Mast cells play pivotal roles in the initiation of the allergic response. To gain an understanding of the functions played by phosphatases in IgE-mediated mast cell activation, a small interfering RNA (siRNA) library that targets all mouse phosphatase genes was screened in a mouse mast cell line, MMC-1. Of 198 targets, 10 enhanced and 7 inhibited FcεRI-induced degranulation. For seven of the strongest hits, four different siRNAs per target were tested, and at least two out of the four single siRNA per target had similar effects as the pool suggesting that these were true hits. Bone marrow-derived mast cells from normal mice further validated these results for six definite positive targets. The mechanism of the reduced mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAPKs and the phosphorylation of protein kinase D/protein kinase Cμ and protein kinase Cδ, which are involved in FcεRI signaling. The screen, therefore, has identified several new molecules that are critical for FcεRI-induced degranulation. Regulating the function of these proteins may be potential targets for the treatment of allergic inflammation. The result also indicates that the system used is efficient for searching molecules implicated in complex receptor-induced signaling.
Mast cells play an important role in allergic reactions and IgE-associated immune responses. Aggregation of FcεRI on these cells initiates a biochemical cascade, which eventually results in mast cell functional responses, such as degranulation and release of inflammatory mediators (1–5). The activation pathway leading from FcεRI stimulation to degranulation is a complex one that involves many molecules, some of which are directly involved in the release of granules, whereas others regulate this process. The role of several molecules has been established by using cell lines that are deficient in one of these molecules. Such cells are usually derived from either in vitro selection of established cell lines or from genetically modified mice that lack those molecules (6–16). However, the development of small interfering RNA (siRNA) technology has made it possible to perform genetic screens in mammalian cell lines, although such screening has not been commonly applied to such complex pathways.
Protein phosphorylation is one of the earliest detectable events after FcεRI aggregation and plays an essential role in this signal transduction pathway. The extent of FcεRI-induced phosphorylation is regulated by the balance between protein kinases and phosphatases. Significant advances have been made recently in understanding the functions of kinases. It is clear now that many kinases, for example, Lyn, Fyn, Syk, Btk, PI3K, MAPK, sphingosine kinase, and different isoforms of protein kinase C are involved in FcεRI signal transduction (17–27). However, there is only limited information about the role of protein phosphatases in mast cells. So far, studies of phosphatases have been restricted to several well-characterized molecules, such as Src homology region 2 domain-containing phosphatase (SHP) 1, SHP2, SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (6, 28–33). We therefore used an siRNA screening approach using a 198-member siRNA library to identify phosphatase genes that are involved in FcεRI-stimulated mast cell degranulation. The screen identified several molecules that regulate FcεRI-induced degranulation; the reduced expression of these molecules either enhanced or inhibited degranulation. Most of these molecules had not been recognized as having a role in this pathway. The results also indicate that this is an efficient system for screening for molecules that are important in complex immune-receptor signaling pathways leading to granular secretion.
Materials and Methods
The HRP-conjugated anti-phosphotyrosine Ab (PY20), anti-protein kinase C (PKC) δ and anti-protein kinase D (PKD)/PKCμ Abs were purchased from BD Biosciences (San Jose, CA). The anti-Syk (N-19), anti-SHIP1, anti-SHIP2, anti-SHP1, anti-SHP2, and anti–phospho-JNK Abs were from Santa Cruz Biotechnology (Santa Cruz, CA); the anti–phospho-p44/42 MAPK, anti-p44/42 MAPK, anti–phospho-p38, anti-p38, anti–phospho-PKCδ (Ser643), anti–phospho-PKCδ (Thr505), and anti–phospho-PKD/PKCμ (Ser916) Abs were from Cell Signaling Technology (Beverly, MA); anti-JNK and anti-PTEN Abs were from Upstate Biotechnology (Lake Placid, NY); anti-calcineurin B Ab was from Abcam (Cambridge, MA). All other Abs used were previously described (34).
