3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition in a rat mast cell line. Impairment of tyrosine kinase-dependent signal transduction and the subsequent degranulation response.

IgE molecules bind mast cells via a heterotetrameric receptor termed Fc epsilon RI. Cross-linking of bound IgE by specific allergen (Ag) initiates a signal transduction cascade resulting in a degranulation response. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in isoprenoid and sterol biosynthesis, by the cholesterol-lowering drug, lovastatin, blocks Fc epsilon RI-dependent [3H] serotonin ([3H]5HT) release from the mast cell line, RBL-2H3. We studied the mode and locus of action of lovastatin in these cells. Lovastatin inhibited Ag-stimulated degranulation, as well as that evoked by ionomycin or by phorbol 12-myristate 13-acetate and ionomycin, stimuli which bypass early receptor events. Inhibition was concentration-dependent, occurred at levels which reduce lipid synthesis, and was reversible by addition of mevalonic acid, the product of the reductase reaction. The effects of lovastatin were not mimicked by treatment with the sterol demethylase inhibitor, econazole, suggesting that nonsterol isoprenoid synthesis is required for the degranulation response. Conversely, tyrosine kinase inhibitors from three disparate chemical classes reduced stimulus-evoked [3H]5HT release in a manner similar to lovastatin, suggesting that these agents share similar loci of action. Accordingly, lovastatin altered the phosphorylation pattern in unstimulated RBL-2H3, and reduced the phosphorylation response to IgE cross-linking. By analogy to 5HT release, this effect was concentration-dependent and mevalonic acid-reversible. The tyrosine kinase inhibitor, geldanamycin, also reduced the phosphorylation response to Ag. Lyn, a Src-related tyrosine kinase activated upon IgE cross-linking, was little influenced by either lovastatin or geldanamycin. Thus, lipid synthesis inhibition by lovastatin results in impaired tyrosine phosphorylation in RBL-2H3. This impairment is reflected in the subsequent exocytotic response. While lovastatin may inhibit tyrosine phosphorylation via an indirect mechanism, our results with tyrosine kinase inhibitors support the concept that multiple tyrosine kinases participate in the Fc epsilon RI-dependent signal transduction process.


3-Hydroxy-3-methylglutaryl-coenzyme A Reductase Inhibition in a Rat Mast Cell
(Received for publication, December 24, 1992, and in revised form, April 2, 1993) Michael P. ShakarjianS, Elisa Eisemanj, Robert C. Penhallow, and Joseph B. Bolen IgE molecules bind mast cells via a heterotetrameric receptor termed Fc,RI. Cross-linking of bound IgE by specific allergen (Ag) initiates a signal transduction cascade resulting in a degranulation response. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in isoprenoid and sterol biosynthesis, by the cholesterollowering drug, lovastatin, blocks Fc.RI-dependent ['HI serotonin (['HISHT) release from the mast cell line, RBL-2H3. We studied the mode and locus of action of lovastatin in these cells. Lovastatin inhibited Ag-stimulated degranulation, as well as that evoked by ionomycin or by phorbol 12-myristate 13-acetate and ionomycin, stimuli which bypass early receptor events. Inhibition was concentration-dependent, occurred at levels which reduce lipid synthesis, and was reversible by addition of mevalonic acid, the product of the reductase reaction. The effects of lovastatin were not mimicked by treatment with the sterol demethylase inhibitor, econazole, suggesting that nonsterol isoprenoid synthesis is required for the degranulation response. Conversely, tyrosine kinase inhibitors from three disparate chemical classes reduced stimulusevoked ['H]6HT release in a manner similar to lovastatin, suggesting that these agents share similar loci of action. Accordingly, lovastatin altered the phosphorylation pattern in unstimulated RBL-2H3, and reduced the phosphorylation response to IgE cross-linking. By analogy to 6HT release, this effect was concentrationdependent and mevalonic acid-reversible. The tyrosine kinase inhibitor, geldanamycin, also reduced the phosphorylation response to Ag. Lyn, a Src-related tyrosine kinase activated upon IgE cross-linking, was little influenced by either lovastatin or geldanamycin. Thus, lipid synthesis inhibition by lovastatin results in impaired tyrosine phosphorylation in RBL-2H3. This impairment As reflected in the subsequent exocytotic response. While lovastatin may inhibit tyrosine phosphorylation via an indirect mechanism, our results with tyrosine kinase inhibitors support the concept that multiple tyrosine kinases participate