INHIBITION OF ANTIGEN AND CALCIUM IONOPHORE INDUCED SECRETION FROM RBL-2H3 CELLS BY PHOSPHATASE INHIBITORS

Cell Biology International 1998, Vol. 22, No. 11/12, 855–865 Article No. cb980332, available online at http://www.idealibrary.com on

INHIBITION OF ANTIGEN AND CALCIUM IONOPHORE INDUCED SECRETION FROM RBL-2H3 CELLS BY PHOSPHATASE INHIBITORS RUSSELL I. LUDOWYKE*, KRYSTINA WARTON and LYNDEE L. SCURR Centre for Immunology, St Vincent’s Hospital, University of New South Wales, Sydney, Australia Received 10 December 1997; accepted 4 November 1998

The role of serine/threonine protein phosphatases PP1 and PP2A in mast cell secretion was investigated using the phosphatase inhibitors okadaic acid and calyculin A. Calyculin A (5–25 n) inhibited antigen-induced secretion from a rat mucosal mast cell line (RBL-2H3) when added in conjunction with the activator. Okadaic acid (250–1000 n) inhibited secretion only when added before activation and did so in a time- and concentration-dependent manner. Both inhibitors caused the cells to become rounder, but only calyculin A induced membrane blebbing and a loss of adherence. Okadaic acid also inhibited secretion induced by the calcium ionophore A23187, in the presence or absence of PMA, indicating that the phosphatase inhibitors act on a component of the secretory pathway downstream of calcium mobilization. Okadaic acid increased the phosphorylation of a number of proteins, as did an analogue methyl okadaate, which also inhibited secretion, but less effectively. Okadaic acid induced the phosphorylation of triton-insoluble proteins of 55, 18 and 16 kDa. The 55 kDa protein was identified as vimentin and okadaic acid induced its partial translocation to the triton-soluble fraction. Our data indicate that full secretory function in mucosal mast cells requires  1998 Academic Press phosphatase activity. K: okadaic acid; PP1; PP2A; phosphorylation; mast cells; calyculin A; vimentin A : PP1 or PP2A, protein phosphatases type 1 or 2A; DNP-BSA, 24 molecules of dinitrophenol conjugated with 1 molecule of BSA as the antigen; [3H] 5-HT, [3H] 5-hydroxytryptamine

INTRODUCTION In studies of regulated granule exocytosis in platelets, neutrophils, chromaffin cells and mast cells, the important role of serine/threonine kinases is well established. To understand fully the role of phosphorylation in the secretory process, we have to also understand the important role of dephosphorylation in controlling the level of phosphorylation of a particular protein. Yet the type or actions of phosphatases playing a part in the secretory process is not established. It is now becoming clear that serine/threonine phosphatases play an active role in regulating a diverse range of biological processes such as secretion, although the mechanisms of action on the secretory process and *To whom correspondence should be addressed at: Centre for Immunology, St Vincent’s Hospital, Victoria Street, Sydney, NSW 2010, Australia. 1065–6995/98/110855+11 $30.00/0

the effects on specific intracellular signals remain unclear. Mammalian serine/threonine protein phosphatases are classified into four major subtypes, protein phosphatase 1 (PP1), PP2A, PP2B and PP2C. Of these, PP1 and PP2A are the predominant forms (Hardie et al., 1991). Okadaic acid and calyculin A are cell-permeable serine/threonine protein phosphatase inhibitors (Haystead et al., 1989). In vitro, okadaic acid inhibits both PP1 and PP2A, although it is more potent against PP2A (PP1, IC50 =10–15 n; PP2A, IC50]0.1 n) (Bialojan and Takai, 1988; Cohen, 1989; Cohen and Cohen, 1989; Hardie et al., 1991). Calyculin A is an equipotent inhibitor of PP1 and PP2A (IC50]1 n). Neither okadaic acid nor calyculin A has significant effects upon PP2B or PP2C in vitro, at concentrations below 5
 (Cohen, 1989; Cohen and Cohen, 1989).  1998 Academic Press