The mouse mast cell line MMC-1 (CXBI-I-CA5) was maintained in DMEM supplemented with 20% heat-inactivated FBS, 2 mM l-glutamine, 5 × 10−5 M 2-ME, 10% NCTC 109 media, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and antibiotics as described previously (35). Cells were subcultured every 2 to 3 d to maintain their high viability and good degranulation response.
Bone marrow cells from C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were cultured in the same medium supplemented with 30 ng/ml IL-3 and 25 ng/ml stem cell factor. Bone marrow-derived mast cells (BMMCs) were used for these experiments after 5–7 wk of culture. The usage of mice has been reviewed and approved by National Institute of Dental and Craniofacial Research institutional review committee (Bethesda, MD).
The phosphatase siRNA library was purchased from Dharmacon (Chicago, IL). The library contains pools of four siRNA duplexes per gene that target 198 mouse enzymes with known or predicted phosphatase activity. The list of genes, Entrez gene ID, locus number, catalog number, and sequences can be found in Supplemental Table I. Negative control siRNA was the siCONTROL nontargeting pool that contains four nontargeting siRNAs, which has minimal targeting of known genes in human, mouse, and rat cells (Dharmacon). Mouse Syk (NM_011518) siGENOME SMARTpool was also from Dharmacon. For transfection, cells were seeded in 96-well dishes and transfected with ∼0.53 μg siRNA/106 cells using the Amaxa Nucleofector 96-well Shuttle System according to the manufacturer’s protocols (Amaxa Biosystems, Cologne, Germany) using the FF-138 program for MMC-1 cells and DC-100 for the BMMCs. In each experiment, there were negative control cells transfected with scrambled siRNA and positive control cells with siRNA for Syk.
Cells were sensitized with 0.3 μg/ml Ag-specific IgE (anti-trinitrophenyl mAb) in culture medium for 24 h. For cell activation, MMC-1 cells were incubated for 45 min at 37°C with DMEM-TB (DMEM containing 0.1% BSA and 10 mM Tris [pH 7.5]) with or without 30 ng/ml of Ag, whereas BMMCs were stimulated with 3 ng/ml of Ag for 45 min. Duplicate wells were used for each treatment with a reaction volume of 200 μl/well. The overview of the procedures used for sensitization and stimulation is summarized in Fig. 1A.
The measurement of degranulation and immunoblotting
Poststimulation, cells were centrifuged, and the supernatants were used for measuring the enzymatic activities of β-hexosaminidase. The cell pellets from wells incubated only with DMEM-TB were solubilized with 1% Triton X-100 in DMEM-TB (blanks) and used to determine the total cellular content of β-hexosaminidase. The cell pellets from extra wells that had been transfected but not further manipulated were combined with those from Ag-stimulated cells, lysed with SDS-PAGE sample buffer, and used for Western blotting to determine changes in the level of expression of proteins (34). The β-hexosaminidase enzymatic activity was measured with p-nitrophenyl N-acetyl-β-d-glucosaminide in 0.1 M sodium citrate (pH 3.5) for 60 min at 37°C. After stopping the reaction by adding 0.4 M glycine (pH 10.7), the released product 4-p-nitrophenol was detected by absorbance at 405 nm. The extent of Ag-induced degranulation was calculated as follows: 4-p-nitrophenol absorbance in the supernatant of Ag-stimulated cells minus 4-p-nitrophenol absorbance in the supernatant of blank cells (spontaneous release), which was then divided by the sum of 4-p-nitrophenol absorbance in the supernatants and cell pellets of blank cells. The release was expressed as a ratio of that induced by scrambled siRNA-transfected cells, and in some graphs this value was subtracted from 1.00.
