains a relatively high degree of rotational diffusion. Acyl chains of GPI-anchored proteins and/or tyrosine kinases would find such a lipid-ordered state well suited for dynamic interaction, and therefore would migrate more easily into the raft compartment. In addition, small amounts of cholesterol have the tendency to localize along the boundaries between sphingolipid-enriched domains (rafts) and fluid-phase phosphoglycerides in an effort to minimize the unfavorable energetic effects created at the junctions. This will change the line tension along the mismatch region, leading to changes in microdomain size, shape and physical characteristics. As a result, smaller rafts would fuse, leading to the co-localization of signaling molecules in rafts. However, in addition to these general theoretical physico-chemical findings of the action of cholesterol in membranes, it remains unclear how and to what extent this lipid modulates signaling in mast cells (6).
Signaling cascades initiated by cholesterol such as the Wnt pathway or, alternatively, the activation of the sterol-responsive element are well-characterized in different cell types. Yet, direct effects of this lipid on immunoreceptor-initiated signaling are only addressed as far as they relate to the above-mentioned physico-chemical characteristics. Lack of cholesterol dramatically changes the physical architecture of sphingolipid domains (rafts), resulting in diminished tyrosine kinase activity. Here we show that elevated exogenous levels of cholesterol activate mouse mast cells by incorporation of this lipid into rafts with a subsequent activation of Lyn and Syk. However, the responsive signaling cascades differ from an IgE/antigen (Ag) trigger exemplified by the dependence on p38 mitogen-activated protein kinase (MAPK). Consequently, a comparative expression profiling using large-scale cDNA microarrays shows that, in particular, the magnitude of gene expression and the time kinetics vary considerably between the two stimuli.
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Methods
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The growth and stimulation of CPII mouse mast cells, reporter gene assays, hexosaminidase release assays, leukotriene C4 (LTC4) ELISAs, cytokine ELISAs, use of FK506, Apigenin, SB203580, isolation of rafts, immunoprecipitation with an anti-FcR
-chain antibody, Western blot analyses, in vitro kinase assays of sucrose fractions, PAGE, transient transfections, gel shift and supershift analyses, isolation of RNA, PCR analyses, and gel electrophoresis were performed as recently described (712). The 5' serial deletion constructs of the human tumor necrosis factor (TNF)-
promoter driving a luciferase reporter gene (pGL2 vector background) have been described previously (13). For activation with cholesterol, the lipid was dissolved at a concentration of 20 mg/ml in water and applied at a final concentration of 550 µg/ml in the initial dose-finding studies; for all further experiments it was used at a final concentration of 20 µg/ml (either alone or in combination with ionomycin).
Reagents
IgE-anti-DNP was from Becton Dickinson (San Diego, CA) and DNP-modified BSA was from Calbiochem (San Diego, CA). Ionomycin and cholesterol coupled on polyethylene glycol (PEG) were supplied by Sigma (St Louis, MO). [14C]Cholesterol was from Amersham Pharmacia (Little Chalfont, UK). Primers specific for murine plasminogen activator inhibitor (PAI) 1 (5'-GGAGCTCATGGGGCCGTG GAAC-3' and 5'-GACGTCATACTCGAGCCCATCGG-3') and murine tissue plasminogen activator (tPA) (5'-CAGCGG CCTGGTACAATGCCAC-3' and 5'-GGCACCAAGGTCTGGC ATCACC-3') were from MWG Biotech (Ebersberg, Germany). Primers for murine ß-actin were from Clontech (La Jolla, CA). Antibodies directed against linker of activated T cells (LAT), phosphatidylinositol-3-kinase (PI3K), FcR
chain and MEKK were obtained from Upstate Biotechnology (Lake Placid, NY). p-Erk-1,2, Erk-1,2 phospho-p38, p38 and c-Jun antibodies were from Cell Signaling (New England Biolabs, Beverly, MA). Antibodies detecting FosB, c-Fos and JunD were from Santa Cruz (Santa Cruz, CA).
RNase protection assays
RNase protection assays were performed with 5 µg total RNA of the mast cell line CPII stimulated at different time points using the Riboquant system (Becton Dickinson). Samples were analyzed on a 6% polyacrylamide/urea gels.