in the Fc.RIdependent signal transduction process. Mast cells are the primary effectors in immediate type hypersensitivity reactions. Sensitization of these cells with allergen (Ag)'-specific IgE confers them with high responsivity to subsequent Ag challenge. Upon exposure to Ag, mast cells undergo rapid secretion of preformed mediators from cytoplasmic granules. These include biogenic amines, ATP, proteases, proteoglycans, and cytokines. Mast cells bind IgE avidly via specific, high affinity Fc receptors, termed Fc,RI. Fc,RI are heterotetrameric complexes comprising an IgE binding a subunit, a /3 subunit, and two y subunits (1). Activation occurs upon cross-linking of surface-bound IgE molecules by multivalent Ag. It is not, however, apparent how the binding event gives rise to an intracellular activation signal. No effector functions intrinsic to the Fc,RI have been elucidated, suggesting that association of the receptor with additional components are necessary for proper signaling. Truncation of the cytoplasmic tails of /3 and y, but not a subunits, leads to loss of Fc,RI signaling capability when aggregated in a whole cell system (2). Moreover, homologies between Fc,RI and signaling components in the Tand B-cell antigen receptor systems have led to the suggestion that effector molecules, such as protein tyrosine kinases, may associate with /3 or y subunits to form a signaling complex (3). Consistent with this idea are recent reports by a number of investigators demonstrating tyrosine phosphorylation responses upon IgE crosslinking in Fc,RI-bearing cells (4-7). Our finding (8) that the Src-related tyrosine kinase Lyn associates with Fc,RI from activated cells provides added strength for this argument.
Another early event detected upon Fc,RI aggregation is the activation of PLC, leading to the breakdown of phosphatidylinositol and the generation of inositol phosphates and diacylglycerol. These products respectively activate protein kinase C and increase intracellular free Ca2+ levels, signals which positively impact upon the degranulation potential of the mast cell (9,10). Investigators using the rat mast cell line, RBL-2H3, have observed that PLCyl is activated via tyrosine phosphorylation, and that activation of this enzyme accounts for the inositol phosphate generation which follows IgE crosslinking (11,12). This result has been confirmed recently in mouse bone marrow mast cells (13). Thus, tyrosine kinase activation is a necessary precedent in Fc,RI-dependent phosphatidylinositol breakdown, analogous to the scenario set The abbreviations used are: Ag, allergen; [3H]5HT, [3H]serotonin; DNP, dinitrophenol-conjugated bovine serum albumin; FceRI, the high affinity receptor for IgE G protein, guanine nucleotide-binding protein; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; Io, ionomycin; MVA, mevalonic acid; PMA, phorbol 12-myristate 13-acetate; PLC, phosphatidylinositol-specific phospholipase C; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-~iperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. forth by receptor tyrosine kinase systems (14).
The HMG-CoA reductase inhibitors are a recently introduced class of cholesterol lowering agents. They effectively reduce circulating cholesterol levels by competitively inhibiting the rate-limiting step in the isoprenoid and sterol biosynthesis pathway (15). Interestingly, Deanin et al. (16) have noted that the HMG-CoA reductase inhibitor lovastatin inhibits Ag-dependent degranulation in RBL-2H3 cells. Although the locus of action of lovastatin is unclear, stimulusinduced calcium fluxes and inositol 1,4,5-trisphosphate generation are inhibited as well, suggesting that this agent was affecting early events in the Fc-RI-dependent signal transduction cascade. Moreover, through a series of replenishment experiments, the investigators showed that the inhibitory action of lovastatin is lipid-dependent, yet cholesterol-independent. They hypothesized that a nonsterol isoprenoid, possibly one utilized in the posttranslational modification of proteins, is essential for proper signaling through Fc,RI. Our interest in understanding the early links between receptor activation and the degranulation response in mast cells led us to consider lovastatin as a tool in deciphering this signal transduction pathway. In the present work we have examined the relationship between tyrosine phosphorylation and lovastatin action. We find that this HMG-CoA reductase inhibitor disturbs cellular signaling at more than one point and impairs tyrosine phosphorylation in a manner which suggests that a lipid-dependent component is required at a very early step in the Fc,RI signal transduction pathway.