856

Okadaic acid and calyculin A, have been used to show the involvement of PP1 and PP2A in a number of regulated granule secretory systems, but with quite variable results. In platelets, the addition of the inhibitors generally leads to the inhibition of aggregation and secretion (Lerea, 1992; Murata et al., 1992; Walker and Watson, 1992; Nishikawa et al., 1994; Yano et al., 1995). In neutrophils, the phosphatase inhibitors increase superoxide release (Bengisgarber and Gruener, 1995; Djerdjouri et al., 1995). In chromaffin cells, okadaic acid decreases catecholamine secretion induced by high K + or carbachol but has no effect on secretion induced by a calcium ionophore (Yanagihara et al., 1991). However in other studies with chromaffin cells and in permeabilized chromaffin cells, phosphatase inhibitors generally increase catecholamine secretion (Wagner and Vu, 1990; Wu and Wagner, 1991; Gutierrez et al., 1995). In mast cells the effects of the phosphatase inhibitors upon the secretion of inflammatory mediators have also proved to be variable. In rat peritoneal (connective tissue) mast cells, okadaic acid enhances the secretory response to fluoride and compound 48/80 (Botana et al., 1992), inhibits IgE-dependent histamine release, but has no effect on secretion induced by the calcium ionophore A23187 (Takei et al., 1993; Estevez et al., 1994). However, in human lung mast cells (mucosal mast cells), okadaic acid and calyculin A not only inhibit IgE-dependent histamine release, but also that induced by A23187 (Peachell and Munday, 1993). The mast cell line RBL-2H3, is homologous to mucosal mast cells (Seldin et al., 1985), therefore it would be expected that the response to phosphatase inhibitors would more closely resemble those of mucosal mast cells. However, in one study, okadaic acid and calyculin A themselves induced mediator release from RBL-2H3 cells (Sakamoto et al., 1994), while two other studies found little effect of okadaic acid on leukotriene generation or mediator release induced by calcium ionophore (Hagmann, 1994; Kitani et al., 1996). In the present study we examined the effects of the phosphatase inhibitors on secretion from RBL-2H3 cells in more detail, and investigated some of the mechanisms through which the phosphatase inhibitors act. We present evidence that clearly shows that the phosphatase inhibitors inhibit both antigen and calcium ionophore A23187 induced secretion from RBL-2H3 cells. We further show that inhibition is coincident with increased protein phosphorylation, which is consistent with the mode of action of protein phosphatase inhibitors.

Cell Biology International, Vol. 22, No. 11/12, 1998

MATERIALS AND METHODS Materials Materials were obtained as follows: phorbol 12myristate 13-acetate, (PMA); A23187; calyculin A and DNP-specific IgE from Sigma, St Louis, MO, U.S.A.; okadaic acid and methyl okadaate from Research Biochemicals International, Natick, MA, U.S.A.; the ammonium salt of okadaic acid used in later experiments was obtained from Alomone Labs, Jerusalem, Israel; radioisotopes from Amersham, Australia. The antigen, DNP24-BSA (24 molecules of DNP conjugated with one molecule of BSA, hereafter referred to as DNP-BSA) was a kind gift of Dr Henry Metzger, NIAMS, National Institutes of Health, Bethesda, MD, U.S.A. Cell culture and radiolabelling RBL-2H3 cells were maintained and activated as adherent monolayer cultures. Labelling with [3H] 5-hydroxytryptamine ([3H] 5-HT) was done as described previously (Maeyama et al., 1988; Ali et al., 1989; Ludowyke et al., 1989; Ludowyke and Scurr, 1994). Labelling with [32P] orthophosphoric acid (100
Ci/ml; 2 h) was done with minor modifications to that described previously (Ludowyke et al., 1989). 1–2106 cells per sample point were labelled in a buffer consisting of 119 m NaCl, 5 m KCl, 5.6 m dextrose, 4 m glutamine and 0.4 m MgCl2 with 25 m PIPES (pH 7.2). The buffer was supplemented with 0.1% BSA and 1 m CaCl2 prior to use. Addition of phosphatase inhibitors In the initial experiments (Figs 1–5), okadaic acid, methyl okadaate and calyculin A were dissolved in dimethylsulphoxide (DMSO). In most experiments the final buffer concentration of DMSO was no more than 0.1%, which was added to control cells and had no effect on the morphology, extent, or rate of secretion. In the experiments on protein phosphorylation, the ammonium salt of okadaic acid was used (okadaic acid-salt; OA-s), and dissolved in buffer prior to use. Cells were preincubated with the inhibitors in labelling buffer at 37C for the designated times before addition of activators. Washing the cells free of excess inhibitors had no effect on the inhibitory capacity. Analysis of secretion Cells were activated in the labelling buffer. Cells, primed overnight with 75 ng/ml DNP-specific IgE,