RNA isolation and quantitative PCR analysis
Total RNA from the cells transfected with scrambled or MTMR4-specific siRNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). The mRNA expression of MTMR4 and Syk (as an internal control) was detected with ABI Prism 7000 Sequence Detection System by using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The primers used for MTMR4 are: forward primer 5′-AAAGACTCTGTCATCAACGTGC-3′ and reverse primer 5′-CCGGCTTAGCCTTGAGAGC-3′. The primers used for Syk are: forward primer 5′-CTACCTGCTACGCCAGAGC-3′ and reverse primer 5′-GCCATTAAGTTCCCTCTCGATG-3′. The results were analyzed using ABI Prism Software (Applied Biosystems), and the expression of MTMR4 mRNA was normalized to that of Syk.
Paired two-sided Student t tests were used to test significance with a comparison with the control without correction for multiple comparisons. The significance for p values between 0.05 and 0.01 is marked with one asterisk (*), values of p between 0.01 and 0.001 are marked with two asterisks (**), and values of p < 0.001 are marked with three asterisks (***). All experiments were repeated between three and six times. In immunoblots, densitometry of the bands was used to calculate the decrease in the protein expression as a percentage of control cells transfected with nontargeting siRNA corrected for gel loading. The annotations in the tables are derived from a search of the Mouse Genome Database at the Mouse Genome Informatics Web site, The Jackson Laboratory; World Wide Web Mouse Genome Database (www.informatics.jax.org) last checked in September 2008.
Screen of mouse phosphatase siRNA library
To identify phosphatases involved in FcεRI signaling, we screened a siRNA library targeting 198 known or predicted mouse phosphatases (Supplemental Table I). Pools of four different siRNA duplexes per targeting gene were used to increase the chance of siRNA-targeted protein knockdown, and IgE-Ag–induced mast cell degranulation was used as a functional readout for each targeted phosphatase. The mouse mast cell line MMC-1 was used as model system, as these cells were found to maintain their high viability and stable degranulation response posttransfection. Efficient transient transfection of these cells was achieved by using the Amaxa transfection system (Amaxa Biosystems); under the optimized conditions, enhanced GFP-expression vector resulted in >80% of the MMC-1 cells becoming fluorescent after 24 h of transfection.
Mouse Syk siRNA pool of four duplexes was used as a positive control to test the screening system, whereas the negative control siRNA was a pool of four nontargeting siRNAs that has been microarray-confirmed to minimally target known genes in human, mouse, and rat cells (Dharmacon). To test this screening system, MMC-1 cells were transfected with Syk or nontargeting siRNA pool in a 96-well plate format (Fig. 1A). Posttransfection, cells were divided into triplicate plates and sensitized with Ag-specific IgE immediately or after 24 or 48 h, respectively. After the 24 h of culture with IgE, the sensitized cells were stimulated by Ag, and the release of β-hexosaminidase was used as a measure of the extent of degranulation. In parallel, as positive controls, the efficiency of the knockdown in Syk protein expression was determined by immunoblotting. Transfection with the Syk siRNA pool effectively reduced Syk protein expression, with the largest reduction observed at 72 h posttransfection (Fig. 1B). As expected from previous reports, Syk protein knockdown decreased FcεRI-initiated mast cell degranulation, and this reduced release was proportionally related to the decrease in Syk protein expression (19).
In preliminary experiments, using siRNA targeting Syk and several other signaling molecules, there were different lag times between siRNA transfection and the maximum decrease in protein expression, which would be expected because of the variability in t1/2 of different proteins. Therefore, in the phosphatase siRNA library screening, the functional readout of FcεRI-induced degranulation was tested repeatedly at 24, 48, and 72 h posttransfection with each siRNA pool (Fig. 1A).
The results of the library screen are summarized in Supplemental Table II, which shows the effects of each siRNA transfection at 24, 48, and 72 h. There was variation in the effects of the different siRNA with >25% inhibition or enhancement by some samples (Fig. 1C), the results having a normal distribution curve (Supplemental Fig. 1). In analyzing the data from all 3 d of testing, 27 out of the 198 siRNA enhanced or inhibited the FcεRI-induced degranulation at >2 SD. These results suggested that siRNA-based target knockdown can be used as an efficient screening tool to study Ag-receptor signal transduction. Furthermore, it suggested that multiple phosphatases were involved in FcεRI-induced mast cell degranulation.