Membrane preparation and fractionation
CPII cells (4 x 106) were stimulated for 1 min before lysing them in a buffer containing 10 mM TrisHCl (pH 8.0), 50 mM NaCl, 10 mM EDTA, 1 mM sodium vanadate, 30 mM sodium pyruvate, 10 mM glycerophosphate, 1 mM PMSF, 0.02 U/ml aprotinin, 0.01% sodium azide and 0.05% Triton X-100 on ice for 10 min. Subsequently the lysate was mixed 1:1 with an 80% sucrose solution [sucrose had been dissolved in 25 mM TrisHCl (pH 7.5), 125 mM NaCl and 2 mM EDTA] before loading onto a sucrose gradient. The gradient was performed stepwise with 2 ml of 80, 60, 40 (containing the cell lysate), 30, 20 and 10% sucrose. Centrifugation was performed using a SW40 rotor at 37,500 r.p.m. for 18 h at 4°C. After centrifugation, 666-µl fractions were collected from the top of the gradient. Protein content of the fractions was determined with a Bradford assay (Bio-Rad, Hercules, CA) according to the protocol provided by the manufacturer.
Kinase assay with sucrose fractions
For the in vitro kinase reaction, 20 and 40% sucrose fractions were pooled (10 µl of each fraction) and diluted to 120 µl kinase buffer [25 mM HEPES (pH 7.3), 150 mM NaCl and 5 mM MnCl2]. Then 10 µCi of [
-32P]ATP (Amersham) was added and the reaction incubated for 10 min at 30°C; 20 µl of each reaction was used for PAGE. After electrophoresis the gel was fixed in 40% methanol and 10% acetic acid before drying. The gel was then subjected to autoradiography.
RNA labeling and expression analysis
For each sample, 2 µg of total RNA was labeled and hybridized to Affymetrix Murine 11KsubA and 11KsubB gene arrays as recommended by the manufacturer (Affymetrix, Santa Clara, CA). Together these gene arrays contain
11,000 murine genes and expressed sequence tag (EST) clusters. Each labeled sample was hybridized in duplicate to Murine 11KsubA and 11KsubB arrays. For each hybridization, Affymetrix genechip analysis software was used to calculate the average difference value for each gene, which represents the average intensity of the perfect match oligonucleotide minus the average intensity of the single nucleotide mismatch oligonucleotide for each gene. Dynamic genes were identified by a combination statistical analysis of absolute expression levels and fold change across all experiments. First, genes with an expression level >200, corresponding to
5 mRNA copies in at least one sample, were identified (4757 genes). Next, ANOVA was used to identify from this list genes that changed between any two groups with a significance cut-off of P < 0.005; this identified 665 changing genes. Next, genes that changed by at least 5 times between any two groups were identified, resulting in the identification of 183 genes. Finally, these genes were grouped by time of peak expression. To visualize the gene expression, the raw average difference data for all hybridizations were uploaded into Genespring (Silicon Genetics, Redwood City, CA) for analysis. Data for each sample (array) and gene were normalized to allow comparison and visualization of the data. The 50th percentile of all measurements for each sample was used as a positive control for each sample; each measurement for each gene was divided by this synthetic positive control, assuming that this was at least 10. The bottom 10th percentile was used as a test for correct background subtraction. This was never less than the negative of the synthetic positive control. Each gene was normalized to itself by making a synthetic positive control for that gene and dividing all measurements for that gene by this positive control, assuming it was at least 0.01. This synthetic control was the median of the genes expression values over all the samples. Lastly, normalized values <0 were set to 0. Genes with similar peak expression were grouped together and displayed using the ordered list function in Genespring. The graphic presents normalized values for each gene.
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Results
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We have recently shown that sphingolipids have the potential to activate mast cells by two completely different mechanisms. Sphingosine-1-phosphate supposedly acts as a second messenger molecule, while galactosylsphingosine enforces raft formation leading to Fc
RI-independent signal initiation (11,14). In an effort to extend these observations, cholesterol, also known as a raft component and as a direct-signaling molecule, was analyzed similarly. Using a TNF-
reporter gene assay as a first surrogate parameter for mast cell activation, doses of 550 µg/ml cholesterol coupled to PEG (for solubility reasons and for mimicking LDL) in combination with ionomycin synergistically activated CPII mast cells, as determined in this reporter gene assay (Fig. 1A, left panel). The carrier alone (PEG) had no effect in such an assay (Fig. 1A, right panel). To test this activation potential on physiologically relevant mast cell parameters, an analysis of the degranulation reaction (hexosaminidase release assay, Fig. 1B), leukotriene synthesis (LTC4 ELISA, Fig. 1C) and transcriptional induction of endogenous TNF-
mRNA (Fig. 1D) was performed (cholesterol used at a final concentration of 20 µg/ml). Analogous to the reporter gene assay, all steps of mast cell activation responded additively/synergistically to a cholesterol/ionomycin stimulation. Compared to IgE/Ag, however, differences in the magnitude existed and the stimulation in general was observed to be more variable with the lipid trigger. This did hold true, in particular, for the degranulation reaction, i.e. hexosaminidase release varied with cholesterol/ionomycin from 16.9 ± 1.8% total (ionomycin alone 8.4 ± 0.21% total, spontaneous release 4.7 ± 0.25% total) to 67.4 ± 4.6% total (ionomycin alone 31.5 ± 5.2% total, spontaneous release 4.9 ± 0.4%).