Cell Biology International, Vol. 22, No. 11/12, 1998

were activated with the specific antigen DNP-BSA at 25–50 ng/ml which was optimal for secretion. Where required, cells were activated with 50 n PMA, 1000 n of the calcium ionophore, A23187 or the combination of 50 n PMA and 500 n A23187. Control samples were incubated with buffer alone. After the designated time, the reaction was stopped by placing the samples on ice and an aliquot of the medium was taken for assay of the release of [3H] 5-HT. The total cellular content of [3H] 5-HT was determined following lysis of unstimulated cultures and the actual release expressed as a percentage of total. Control release was that in the presence of the buffer alone. Analysis of phosphorylated proteins Cells prelabelled with 32Pi were washed to remove unincorporated radiolabel and then preincubated with the ammonium salt of okadaic acid (OA-s). This form of okadaic acid inhibited secretion to the same extent as the free acid form (OA) but inhibition was delayed in onset. After antigen activation when required, supernatants were removed from the cells and either Laemmli sample buffer or an ice-cold cytoskeletal extraction buffer added to the wells. With Laemmli sample buffer, the total cell lysate was scraped into microcentrifuge tubes and heated at 100C for 5 min. The cytoskeletal buffer consisted of 2 m MgCl2, 50 m KCl, 5 m EGTA, 20 m PIPES pH 6.8 and 0.5% Triton (Ludowyke et al., 1994). Additionally, 1 m ATP and 25 m sodium pyrophosphate were added as phosphatase inhibitors with 10
g/ml of pepstatin, leupeptin and aprotinin as protease inhibitors. After 15 min on ice, the cell lysates were scraped into microcentrifuge tubes and the triton-insoluble cytoskeletal pellets separated from supernatants by microcentrifugation at 4C for 20 min. Proteins in each fraction were then solubilized in Laemmli sample buffer. Proteins were separated by SDS electrophoresis under reducing conditions on gels ranging from 7.5% to 15% polyacrylamide. Initially, proteins were visualized by staining with Coomassie Brilliant Blue and scanned using a Molecular Dynamics (Sunnyvale, CA, U.S.A.) laser densitometer to equilibrate protein loadings for later gels. Owing to the cytoskeletal extraction system, proteins in the supernatant fraction were more dilute than those in the pellet fraction. Radiolabelled proteins were visualized by autoradiography for various times, using Kodak film or by a Molecular Dynamics phosphorimager.

857

Western immunoblotting This was done as described previously, separating proteins on 10% SDS gels and transferring to PVDF membranes (Kawasugi et al., 1995). The anti-vimentin monoclonal antibody (Amersham, Australia) was diluted 1/5 as per the manufacturers instructions. The horseradish peroxidase conjugated secondary antibody diluted 1/3000 (Caltag Laboratories, Burlingame, CA, U.S.A.) was detected using chemiluminescence (NEN, Life Sciences, Australia) or, in the presence of radioactively labelled cell lysates, using colour detection with 4-chloro-1-naphthol (Bio-Rad, Hercules, CA, U.S.A.). RESULTS Effects of phosphatase inhibitors on secretion Okadaic acid and calyculin A inhibit PP1 and PP2A at 0.1–10 n in vitro, but higher concentrations were expected to be required for effects in intact cells (Hardie et al., 1991). Increasing concentrations of okadaic acid up to 1000 n were added simultaneously with the antigen, DNP-BSA, to IgE-primed RBL-2H3 cells but this had no effect upon secretion (Fig. 1A). However, when the cells were preincubated with okadaic acid before addition of the antigen, a striking inhibition of secretion was observed (Fig. 1B). Under these conditions, okadaic acid inhibits antigen-induced secretion in a time- and concentration-dependent manner. In contrast to okadaic acid, when calyculin A was added to the cells simultaneously with the antigen, secretion was inhibited. Inhibition occurred in a concentration-dependent manner with an IC50 of approximately 50 n (Fig. 2A). Preincubation with low concentrations (5–25 n) of calyculin A also led to inhibition (723.6% inhibition with 25 n after 20 minutes; data not shown). As calyculin A had a much faster inhibitory effect on secretion, we investigated its effects on the time course of secretion. 100 n calyculin A reduced the rate of [3H] 5-HT release by 2.5 minutes post-stimulation, with total abolition of further release by 10 minutes (Fig. 2B). No effect was seen with 25 n calyculin A until 5 minutes post-stimulation and additional release was inhibited after 15 minutes. Calyculin A (1 n) had no effect upon secretion. The inhibitory effect induced by okadaic acid or calyculin A was not easily reversible as removal of