Confirmation of positive siRNA pools
The same siRNA gave similar results on the three different test days, suggesting the reproducibility of the results and that the knockdown had effects for several days (Supplemental Fig. 2). As outlined in Fig. 1A, the screening method used in the current experiments involved multiple handling procedures. To eliminate the possibility that false positives are due to experimental errors, we retested the siRNA pools that enhanced or inhibited degranulation at >1.5 SD. The results of the two separate experiments were closely related, as shown by the correlation coefficient of 0.94 (Fig. 2A). Supplemental Table III shows the results of the original and repeat determinations for all the siRNA that induced >2 SD effects in the original screen; for these, the correlation coefficient was 0.90. These results demonstrate that the majority of the hits from the original screen still remained positive in the repeat experiments, which strongly suggests that the observed effects were the true results of the siRNA transfections.
The targets that were positive on original screening that were confirmed on retesting to show >30% change in degranulation are in Table I. Among the 17 positives, 10 enhanced and 7 inhibited degranulation. The majority of these were poorly characterized molecules that on the basis of their sequence had been classified as protein tyrosine, serine, or dual-specific phosphatases. However, there were also some well-characterized proteins that inhibited degranulation [e.g., the siRNA that targeted both the regulatory (Ppp3r1) and the catalytic (Ppp3cc) subunit of calcineurin and PTEN].
Western blotting was then used to investigate if the observed functional changes were indeed due to siRNA-targeted decrease in protein expression. Different Abs for the eight positive hits for which commercial Abs were available were tested. Unfortunately, only anti-PTEN and anti-calcineurin B (another name for Ppp3r1) out of the >10 tested Abs detected the specific proteins. Fig. 2B shows the results of immunoblotting for the cells transfected with Pten or Ppp3r1 siRNA, which indicated that in these cells, the decreased mast cell degranulation was directly related with the reduced expression of targeted proteins.
Some siRNAs have off-target effects due to partial homology to other transcripts that could result in the inhibition or enhancement of degranulation. A recommended strategy to preclude this problem is to test four independent siRNAs against the same transcript; if at least two of these are positive, then it is unlikely that two independent siRNAs against the same gene target a common off-target transcript for an effect. Therefore, each of the four different siRNAs included in the original siRNA pools were separately tested for five genes that showed the strongest inhibition and two genes that enhanced. These were Inpp5b, Ppp3r1 (calcineurin B), Pten, Mtmr4, Ptpn4, Ptpn9, and Ptpn14. Their effects on IgE-Ag–induced degranulation of each single siRNA from the chosen pools was examined several times; the summary of these results is shown graphically in Fig. 3A, and the values are in Supplemental Table IV. Out of the seven tested as single siRNAs, at least two out of the four single siRNA showed similar effects as their pool, suggesting that these were true hits.
Additionally, as shown in Fig. 3B, the on-target effects of the single siRNA for Pten and Ppp3r1 (calcineurin B) were further confirmed by the specifically decreased protein expression. For Mtmr4, the on-target effects of single siRNA were confirmed by real-time PCR (Supplemental Fig. 3).
Positive target validation by using BMMCs
To test if the knockdown of these positive targets had the same effect in primary cells, BMMCs grown from normal C57BL/6J mice were used for further validation. Syk siRNA was used as a positive control to test the BMMC transfection system. As shown in Fig. 4A, the transfection of BMMC with Syk siRNA specifically reduced the Syk protein expression, which correlated with the decreased FcεRI-induced degranulation.