To exclude that the cholesterol-induced activation is an abnormality of our mouse mast cell line, we extended our investigations to primary bone marrow-derived mast cells (BMMC). In an analogous experimental setting we analyzed the degranulation reaction as well as the transcription and secretion of two cytokines after cholesterol/ionomycin stimulation by hexosamindase release, PCR and ELISA respectively. As shown in Fig. 2, cholesterol/ionomycin-stimulated BMMC degranulated (spontaneous release 2.4 ± 0.4% total, cholesterol/ionomycin 44.7 ± 11.0% total, PEG/ionomycin 29.3 ± 4.8% total and IgE/Ag 86.0 ± 13.2% total) and produced cytokines such as TNF-
(non-stimulated 26 ± 36 pg/ml, cholesterol/ionomycin 947 ± 278 pg/ml, PEG/ionomycin 373 ± 29 pg/ml and IgE/Ag 3100 ± 70 pg/ml) and IL-6. While the induction of IL-6 was considerable and detected by both techniques applied, TNF-
was only moderately induced at the PCR level (Fig. 2B and C), as TNF-
mRNA is already present in non-induced cells. Taken together, these results excluded that the cholesterol-induced response is an abnormality of the cell line and therefore that primary BMMC have the capacity to react to cholesterol, but in a distinctly different manner than after IgE/Ag triggering. In addition, BMMC and CPII cells responded identically to the stimulus with respect to the investigated parameters and therefore the cell line was regarded as a suitable model system for further analyses at the molecular level.
Cholesterol and glycosphingolipids are characteristic components of rafts (glycosphingolipid-enriched domains), a membrane compartments thought to have a crucial role in the activation of the tyrosine kinases Lyn and Syk (11). Analogous to our recent findings with exogenously added galactosylsphingosine, we speculated that cholesterol might also accumulate in this membrane compartment. Radiolabeled cholesterol (used either alone in negligible amounts or as a spike into a final concentration of this lipid of 20 µM) was applied to follow the intracellular distribution and accumulation of this lipid under these two circumstances. A sucrose gradient, which separated the bulk of cytoplasmic proteins (40% sucrose; marker protein PI3K) from the raft fraction (20% sucrose; marker protein LAT), was employed and Western blot analysis showed successful separation (Fig. 3A, inset). Radiolabeled cholesterol (when used as a spike in a concentration of 20 µM non-radiolabeled material) applied 2-min prior to cell lysis clearly peaked in the 20% sucrose fraction indicating that the primary entry sites were rafts (Fig. 3A). At this (high) concentration a strongly enhanced incorporation of this lipid into the 20% raft fraction was observed when comparing it side by side to the same amount of radioactive cholesterol alone (low concentration). However, no such difference in uptake was observed for the 40% (cytosolic) fraction. The specificity of this uptake is further underlined by the fact that significantly higher amounts of radioactive material were detected in the 10 and 60% sucrose fractions at a low cholesterol concentration. In an in vitro kinase assay of the raft fraction (20%), accumulated cholesterol (at the high concentration) led to an activation/phosphorylation of 56 and 72-kDa proteins (most likely p56lyn and p72syk; Fig. 3B, long exposure). While the de novo phosphorylation after cholesterol/ionomycin stimulation of the p56 band was relatively strong, the phosphorylation of the p72 band was only slightly induced compared to non-stimulated and IgE/Ag-triggered cells, suggesting differences in the initiation of the signal between the two stimuli. Still, MAPK (exemplified by Erk-1,2) were phosphorylated/activated after both activation conditions in an identical manner as shown with the 40% cytosolic fraction (Fig. 3B, 40% fraction). Both stimuli also provoked a nearly identical phosphorylation of 30- and 10-kDa proteins in the raft fraction (Fig. 3B, 20% fraction, long and very long exposure) that most likely corresponded to the signal-transducing ß and
chains of the Fc
RI respectively. To identify these proteins, immunoprecipitation with an anti-FcR
chain antibody using an in vitro kinase reaction of the 20% fraction was performed. The 10-kDa protein (FcR