EXPERIMENTAL PROCEDURES
Chemicals-Lovastatin and its water-soluble sodium salt were synthesized by the Department of Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute. Econazole diacetate was kindly provided by Dr. Joseph OSullivan of Bristol-Myers Squibb. Phorbol 12-myristate 13-acetate (PMA) and genistein were purchased from LC services, ionomycin (Io) from Calbiochem, geldanamycin from Life Technologies, Inc., and tyrphostin 47 (RG 50864) from Biomol Research Labs. All of the above agents were dissolved in Me2S0 (Sigma), save lovastatin sodium and genistein. Genistein was prepared by addition of Me2S0, H20, and NaOH. MVA, obtained from Sigma as mevalonolactone, was diluted in 10 mM NaAc, pH 4.5, and kept as a 1 M frozen stock. Neither genistein nor MVA treatments altered the final pH when added to cell cultures.
Immunological Reagents-The polyclonal antibody to Lyn was prepared as described (17). Mouse monoclonal anti-dinitrophenol IgE was generated from the hybridoma cell line TIB-142 (ATCC) and used as a 1:lOOO dilution of the ascites. Dinitrophenol-conjugated bovine serum albumin (DNP) was a gift from Dr. Henry Metzger, NIAMS.
Culture of RBL-ZH3"The RBL-2H3 cell line was kindly provided by Dr. Henry Metzger. Cells were grown as adherent monolayers in Eagle's minimal essential medium with Earle's salts (Life Technologies, Inc.), supplemented with 16% fetal bovine serum, penicillin/ streptomycin, and 2 mM glutamine. They were maintained at 37 "C in tissue culture flasks with closed caps. At time of passage, cell monolayers were detached by a 5-min treatment with trypsin-EDTA, followed by gentle physical dislodgement. Cells were diluted and centrifuged at 250 X g. The resulting cell pellet was resuspended and utilized for replating.
PHISerotonin (5HT) Release-RBL-2H3 cells were plated at a concentration of 2 X lo5 cells/well in 24-well culture plates. After 4- bovine serum albumin) containing appropriate treatments. Sensitization of cells with anti-DNP IgE was performed at least 1 h prior to washing. Cells were warmed briefly, treated with stimuli or vehicles, and incubated at 37 "C. Stimulation was generally allowed to progress for 1 h, unless otherwise indicated. Incubation was terminated by removal of supernatant from the wells. Pellets were harvested by solubilization in 1% Triton X-100. The 3H in each fraction was counted by liquid scintillation spectroscopy. Specific release was defined as that portion of the total radioactivity recovered from the supernatant, after subtraction of spontaneous release. The percentage inhibition was then calculated from these values. Incorporation of r4C]Acetate into CholesteroE and Precursors-Cells grown in 6-well culture plates were maintained for 24 h in complete medium supplemented with 5% lipid-free fetal bovine serum. Drugs or vehicle were added to appropriate wells, followed by addition of 15 gCi of [I4Clacetate (40-60 mCi/mmol, ICN), and unlabeled acetate to attain a final concentration of 100 +M. Culturing continued for a period of 20 h. Wells were then washed twice with ice-cold phosphate-buffered saline and treated with 1 ml of 0.1 M NaOH. The contents of each well were transferred to tubes containing 3 ml of ethanol and 0.5 ml of 40% KOH. A recovery standard consisting of 35 nCi of [3H]cholesterol (20-40 Ci/mmol, Du Pont-New England Nuclear) and unlabeled squalene, cholesterol, and lanosterol was added at this time to aid in identifying products and monitoring yield. Samples were heated at 65 "C for 45 min to saponify lipids. Three extractions with petroleum ether followed. The reserved organic phases were pooled for each sample and dried under N2. The extract was reconstituted in MeC1:CHC13 (1:l) and spotted on silica gel thin layer plates (PE SIL G, Whatman Ltd). Plates were chromatographed for 1 h in a CHCl3 mobile phase. Separated lipid was analyzed by liquid scintillation counting and autoradiography/phosphorimaging (Molecular Dynamics). Inhibition of lipid synthesis was determined by comparing lipid profiles between drug-and vehicletreated cell lanes. As the econazole block resulted in accumulation of lanosterol and its nonsaponifiable precursors on the TLC plate, the contribution of these bands was subtracted from the total lipid profile for all treatments and was not included in our analysis.