It has been reported that PTEN deficiency enhances the IgE-Ag–induced mast cell degranulation (33). However, in MMC-1 cells, reduced expression of PTEN resulted in decreased release (Figs. 2, 3). To clarify these discordant results, BMMCs were transfected and tested for FcεRI-induced degranulation and PTEN protein expression with the same scrambled and Pten siRNA that had been used in MMC-1 cells. Compared to control cells, the transfection of Pten siRNA reduced PTEN protein expression in BMMC and slightly enhanced IgE-Ag–induced degranulation (Fig. 4B). The discrepancy between MMC-1 and BMMC in the degranulation response to the siRNA-induced decrease in PTEN expression is probably due to a difference in the status of PI3K activation in the two cell types. Indeed, it had been observed that MMC-1 cells have a high basal PI3K activity as indicated by the increased Akt phosphorylation even in nonstimulated cells (E.A. Barbu, J. Zhang, and R.P. Siraganian, unpublished observations).
All of the positive hits shown in Fig. 3A were retested with BMMC. Except for PTEN, transfection of these positive pool siRNA in both BMMC and MMC-1 cells had similar effects on FcεRI-induced mast cell degranulation (Fig. 5A). Thus, Inpp5b or Ptpn9 siRNA transfection enhanced IgE-Ag–initiated degranulation, whereas Ppp3r1, Mtmr4, Ptpn4, and Ptpn14 inhibited this response. Furthermore, immunoblotting with specific anti-PPP3R1 Ab (anti-calcineurin B) indicated that Ppp3r1 siRNA specifically reduced its target protein expression in both MMC-1 and BMMC cells (Fig. 5B).
Knockdown of calcineurin B impairs FcεRI signaling in mast cells
To understand the mechanism by which calcineurin B (Ppp3r1) deficiency impaired IgE-Ag–initiated degranulation, cellular protein phosphorylation was examined. Fig. 6 compared the FcεRI-induced total cellular protein tyrosine phosphorylation in MMC-1 cells transfected with control scrambled siRNA to those with calcineurin B siRNA. The reduced expression of calcineurin B slightly decreased total cellular protein tyrosine phosphorylation.
Calcineurin B is regulated by calcium influx. Therefore, it was expected that its deficiency may have some effect on the downstream part of the FcεRI signaling pathway. This was observed when the IgE-Ag–induced phosphorylation of several MAPKs was examined. As shown in Fig. 6, the reduced expression of calcineurin B clearly decreased FcεRI-initiated phosphorylation of p42/44 Erk and p38 MAPKs, especially at late time points. In the cells transfected by calcineurin B siRNA, the FcεRI-induced phosphorylation of JNK was also reduced, but its extent was much less.
PKC plays an important role in mast cell degranulation. We therefore also measured the effect of calcineurin B deficiency on the phosphorylation of PKCs. The reduced calcineurin B expression decreased FcεRI-induced PKCδ phosphorylation at Thr505 and Ser643 sites, as shown in Fig. 6. PKD/PKCμ is a distant relative of the PKC isozymes, which function downstream of PKCs (36). Phosphorylation of Ser916 of PKD/PKCμ is directly related with its enzymatic activity (37). The calcineurin B deficiency inhibited IgE-Ag–initiated phosphorylation of Ser916 of PKD/PKCμ (Fig. 6). Altogether, these results suggest that calcineurin B is involved in the regulation of FcεRI pathways at multiple steps.
In this study, we demonstrated the utility of an siRNA functional screen to identify phosphatases involved in the regulation of FcεRI signaling. Following siRNA transfection, among 198 known or predicted mouse phosphatases, there were 17 that on retesting were confirmed to enhance or inhibit the IgE-Ag–induced mast cell degranulation (Table I). This number of positives is probably due to the fact that a phosphatase library was used in this screen of a signaling pathway in which a critical event is phosphorylation. A subset of the positive hits was then validated by testing separately each of the four different siRNAs included in the original siRNA pool, which confirmed the on-target effect of these positive hits. In addition, BMMCs grown from normal C57BL/6J mice were used for the further validation of the positive pools. Except for Pten, all of the other six positive hits including Inpp5b, Ppp3r1, Mtmr4, Ptpn4, Ptpn9, and Ptpn14 had similar effects on FcεRI-induced mast cell degranulation in both MMC-1 and BMMC cells.