chain) and to a lesser extent the 30-kDa protein (co-precipitation of the FcR ß chain) were specifically enriched by this method using a MEKK antibody as specificity control (Fig. 3C). To exclude that the activation of Lyn was an epiphenomena after cholesterol stimulation, PP2, a Lyn inhibitor, was applied. This inhibitor strongly reduces the tyrosine phosphorylation observed in induced mast cells after stimulation at the level of the ß chain (data not shown) and totally abrogates
chain phosphorylation (see Fig. 3D, inset; left:
chain phosphorylation; right: reporter gene assay after IgE/Ag stimulation). In line with such an inhibition and a function of Lyn tyrosine kinase in the cholesterol stimulus, PP2 also abrogates the cholesterol-induced TNF-
induction at an even lower concentration as after IgE/Ag triggering, underlining that Lyn is critically involved in this lipid-initiated signaling (Fig. 3D). The implication of Syk activation by cholesterol was addressed in an analogous setting. As a readout, we used the prototype substrate LAT of this kinase in mast cells after immunoprecipitation, in conjunction with an in vitro kinase assay, to measure the activity increase of co-precipitated Syk of IgE/Ag-triggered and cholesterol/ionomycin-stimulated cells versus non-stimulated ones. LAT was recently shown to be central to mast cell activation after becoming tyrosine phosphorylated by this ZAP 70 kinase family member (15). Furthermore, effects of the experimental inhibitor Piceatannol, a Syk kinase antagonist, on this phosphorylation step as well as on mast cell activation have been demonstrated recently (11). Therefore, equal amounts of LAT were precipitated (Fig. 4A) and subjected to a kinase reaction showing that LAT becomes phosphorylated to a similar extent after both types of activation (IgE/Ag and cholesterol/ionomycin). Furthermore, this in vitro reaction is totally abrogated by using 10 µM Piceatannol as an inhibitor. To support these findings further, Piceatannol was used in a TNF-
reporter gene assay on cells directly. It also abrogated the cytokine induction in vivo by cholesterol/ionomycin with the same IC50 values as determined for the IgE/Ag signal (Fig. 4B).

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Fig. 3. Cholesterol specifically accumulates in rafts and leads to chain phosphorylation. (A) Distribution of exogenously added [14C]cholesterol in CPII cells in a sucrose gradient after a 2-min incubation. Corresponding concentration of sucrose (in percent) of each fraction (20% = rafts, 40% = cytosolic fraction) given at the x-axis; percent of cell-bound cholesterol, distributed in those fractions is indicated on the y-axis. Inset shows Western blot analyses with the 20 and 40% fractions, and antibodies to LAT and PI3K to prove the successful separation. (B) Kinase assay of the fractions corresponding to the 20% (rafts) sucrose of either non-stimulated (nst) or stimulated cells as indicated on the top. Different exposures of the autoradiogram are given to visualize all phosphorylated proteins (1 h, lanes 13; 6 h, lanes 46; overnight, lane 7). Molecular weight standards are given on the left. Western Blot analysis of pooled fractions corresponding to the 40% (cytosolic proteins) sucrose using p-Erk and Erk antibodies to check for stimulation. Stimuli are given on the top and antibodies used are indicated on the right. (C) Immunoprecipitation with antibodies directed against the common chain (lanes 3 and 4) and MEKK-1 (antibody specificity control, lanes 5 and 6) from an in vitro kinase reaction (lanes 1 and 2) with the 20% fraction. Stimulation conditions are given on the top and mol. wt standards are shown on the left. (D) TNF- reporter gene assay after cholesterol/ionomycin stimulation using different concentrations of the Lyn kinase inhibitor PP2 as indicated on the x-axis. Small inset, left: immunoprecipitation with anti-pTyr antibody, coupled to a Western blot analysis with an anti- chain antibody, indicating the abrogation of chain phosphorylation after PP2 treatment; right: bar graph showing a TNF- reporter gene assay for the IgE/Ag stimulus as a reference. Lines indicate non-stimulated values (nst) and maximally stimulated values (chol./iono. or IgE/Ag). Squares indicate a solvent control (inhibition) curve (DMSO) and diamonds show an inhibition curve with the PP2 inhibitor, both at concentrations outlined on the x-axis.