Anti-phosphotyrosine Immunoblotting-Cells were treated with appropriate drug, stimulus, or respective vehicle for the prescribed time period. All cells were lysed by the addition of ice cold lysis buffer (50 mM Tris, pH 8.0, 150 mM NaC1, 1% Nonidet P-40, 2 mM EDTA, 1 mM NaaV04, 5 mM NaF, and 10 pg/ml aprotonin and leupeptin). The samples were centrifuged at 14,000 X g for 4 min. Aliquots of each supernatant containing 50 pg of protein were heated in 2 X loading buffer and subjected to SDS-PAGE (18). Separated proteins were transferred to nitrocellulose membranes. Western blotting of membranes was performed with polyclonal anti-phosphotyrosine primary antibody (Upstate Biotechnology Inc.) followed by 1251-labeled protein A (30 mCi/mg, Amersham Corp.). Reactive bands were visualized by autoradiography and phosphorimage analysis.
Immune Complex Kinase Assay-This procedure has been described in detail elsewhere (19). Briefly, detergent lysates (250 pg of protein) were incubated 1 h at 4 "C with anti-Lyn antibody. Pansorbin (Calbiochem) was then added, and tubes were rotated for 30 min at 4 "C. The immune complexes were collected by centrifugation, washed five times with 1 ml of lysis buffer, and once with 1 ml of kinase buffer (20 mM MOPS, pH 7.0, 5 mM MnC12, and 1 mM Na3V04). A reaction mix composed of [Y-~*P]ATP (3000 Ci/mmol, Dupont-New England Nuclear), 10 p~ unlabeled ATP, 0.5 mg/ml rabbit muscle enolase, and kinase buffer was used to resuspend the pellet in a final volume was 25 pl. The kinase reaction proceeded for 1 min at room temperature, with agitation, and was stopped by addition of 2 X SDS-PAGE sample buffer. The reaction products were fractionated on 8% SDS-polyacrylamide gels. Pretreatment with lovastatin was found to reduce IgE-dependent [3H]5HT release over a concentration range of 0.1 to 10 p~. Degranulation responses to Io or PMA/Io stimulation were susceptible to the actions of lovastatin as well. A consistent rank order of sensitivity to lovastatin was observed, with the IgE/DNP response most susceptible, and the Io response least susceptible. Degranulation induced by the PMA/Io combination was inhibited by lovastatin intermediate to that of the above stimuli, suggesting that PMA conferred sensitivity to the inhibitory effects of lovastatin. Lovastatin-treated cultures displayed no change in viability or cellularity detectable by enumeration after trypan blue staining. Nor was there any significant alteration in cellular protein synthesis, as measured by [3H]leucine incorporation into trichloroacetic acid precipitable fractions (20), under the conditions used in this study (data not shown).

Lovastatin Inhibits Degranulation Evoked by Various Stim-
We next examined the specificity of lovastatin action by determining whether its inhibitory effect upon the RBL-2H3 degranulation response is due to the inhibition of lipid synthesis. By providing the cells with MVA, the product of the HMG-CoA reductase reaction, it is possible to bypass the block imposed by lovastatin and reverse any actions of lovastatin specific to HMG-CoA reductase inhibition. As shown in Fig. lC, addition of 1 mM MVA four h prior to cell stimulation rescued the degranulation response from the inhibitory actions of lovastatin for each of the stimuli tested. MVA treatment had no influence upon spontaneous release, nor did it alter stimulus-evoked release in the absence of lovastatin (data not shown). We infer from these experiments that lovastatin inhibits the degranulation response as an extension of its action upon HMG-CoA reductase.