Although degranulation is a complex event that depends on many cellular molecules, it can be determined by a robust and specific assay. This assay is quantitative and allows for identifying of both weak and strong hits; however, weak phenotypes can be due to partial knockdown of a gene with a strong effect or a strong knockdown of a gene with a weak effect. Among the positive hits, there was approximately an equal number that inhibited or enhanced degranulation (Table I). There was a >40% decrease or increase in secretion by these siRNAs, which indicates that these are important regulators of this signaling pathway. The putative phosphatases included molecules that act on phosphoinositides or on proteins phosphorylated on tyrosine, serine, or threonine residues.
siRNA library screening has been used as an efficient and useful approach to identify targets involved in different cell functions, although most of these have been with simpler assays (38–40). However, the utility of such siRNA screens for studies of Ag receptor signaling has been hampered by the difficulty of transfecting differentiated immune cells and the complexity of Ag-receptor signaling. In preliminary experiments, we tested different mast cell lines and siRNA delivery systems in attempts to establish a suitable transfection system that would work for large-scale screening. The virus-based transfection that has obvious advantages was tested by using lentiviral transduction particles. However, the results with both GFP and Syk short hairpin RNA transduction suggested that the transfection efficiency of the lentiviral system was too low and not efficient in decreasing the target protein expression in transient transfection systems (J. Zhang and R.P. Siraganian, unpublished observations). To increase transfection efficiency, small chemical synthesized siRNA was tested using different delivery methods followed by immunoblotting to monitor the targeted protein knockdown and FcεRI-induced degranulation to evaluate the effect of transfection on mast cell function. The protocol that was eventually developed for the current study yielded >80% transfection efficiency, and, as demonstrated with control and Syk siRNA, it dramatically decreased targeted protein expression without having nonspecific effects on mast cell function. As compared with stable transfected cell lines, this transient transfection system is suitable for large-scale screening, and it also eliminated the possibility of clonal variation.
Because the MMC-1 line was derived from transformed tumor cells that grow independent of growth factors, BMMC obtained from normal mice were used to further validate the seven positive hits; six out of the seven positive pools had very similar effects in both cell types, suggesting that the observed changes were due to the siRNA treatment. The positive hit that showed discrepant results in the two cell types was PTEN; transfection of Pten siRNA strongly inhibited FcεRI-induced degranulation in MMC-1 cells, whereas it slightly enhanced it in BMMC. Immunoblotting confirmed the specific PTEN protein knockdown by siRNA in both cell types. The knockdown of PTEN mediated by lentivirus transfection of short hairpin RNA was reported to increase FcεRI-induced degranulation in CD34+ peripheral blood-derived human mast cells (33). PTEN regulates phosphatidylinositol (PI) phosphate levels in cells by dephosphorylating several of the PIs at the 3′ position. It dephosphorylates PI 3,4,5-trisphosphate [PI(3,4,5)P3], which would increase PI 4,5-bisphosphate [PI(4,5)P2] levels and decrease the level of PI(3,4,5)P3. The intracellular levels of both PI(4,5)P2 and PI(3,4,5)P3 are important in mast cell signaling that results in degranulation. Receptor aggregation results in phospholipase C stimulation, which cleaves PI(4,5)P2 to form inositol (1,4,5)P3 and 1,2-diacylglycerol that release intracellular calcium and activate PKC, respectively, which are critical for FcεRI-induced degranulation. The difference in response to PTEN protein knockdown between MMC-1 and BMMC cells could be explained by the different activation status of PI3K in these cells. In MMC-1 cells, the increased basal PI3K activity would shift the balance between PI(4,5)P2 and PI(3,4,5)P3 with an increase in PI(3,4,5)P3 levels. The decreased expression by siRNA of PTEN in these cells would further enhance this shift and result in less PI(4,5)P2 substrate for phospholipase C, which could explain the reduced degranulation, whereas in BMMC, the balance between PI(4,5)P2 and PI(3,4,5)P3 is different because of the lower basal activity of PI3K in these cells. The decrease by siRNA in PTEN in BMMC would then shift the balance toward the accumulation of PI(3,4,5)P3 and result in enhanced secretion.