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Fig. 4. Cholesterol/ionomycin activation requires Syk function. (A) Immunoprecipitation(s) coupled to in vitro kinase assay(s) of LAT with and without Piceatannol (Pic., 10 µM) as an inhibitor of Syk. Shown is an autoradiogram measuring the intrinsic kinase activity in the precipitate (indicated by an arrow and kinase assay on the right). A Western blot analysis of an aliquot of the immunoprecipitated LAT is given underneath for normalization of the immunoprecipitation (indicated by Western blot on the right). Stimulation conditions of the cells are shown above the lanes. (B) TNF- reporter gene assay after cholesterol/ionomycin stimulation using different concentrations of the Syk kinase inhibitor Piceatannol as indicated on the x-axis. Small inset bar graph shows an identical experiment for the IgE/Ag stimulus as a reference. Lines indicate non-stimulated values (nst) and maximally stimulated values (chol./iono. or IgE/Ag). Squares indicate a solvent control (inhibition) curve (DMSO) and diamonds show an inhibition curve with Piceatannol (Pic.), both at concentrations outlined on the x-axis.
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To identify the downstream signaling cascades which transmit this receptor-cross-linking-independent activation into the nucleus, we employed known signaling inhibitors in a TNF-
reporter gene assay. FK506, a calcineurin inhibitor, was applied to check if the transcription factor NF-AT, a target of this phosphatase, was required for TNF-
transcriptional induction after cholesterol/ionomycin stimulation (8). As shown in Fig. 5(A), both IgE/Ag- as well as cholesterol/ionomycin-treated cells were equally sensitive to this drug, suggesting that this transcription factor was employed for cytokine induction after both stimuli. NF-AT binding and activity, however, depend on co-factor binding, adjacent to this transcription factor at the TNF-
promoter. After IgE/Ag stimulation, two branches of the MAPK pathway (Erk and Jnk dependent) induce the activation of activation protein 1 (AP1) family members, which cooperate with NF-AT. The third branch of this pathway, the p38 MAPK, in contrast, is dispensable for TNF-
induction after IgE/Ag triggering (7). To investigate the contribution of these signaling cascades to cholesterol/ionomycin-mediated cytokine induction, two MAPK pathway inhibitors were applied on CPII mouse mast cells. Apigenin abrogated the Erk- and Jnk-dependent cascades, and SB203580 served as a specific p38 MAPK inhibitor (7,16). Apigenin was effective after both stimulation conditions (Fig. 5B), a finding which corresponded well with the observed identical phosphorylation of Erk-1,2 detected in Western blot analyses (compare Fig. 3B, 40% fraction). SB203580 only affected cholesterol/ionomycin-stimulated cells (Fig. 5C). This finding was consistent with a p38 Western blot analysis of total cellular extracts of mast cells activated with cholesterol/ionomycin. Phosphorylated/activated p38 was strongly induced after 5 min of induction and remained active up to 1 h (Fig. 5D). After IgE/Ag, in contrast, no changes in the phosphorylation pattern of this kinase were detected, as published previously (7). Taken together, the data reveal a clear difference between cholesterol/ionomycin versus allergic stimulation with respect to downstream signaling pathways responsible for NF-AT cofactor activation.
With these differences in mind, we asked the question how these changes translate further into promoter elements and/or distinct subsets of interacting transcription factors. Three characteristic 5' deletion constructs of the TNF-
promoter were therefore analyzed in allergically and cholesterol/ionomycin-stimulated mast cells side by side (13). As shown in Fig. 6(A), a comparable pattern was observed for both stimuli, confirming that the formerly defined extended
3 site after an IgE/Ag stimulus is critical for transcriptional induction of TNF-
after both types of stimulation. In a gel shift analysis using this particular DNA element as a radiolabeled probe, induced complex formation was detected in both cholesterol/ionomycin- and IgE/Ag-triggered mast cells versus non-stimulated cells (Fig. 6B). However, with respect to the AP1 complex at this probe, which previously had been identified after IgE/Ag and PMA/ionomycin stimulation (Fig. 6B, middle complex) (7,13), the pattern after cholesterol/ionomycin activation appeared to be different. This suggested that the repertoire of activated AP1 factors might vary depending on the stimulusa finding in agreement with the differences detected in MAPK pathway activation. Therefore, we performed a gel shift/supershift analysis using a consensus AP1-binding site and antibodies specific for AP1 family members. Both conditions resulted in the induction of a specific subset of AP1 family members with a clear and prominent shift from c-Fos (after IgE/Ag) to FosB (after cholesterol/ionomycin; Fig. 6C), concomitant with a significant reduction in c-Jun. These differences in binding are reflected by the different levels of expression of these proteins as visualized in a Western Blot analysis (Fig. 6D). Altogether the data indicated that cholesterol/ionomycin induced all three phases of mast cell activation (degranulation, leukotriene synthesis and cytokine induction). However, due to differences in the signaling process and consequently the transcription factors being induced, the magnitude varies between the two alternative stimuli.