To determine whether a depletion of cholesterol itself could account for the inhibitory effects of lovastatin upon mast cell degranulation, we blocked cholesterol synthesis by an alternative method. We employed the sterol 14a-demethylase inhibitor, econazole, which imposes a block in the latter stages of cholesterol synthesis, sparing the events leading to synthesis of lanosterol. Cells were pretreated with lovastatin, econazole, or vehicle. Sterol synthesis was assessed by monitoring the incorporation of [ '*C]acetate into nonsaponifiable lipids. Parallel cultures were loaded with [3H]5HT to measure degranulation. As seen in Table I  the impairment of the mast cell secretory response by lovastatin. Effect of Tyrosine Kinase Inhibitors upon Stimulus-evoked Degranulation-We also compared the sensitivity of the various degranulating stimuli to tyrosine kinase inhibitors. Tyrosine kinase inhibitors representing three disparate chemical classes were utilized. Depicted in Fig. 2  tyrphostin 47 (RG 50864), and the ansamycin antibiotic geldanamycin, an analogue of herbimycin A. All effectively reduced degranulation responses after a preincubation of 90 min. As observed with lovastatin treatment, IgE/DNP-evoked degranulation was most sensitive to tyrosine kinase inhibition. Degranulation responses initiated by Io or PMA/Io were more resistant to pretreatment with the tyrosine kinase inhibitors. Nevertheless, the partial sensitivity displayed by these latter stimuli suggested that tyrosine phosphorylation might mediate some of their effects as well.
Relative Abilities of Stimuli to Induce Tyrosine Phosphorylation in RBL-2H3-1n order to further understand the relationship between tyrosine phosphorylation and the secretory response in RBL-2H3 cells, we compared the abilities of the stimuli of interest to induce protein tyrosine phosphorylation. Treatment of cells with IgE/DNP resulted in a vigorous phosphorylation response (Fig. 3A). Protein bands that were phosphorylated over the 10-min time course included a band corresponding to 42 kDa, multiple bands in the 50-56-kDa range, a prominent 72-kDa band, and others at 85, 100, and 135 kDa. Interestingly, PMA/Io stimulation of RBL-2H3 cells also resulted in a tyrosine phosphorylation response (Fig. 3A). ting were a subset of those identified by IgE/DNP stimulation, with the 72-kDa band conspicuously absent. Addition of PMA, at 0.1 PM, produced little if any change in the resting tyrosine phosphorylation pattern of RBL-2H3 (Fig. 3B). This is consistent with the inability of PMA, when added singly, to evoke a secretory response in these cells. Addition of 2 PM Io resulted in a weak but detectable tyrosine phosphorylation response, evident at early time points, after which the response waned (Fig. 3B). As with the PMA/Io combination, no phosphorylation in the 72-kDa range was observed.

The bands detectable by anti-phosphotyrosine immunoblot-
Lowastatin Modifies the Tyrosine Phosphorylation Pattern in RBL-2H3-In light of the observations that inhibition of [3H]5HT release by lovastatin resembles that of tyrosine kinase inhibitors and appears to parallel, in part, the ability of stimuli to induce tyrosine phosphorylation, we directly examined the effect of this HMG-CoA reductase inhibitor upon IgE/DNP-stimulated tyrosine phosphorylation. As shown in Fig. 4A, the actions of lovastatin were 2-fold. In the absence of stimulus, lovastatin pretreatment typically led to a shift in the phosphorylation of 135-and 150-kDa protein bands, with phosphorylation diminishing in the upper band and increasing in the lower band. Furthermore, lovastatin reduced the tyrosine phosphorylation response to IgE/DNP stimulation. These changes occurred under conditions identical to those which result in 100% inhibition of the degranulation response. The effects of lovastatin upon tyrosine phosphorylation in RBL-2H3 were concentration-responsive (Fig. 4B). It should be noted, however, that although this response to IgE/DNP was progressively diminished, it was not entirely eliminated, even at concentrations of lovastatin supramaximal for the inhibition of secretion. As indicated in Fig. 4C, the effects of lovastatin upon both resting and stimulated tyrosine phosphorylation were reversible with MVA pretreatment. Although the shift in phosphorylation of 135and 150-kDa bands was less pronounced in this experiment, one can observe a reduction in the phosphorylation of p135 concomitant with restoration of the Ag-induced response by MVA. These actions of MVA were concentration-responsive. Thus tyrosine phosphorylation, like [3H]5HT release, is altered by lovastatin as a function of its inhibition of HMG-CoA reductase.