Calcineurin is a calcium-dependent serine/threonine protein phosphatase consisting of catalytic and regulatory subunits. The siRNA that targeted both the regulatory and the enzymatic subunits of calcineurin inhibited degranulation (37% for the regulatory Ppp3r1 and 31% for the enzymatic Ppp3cc). Calcineurin B (Ppp3r1) is the 19-kDa regulatory subunit of calcineurin and plays an important role in the activation of calcineurin phosphatase activity. Increased intracellular calcium activates calcineurin, which binds and dephosphorylates proteins, including the transcription factor NFAT, resulting in its nuclear translocation and subsequent gene expression. However, the function of calcineurin B in cells has not been easily defined because germ line mutation of the calcineurin B gene results in lethal defects in vascular patterning (41). Calcineurin enzymatic activity is inhibited by the cyclosporin A (CsA), which has been used to study the function of calcineurin in mast cells. CsA inhibits the FcεRI-dependent degranulation of bone marrow-derived mouse mast cells, human lung mast cells, or RBL-2H3 cells and blocks anti-IgE–induced histamine and cytokine releases from basophils (42–44). CsA binds to the cytosolic protein cyclophilin, which inhibits calcineurin. However, as cyclophilin is also present in mitochondrial membrane, CsA may also have an effect on mitochondria, such as preventing the formation of mitochondrial permeability transition pore. In the present experiments, siRNA knockdown of calcineurin B expression indicates a specific function of this protein in regulating FcεRI-induced degranulation.
The mechanism by which calcineurin controls NFAT activation has been extensively studied, but how this protein regulates mast cell degranulation is unclear. A possible mechanism could be by the regulation of PKC. AKAP79, a mammalian scaffold protein, coordinates the formation of a complex of PKC and calcineurin B (45). In the current study, we observed that decreased expression of calcineurin B reduced the phosphorylation of PKCδ and PKD/PKCμ, a substrate of PKC. Because PKC activation is required for mast cell degranulation, the reduced PKC activity could be responsible for the decreased degranulation in the calcineurin B-deficient cells. In thymocytes, calcineurin B deficiency reduced CD3/CD28 initiated Erk phosphorylation during thymocyte development (46). Similarly, we observed that the knockdown of calcineurin B reduced IgE-Ag–induced Erk and p38 phosphorylation in mast cells. Therefore, calcineurin B is implicated in the regulation of multiple molecules involved in FcεRI signaling.
In summary, these experiments demonstrated the utility of siRNA-based functional screening to identify molecules that positively or negatively regulate degranulation. Many of the phosphatase genes that were found to regulate this complex event have not been previously associated with degranulation. Further analysis of how they regulate these signaling pathways should provide new insights into this important basic cellular function. Furthermore, these studies have identified several new molecules that are critical for FcεRI-mediated mast cell degranulation, which may serve as potential therapeutic targets for the treatment of asthma and allergic diseases.
We thank Dr. Nicholas Ryba for discussion and reviewing the manuscript.
Disclosures The authors have no financial conflicts of interest.
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Dental and Craniofacial Research.
The online version of this article contains supplemental material.
Abbreviations used in this paper:
- bone marrow-derived mast cell
- cyclosporin A
- phosphatidylinositol 3,4,5-trisphosphate
- phosphatidylinositol 4,5-bisphosphate
- protein kinase C
- protein kinase D
- phosphatase and tensin homologue deleted on chromosome 10
- Src homology region 2 domain-containing phosphatase
- small interfering RNA.
- Received December 30, 2009.
- Accepted April 9, 2010.
- Copyright © 2010 by The American Association of Immunologists, Inc.