Based on these findings we hypothesized that a broad survey could unravel genes or gene sets (pathophysiological parameters) that are even more strongly differentially expressed between the two types of stimulation. This could link changes in signaling cascades to a particular gene expression pattern. To address this issue, an Affymetrix chip analyzing
11,000 known mouse genes and ESTs in parallel was used to create a characteristic fingerprint after the two stimuli. A comparison of 30-min and 2-h activated cells, either by IgE/Ag or cholesterol/ionomycin, was performed. This analysis resulted in 4757 genes which were expressed above our detection level in CPII mouse mast cells (cut-off: 5 mRNA copies). From these, 78 genes were detected as induced (cut-off: 5-fold induced; PANOVA = 0.005) and 105 as repressed (cut-off: 5-fold repressed = 80%; PANOVA = 0.005) after one or the other stimulus. The overall validity of the analysis is demonstrated by the representative analysis of four known inducible genes (Fos, Jun, IL-13 and TNF-
) for the IgE/Ag stimulus (Fig. 7A) (10,11,17).

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Fig. 7. Gene chip analysis. (A) Table giving fold induction of four marker genes after the IgE/Ag stimulus at time points indicated to validate the analysis. (B and C) Graphs representing the 105 genes down-regulated (B) and the 78 genes induced (C) (four panels) that fluctuate in expression >5-fold. The latter is subdivided into preferentially induced by IgE/Ag at 30 min (top left) and at 2 h (bottom left), and preferentially induced by cholesterol/ionomycin at 30 min (top right) and at 2 h (bottom right). Expression values determined by the gene chip analysis in duplicates are given on the y-axis, and stimuli and time points of stimulation of the CPII cells that served as a source for the RNA are shown on the x-axis.
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Among the 105 repressed genes, a remarkable imbalance in magnitude of repression between cholesterol/ionomycin and IgE/Ag was seen with overall much stronger repression by the lipid stimulus (Fig. 7B). The group of induced genes can be further subdivided into four classesthose which were more efficiently induced by IgE/Ag (either at 30 min or at 2 h) and those which responded better to cholesterol/ionomycin (again either at 30 min or at 2 h) (Fig. 7C). In three of the four groups, the differences in expression were purely quantitative (i.e. better induced by one or the other stimulus). The most prominent example with the highest discrepancy in expression between the two stimuli was found in the preferentially induced by cholesterol/ionomycin after 2 h group and was PAI1 (accession no. NM_008871) (Fig. 8A). This gene was
16-times stronger up-regulated by the lipid stimulus than by IgE/Ag. As PAI1, a protein involved in hemostasis, is implicated in atherosclerosis, a disease where high cholesterol levels are a risk factor, we further wanted to verify this finding. A RT-PCR with primers specific for PAI1 and its natural counterpart tPA proved that the detected quantitative differences by the gene chip analysis can indeed be reproduced (Fig. 8B). That this relates to the cholesterol trigger is shown in the additional RT-PCR analysis given in Fig. 8(C), where the cholesterol stimulus and the ionomycin stimulus are further split up. This analysis not only further validated the results of the gene chip analysis, but shows that the pro-thrombotic PAI1 is more or less exclusively induced in an additive/synergistic manner after a cholesterol/ionomycin stimulation in mast cells, supporting speculations about the involvement of this cell type in the pathogenesis of atherosclerosis.

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Fig. 8. PAI1 induction by cholesterol/ionomycin stimulus. (A) Expression values as determined by the gene chip analysis for PAI1. For legend, see Fig. 7. (B) RT-PCR with primers for mouse ß-actin (as control), mouse PAI1 and mouse tPA. Time points of stimulation and stimuli are shown above the lanes. Control gives an RT-PCR reaction at a plasmid harboring ß-actin sequences. (C) RT-PCR with primers for mouse ß-actin (as control) and mouse PAI1 to show the additive/synergistic influence of cholesterol and ionomycin. Stimuli are shown above the lanes. A densitometry scan of the above gel (bar graph) representing PAI1 expression values (normalized to the ß-actin expression values) is shown underneath.
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In the group of genes, which at 30 min were more strongly induced by IgE/Ag, a clear qualitative difference was detectable (see Fig. 7C, top left panel). Here four out of a group of six immediate early genes induced by IgE/Ag are, on the contrary, repressed below their basal expression by cholesterol/ionomycin (see Discussion).