Inhibition of the Antigen-ewoked Tyrosine Phosphorylation Response in RBL-2H3 by Geldanamycin-We examined the actions of the tyrosine kinase inhibitor, geldanamycin, upon Ag-induced tyrosine phosphorylation in RBL-2H3, as a basis of comparison with those actions of lovastatin (Fig. 5). Sensitized cells were pretreated 90 min with various concentrations of geldanamycin. Geldanamycin inhibited basal phosphorylation at the higher concentrations tested (Fig. 5A). In addition, this agent reduced phosphorylation in DNP-treated cells in a concentration-dependent manner. Both basal and stimulated phosphorylation were diminished by 1 PM geldanamycin pretreatment (Fig. 5B), under conditions where Agevoked [3H]5HT release is completely abolished. Nevertheless, the phosphorylation response to DNP was not entirely eliminated.
Effects of Lowastatin and Geldanamycin upon Lyn Kinase Activity-As the Src-related tyrosine kinase Lyn associates with Fc,RI and is activated after IgE/DNP addition (8), we investigated whether lovastatin or geldanamycin influenced the activity of this enzyme. Cells were stimulated with IgE/ DNP in the presence or absence each agent and lysed at the appropriate time points. Immune complex kinase assays were then performed with immunoprecipitated Lyn kinase. Lovastatin caused an increase in basal Lyn kinase activity, observable with 1 PM treatment, as depicted in Fig. 6A. This agent  03 0.1 0.3 1 0 0.03 0.1 0. also appeared to retard the time course of enzyme activation. Nonetheless, there was no dramatic reduction in stimulated kinase activity, even at 70 PM. Geldanamycin, likewise, had no dramatic effect upon Lyn kinase (Fig. 6B). Basal activity appeared to be increased with this drug as well, and although the results may suggest the occurrence of a kinetic shift of enzyme activation toward earlier time points, the effects of geldanamycin upon this enzyme appear minor.

DISCUSSION
In accordance with the results of Deanin et al. (16), we have demonstrated that the HMG-CoA reductase inhibitor lovastatin potently inhibits Ag-dependent degranulation in the mast cell line RBL-2H3. The inhibition is concentrationresponsive and occurs under treatment conditions where the activity of the cholesterol biosynthesis pathway is also reduced. We examined the effect of lovastatin on the actions of other secretogogues so to ascertain at what level in the signal transduction pathway this agent interacts. The Ca2+ ionophore, Io, and the protein kinase C activator, PMA, respectively, mimic the actions of inositol 1,4,5-trisphosphate and diacylglycerol, products of phospholipase C activation. Therefore, events leading to the activation of PLC in the Fc,RI pathway are bypassed by these agents. Degranulation evoked by Io or the PMA/Io combination was also reduced by lovastatin. However, neither Io-nor PMA/Io-stimulated [3H]5HT release were as sensitive to lovastatin action as was Ag- stimulated release. The determined rank order of sensitivity to lovastatin action, IgE/DNP > PMA/Io > Io, allows us to conclude that lovastatin can disturb the pathway leading to the secretory response from Fc,RI at two levels. First, lovastatin can inhibit an event that occurs prior to, or independent of, the increase in cytosolic free Ca2+ levels and the activation of protein kinase C. Second, lovastatin can inhibit secretion at a point beyond the generation of these two signals. In the case of each of the stimuli tested, secretory responses were restorable by addition of MVA, the product of HMG-CoA reductase. These results indicate that the inhibitory effects of lovastatin are a consequence of its action upon lipid synthesis. Taken together, the data provide evidence for two potential points in the Fc,RI signal transduction cascade that require activity of the cholesterol biosynthesis pathway.
We performed experiments with tyrosine kinase inhibitors in order to understand what processes might be targeted by lovastatin action. These agents inhibited [3H]5HT release with activity profiles surprisingly akin to that displayed by lovastatin. Deanin et al. (21) made a similar observation when comparing the effects of tyrosine kinase inhibitors and lovastatin on GTP+-or Ag-stimulated responses in permeabilized cells. We found the Ag-dependent degranulation response to be most sensitive to the actions of the tyrosine kinase inhibitors, in accordance with evidence that tyrosine kinase activation is an early consequence of Fc,RI signaling (4-8). However, secretion induced by Io and the PMA/Io combination displayed a finite degree of sensitivity, suggesting that secretion initiated by these stimuli may be dependent upon tyrosine kinases as well. Recent studies by Benhamou et al. (22) and Yu et al. (7), in RBL-2H3 cells, are in agreement with this result. By use of anti-phosphotyrosine immunoblotting, each group showed that an increase in tyrosine phosphorylation accompanies Ca2+ ionophore-induced degranulation. Moreover, tyrosine kinase inhibitors reduced secretion and tyrosine phosphorylation in parallel. Our results (Fig. 3) confirm the stimulatory actions of Ca2+ ionophore upon tyrosine phosphorylation. Furthermore, we extend this observation to show that PMA enhances ionomycin-induced tyrosine phosphorylation, thus revealing an additional outcome of concerted protein kinase C activation and Ca2+ influx.
Interestingly, the capacity of the stimuli tested to induce tyrosine phosphorylation in RBL-2H3 cells closely parallels their sensitivity to the effects of lovastatin upon [3H]5HT release. A basis for this relationship was revealed by observing the effect of lovastatin upon Ag-induced tyrosine phosphorylation (Fig. 4). Lovastatin diminished the phosphorylation response in cells treated with IgE/DNP, a stimulus highly sensitive to the actions of this drug as assessed by [3H]5HT release. From these experiments, it may be inferred that lovastatin somehow disrupts the Fc,RI signaling mechanism a t or prior to activation of a tyrosine kinase. Lovastatin also induced discrete alterations in the phosphotyrosine pattern of unstimulated RBL-2H3 cells, as evidenced by a shift in intensities of two protein bands. It is unclear whether this shift is responsible for, or independent of, the diminished ability of the cells to respond to the IgE/DNP stimulus. One Ag-dependent phosphorylation event clearly susceptible to the actions of lovastatin is that of a prominent 72-kDa protein band. Phosphorylation of this band appears indicative of an early response to Ag stimulation, as neither Io nor PMA/Io treatments induce its phosphorylation in RBL-2H3. It is thus likely that lovastatin acts at a point in the signal transduction cascade leading from Fc,RI that is not shared by Io or PMA/ Io. A possible target of action is the Src-related tyrosine kinase, Lyn. As we have previously shown, Lyn coprecipitates with Fc,RI and becomes activated upon receptor cross-linking (8). Association of Src-related tyrosine kinases with analogous receptor systems has been demonstrated as well (23). However, lovastatin, at biologically relevant concentrations, was found to cause only minor changes in the activity of Lyn. Thus, Lyn does not appear to be an important target of lovastatin action. Although the activation of other kinases might be affected, it is equally likely that lovastatin somehow alters access of substrates to kinases, thereby diminishing the efficiency of the phosphorylation process.
The results from our studies with geldanamycin deserve a somewhat different explanation. This agent, like the other tyrosine kinase inhibitors tested, selectively inhibited [3H] 5HT release stimulated by Fc,RI cross-linking. In accord with these results, Ag-stimulated tyrosine phosphorylation was also diminished. Nevertheless, geldanamycin had an equally dramatic influence upon tyrosine phosphorylation in the absence of stimulus. Thus, under our experimental conditions, geldanamycin apparently inhibits kinases responsible for both constitutive and stimulus-evoked tyrosine phosphorylation. It is important to note that the reduction in immunoreactivity to anti-phosphotyrosine antibody did not, in our hands, directly correlate with inhibition of secretion by geldanamycin. For instance, at 0.1 PM, geldanamycin reduces [3H]5HT release by 75%, while producing only small reductions in antiphosphotyrosine immunoreactivity. This observation is not isolated to geldanamycin, but has been noted with other tyrosine kinase inhibitors tested,' as well as with lovastatin. We conclude that discrete phosphorylation events involved in degranulation may not be faithfully reflected in a whole cell anti-phosphotyrosine blot. One explanation is that this readout is a composite of multiple events, varying in importance * M. P. Shakarjian and J. B. Bolen, unpublished results.