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Discussion
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The transcriptomes (identity and multiplicity of mRNAs expressed in a specific cell type) have not yet been established for many immunologically important cell types and reactions. Therefore, only a few dozen or so identified genes are widely applied as molecular probes after different stimuli to investigate and characterize various immunological processes. In mast cells, in particular, DNA and RNA probes for cytokines and for the different AP1 family members of the Fos/Jun class are used to characterize and define the so-called late phase reaction at the gene level. This resulted in the description of some transcription factors and several cytokines in a primarily Th2-type pattern (11,18,19). The complex interplay of genes in a triggered mast cell, however, has remained ambiguous using these approaches. Groundbreaking analysis by Metzgers group using SAGE technology overcame this partially by analyzing 11,300 genes of non-stimulated and 3.5-h IgE/Ag-triggered RBL cells (20). This analysis laid the groundwork of mRNA complexity and abundance with a couple of striking findings such as preprorelaxin expression in mast cells. However, the rat background as well as the use of one time point and one stimulus prevents (i) the identification of the majority of the induced genes (only eight identified from 31 genes) and (ii) insight into the plasticity of a mast cells transcriptional response. In addition, several cytokines characterizing the mast cell late phase were not found at all. Very recently, a first gene chip analysis from human umbilical cord blood-derived mast cells was completed. Here, 31 different cytokines/chemokines were detected to fluctuate after Fc
RI triggering and thereby this analysis overcame some of the shortcomings of the previous data (21). In particular, the finding of enhanced IL-11 expression together with previous reports of mast cells expressing myelin basic protein sheds new light on the role of this cell type in allergic asthma/lung damage (22). It demonstrates the validity of such gene chip analyses and the establishment of transcriptomes for clarifying the specific detailed contribution(s) of a given cell type to the overall pathological picture observed (21,22). Our analysis follows this idea by comparing two pathophysiological stimulations (the IgE/Ag trigger and the lipid-driven cholesterol/ionomycin stimulus) side by side on mast cells. The inclusion of an early time point (30 min) thereby allows us to monitor specific differences in the initial cell activation (signaling) phase which is complemented by our biochemical/molecular analysis. From these data it becomes clear that considerable differences exist in the MAPK pathway (substitution of Jnk activation after IgE/Ag by p38 after cholesterol/ionomycin) with major consequences on the composition of the AP1 components (shift from c-Fos after IgE/Ag to FosB after cholesterol/ionomycin). Furthermore the gene chip analysis reveals qualitative differences (see the gene set preferentially induced by 30-min IgE/Ag) at two other levels of regulation: (i) in the protein degradation/ubiquitination pathway exemplified by DUB-1 (accession no. C80752/XM 148584) and proteasome regulatory subunit S1 (accession no. AK010596) that are both transcriptionally shut off after cholesterol/ionomycin while contrary being constantly expressed/induced by IgE/Ag, and (ii) in the translation facilitating machinery exemplified by a DEAD-like helicase (accession no. C77285/NM_026360) that by itself is an enzyme involved in the unwinding of secondary (inhibitory) structures at the 5'-end of mRNAs (see also Table 1). That these enzymes comprise prototypes of their respective classes that are broadly differentially regulation by the two activating stimuli is seen by the data in the group of the repressed genes. Thirteen out of the 105 annotations here are hits for genes involved in the ubiquitination pathway, translation initiation/elongation and ribonucleoproteins (much more and specifically down-regulated at 30 min by the cholesterol/ionomycin stimulus) (see Table 1). Recently, structural and functional homologies between the 19S lid component of the ubiquitin-proteasome pathway, the COP9 signalosome and eukaryotic initiation factor 3 were described, implying that enzymes of these two pathways act in a concerted manner in regulating some of the early activated transcription factors such as IRF, NF
B, HY5, ID or AP1. In the light of these findings it is particularly striking that we see gross differences in the two AP1 components Fos and Jun. These early events than translate into some quite dramatic quantitative differences in gene expression in the effector phase (2-h time point)best illustrated by PAI1. Given the nature of PAI1 as a major risk factor in atherosclerosis and a link of this disease to high cholesterol levels, it is fair to assume that this difference will translate into pathological conditions. In this respect it is important to note that mast cells (with one exception) have so far been recognized as producers of tPA, but not its counterpart. As PAI1, after adjusting for other risk factors, is directly responsible for elevated systolic and diastolic blood pressure and pulse wave velocity of the aorta, all three being subclinical indicators for atherosclerosis, these findings support an involvement of mast cells in this disease (23,24).