to the response of interest. Our inability to discern any significant inhibitory effect of geldanamycin upon Lyn kinase activation is consistent with this notion. It suggests that more than one kinase is involved in Fc,RI signaling. For instance, by reducing constitutive phosphorylation, geldanamycin may provide conditions unfavorable for receptor activation. Alternatively, this inhibitor may affect another tyrosine kinase which participates in the early steps of activation. One possible candidate is a 72-kDa tyrosine kinase found in association with Fc.RI and suspected to be Syk or a related kinase (24,25). This kinase may in fact be the identity of the aforementioned 72-kDa tyrosine phosphorylated protein which is a major landmark of Fc,RI-dependent stimulation. Furthermore, a putative 44-kDa tyrosine kinase has recently been claimed to associate with PLC-yl upon IgE cross-linking in mouse bone marrow mast cells (13). While it is likely that geldanamycin lacks affinity for Lyn at the concentrations tested, the complex regulation of the activity of this kinase, like others of the Src family, allow for an indirect action of the inhibitor upon Lyn. Activity of the Src-related kinases is regulated not only by autophosphorylation, but also by an additional phosphorylation event at a conserved C terminal tyrosine residue. A kinase with this capacity has been isolated and described as c-Src kinase (Csk). Inhibition of this regulatory kinase can, in fact, enhance the activity of Src-related kinases such as Lyn (26). This may explain the slightly increased basal level of Lyn and the earlier activation of this kinase in geldanamycin-treated cells. In support of this possibility, we have evidence indicating the presence of stable transcripts of Csk in RBL-2H3. 3 It should be apparent from this work that neither geldanamycin nor lovastatin impair Fc,RI signaling by directly reducing Lyn activity. While it is likely that geldanamycin acts by inhibiting other tyrosine kinases, alternative explanations deserve consideration in the case of lovastatin. As MVA, but neither dolichol nor cholesterol, restores Ag-dependent degranulation in lovastatin-treated cultures, it has been suggested that this drug acts by depleting the cell of isoprenoids utilized in the posttranslational modification of protein (16). Consistent with this notion are our findings from experiments with econazole. This agent, which selectively inhibits the final steps of cholesterol biosynthesis and spares nonsterol isoprenoid synthesis, is not an inhibitor of mast cell degranulation. Isoprenoid addition, in the form of 15-(farnesyl) and 20-(geranylgeranyl) carbon chains, is directed to cysteine residues near the carboxyl-terminal end of certain nascent polypeptides. Proteolytic removal of the remaining carboxyl-terminal amino acids and carboxyl methylation of the prenylated cysteine typically follows (27). This set of modifications enhances the affinity of proteins for biological membranes. A number of proteins involved in signal transduction are so modified, and include members of the Ras superfamily of low molecular weight G proteins, the y subunit of the heterotrimeric G proteins, LY subunits of retinal cGMP phosphodiesterases, and rhodopsin kinase (27,28). Our results indicate that prenylated proteins may be required at two levels of the Fc,RI signal transduction cascade: during the early activation of tyrosine kinases by Fc,RI cross-linking, and beyond the stimulation of Ca2+ fluxes and protein kinase C. Such proteins might possess an intrinsic effector function or merely serve as a membrane anchor for the association of molecules essential in the propagation of the signal produced by IgE crosslinking.
Thus, we have found lovastatin to be a valuable tool in understanding the transductional events involved in mast cell R. C. Penhallow and J. B. Bolen, unpublished results. degranulation. Degranulating stimuli display sensitivity toward lovastatin analogous to their sensitivity to tyrosine kinase inhibitors and their ability to induce tyrosine phosphorylation in RBL-2H3 cells. The inhibitory action of lovastatin upon Ag-stimulated tyrosine phosphorylation provides an explanation for its effects upon degranulation and suggests that a very early step in the signal transduction process is perturbed by this agent. By attributing this perturbation to an inhibition of protein prenylation, one invokes, by extension, the importance of specific proteases and methyltransferases in constitution of the signal transduction cascade. Such enzyme activities have been previously considered to play regulatory roles in the Fc,RI signaling process (29). In fact, we have found that 5'-methylthioadenosine, a general methyltransferase inhibitor, reduces Ag-induced degranulation and tyrosine phosphorylation in a manner analogous to that of lovastatin.' We are presently using more specific means to assess the importance of protein modifications, such as prenylation and carboxyl methylation in mast cell responses to IgE cross-linking.