As cholesterol primarily accumulates in the raft fraction of mast cells, the question remains how this lipid generates the signaling process leading to the phosphorylation of the common
chain and hence the subsequent activation of the MAPK pathway. The phosphorylation of the ß and
chains of the Fc
RI argues against participation of known cholesterol-dependent signaling pathways such as the Wnt pathway or alternatively sterol-responsive element-binding protein (SRE-BP). Findings in colon carcinoma cells that Wnt signaling represses c-Fos and FosB equally also against argue the first (25). We therefore currently speculate that lipid physico-chemical parameters and changes in the membrane (phospho) lipid content conveyed by the incorporation of cholesterol (into rafts) form the basis of this signaling. While the ratio of cholesterol:phospholipid in hematopoetic cells is
0.5 (26), treatment of RBL-2H3 cells with a 1:1 mixture of phosphatidylcholine:cholesterol was shown to increase this value considerably to >0.6. As the amount of lipid order is in part a function of the cholesterol content, it is conceivable that a higher concentration of this natural sterol will increase this parameter, particularly in the raft (detergent-resistant membrane) fraction. Using fluorescence anisotrophy measurements, the lipid order in isolated rafts of RBL cells was estimated to be 60% and that of whole plasma membrane vesicles to be 40% in relation to a standard of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine:cholesterol (2:1) liposomes (set as 100%, i.e. a fully ordered Lo state) (27). Therefore, one has to assume that the lipid order in rafts (and probably other microdomains usually isolated in the total bulk of the 40% sucrose fraction) increases beyond this 60% (or 40%). This might create what was very recently described as superrafts, highly detergent resistant membranes, or convert large parts of the Ld membrane into a more ordered structuresimilar to an Lo state, forming a kind of pseudo raft along the cell membrane (28). This and the tendency of high cholesterol levels shown at model membranes to overcome the inherent lateral phase separation of certain phospholipids might lead to a IgE/Fc
RI-independent fusion of Lyn-containing rafts with the Fc
RI that is located just outside of the raft domain at the (Lo + Ld) phase coexistence region. In this hypothesis the primary mechanism of action of cholesterol would be via enhancing the lipid order and/or miscibility of the surrounding Fc
RI annulus (motionally restricted lipid shell). Therefore, its first action would be outside the raft fraction and not directly at the kinase level itself. Despite this fact, the question remains when cholesterol simply mimics/facilitates the coalescence of Fc
RI- and Lyn-containing lipid islands to elicit its signaling, why does IgE/Ag triggering and cholesterol/ionomycin stimulation exhibit differences in the activation of the MAPK pathway (p38 activation) and a shift from c-Fos to FosB. Recent findings for two ras proteins might provide an answer. Hancock and colleagues reported that H-ras, but not K-ras, is found at the inner leaflet of the rafts in the non-activated state (29,30). Its interaction is cholesterol dependent and based on the unique hypervarible C-terminal region conferring membrane tethering. In the GTP (activated) state, H-ras then leaves the rafts and localizes to specific domains outside the rafts that are, however, distinct from the domain where K-ras is found. It is conceivable that a higher cholesterol content might influence this migration (positively or negatively), thereby allowing or preventing one or the other ras proteins to properly activate the downstream MAPK pathway. Some further mechanisms of triggering can be also envisaged that are not based on a primary action of the lipid on the Fc
RI and/or Lyn motility/domain localization. The presumed higher lipid order state triggered by cholesterol incorporation could, for example, displace a tyrosine phosphatase that usually keeps the balance towards the unactivated status in non-triggered cells. In favor of this assumption is a recent finding by Baird and colleagues that Lyn in rafts is hyperphosphorylated at the active site loop of the kinase domain compared to the Lyn fraction outside the rafts (31) which goes parallel to an increased kinase activity. The authors speculate on a phosphatase that is usually excluded to some extend from the raftshigher cholesterol levels could pronounce this exclusion even further and shift the balance to an activated Lyn kinase, initiating the whole cascade of cell activation/triggering.
This physico-chemical distortion may conversely also account for the down-regulation of a large subset of genes after cholesterol application. As CPII cells are still growth factor dependent (cultivated with 10% WEHI supernatant), these respective signaling cascades could be disturbed and thereby transiently switched off.
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Acknowledgements
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We thank all our colleagues at NFI who have supported our work and, in particular, R. Ponting for critical reading of the manuscript.
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Abbreviations
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AP1activator protein 1
BMMCbone marrow-derived mast cell
ESTexpressed sequence tag
IgE/AgIgE plus antigen
LATlinker of activated T cells
LDLlow-density lipoprotein
LTC4leukotriene C4
MAPKmitogen-activated protein kinase
PAIplasminogen activator inhibitor
PI3Kphosphatidylinositol-3-kinase
PEGpolyethylene glycol
TNFtumor necrosis factor
tPAtissue plasminogen activator
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