858

Cell Biology International, Vol. 22, No. 11/12, 1998

60

60

(A)

50

40

40

30

30

20

20

10

10

0

200

400 600 800 Okadaic acid (nM)

1000

(B)

50

Release (%)

Release (%)

50

60

(A)

0 60

50

150 100 Calyculin A (nM)

200

(B) DNP-BSA

50

1 nM

40

40

250 nM

25 nM

30

30

20

20

500 nM

100 nM

10

10 1000 nM

0

20 40 Preincubation time (min)

60

Fig. 1. Effects of okadaic acid on [3H] 5-HT release from RBL-2H3 cells. (A) Increasing concentrations of okadaic acid were added simultaneously with the antigen, 25 ng/ml DNPBSA. (B) Cells were preincubated for up to 60 min with the indicated concentrations of okadaic acid before addition of 25 ng/ml DNP-BSA. Release is given as a percentage of the total [3H] 5-HT present in the cells and results are meansSE of triplicates from at least three experiments.

the inhibitors for up to 2 hours prior to antigen activation did not restore secretion (data not shown). Effects of phosphatase inhibitors on cell morphology We observed by light or phase microscopy that both okadaic acid and calyculin A caused the cells to become rounder, losing the long processes that exemplified the untreated cells (Fig. 3A, D). This effect was evident within 5 minutes of addition of calyculin A (Fig. 3C) but was slower in onset with

0

10

20 Time (min)

30

Fig. 2. Effects of calyculin A on [3H] 5-HT release from RBL-2H3 cells. (A) Inhibition of [3H] 5-HT secretion by increasing concentrations of calyculin A added simultaneously with the antigen, 25 ng/ml DNP-BSA. (B) 1, 25 and 100 n concentrations of calyculin A were added simultaneously with 25 ng/ml of DNP-BSA and release of [3H] 5-HT determined from 1 to 30 min thereafter. The time course of DNP-BSAinduced release in the absence of inhibitor is also given (solid line). Results are meansSE of triplicates from at least three experiments.

okadaic acid (Fig. 3E). Unfortunately, calyculin A also caused blebbing of the plasma membrane within the same time frame (Fig. 3C) and reduced the adhesion of the cells as many became detached from the surface of the wells. These effects of calyculin A made interpretation of the effects upon secretion more difficult as optimal secretion requires cell adherence (Hamawy et al., 1992). However, neither inhibitor affected cell viability during or after the preincubation time, as assessed by trypan blue or ethidium bromide staining. In

Cell Biology International, Vol. 22, No. 11/12, 1998

859

Fig. 3. Morphological changes induced by okadaic acid and calyculin A. Calyculin A (25 n) or okadaic acid (1000 n) were added to RBL-2H3 cells and images recorded under a phase contrast microscope. A, B and C are images taken after 5 min and D, E and F after 45 min at 37C. A and D show cells incubated in buffer alone whilst B and E, cells incubated with okadaic acid and C and F, cells incubated with calyculin A. Rounding and blebbing of the cells is visible after 5 min with calyculin A, yet only rounding is apparent after 45 min with okadaic acid. Original magnification 200, reproduced at 85%.

our hands okadaic acid did not cause the cells to detach from the surface of the wells although this has been experienced by others using higher concentrations of okadaic acid (Kitani et al., 1996). We therefore confined our further experiments to the use of okadaic acid. Antigen activation induces apical membrane ruffling and cell spreading which can be clearly seen with phase contrast microscopy. We have noted, in contrast to others (Kitani et al., 1996), that cells pretreated with okadaic acid did not exhibit these morphological changes when activated by antigen, but remained rounded. Inhibition of secretion induced by the calcium ionophore A23187, and PMA+A23187 To further investigate the role of phosphatases upon secretion we assessed the effect of okadaic acid on activators which bypassed the requirement for IgE aggregation, tyrosine kinase activation and inositol phospholipid hydrolysis. The calcium

ionophore A23187 induces secretion from mast cells by directly increasing intracellular calcium concentrations and activating a number of protein kinase C isozymes (Ludowyke et al., 1996). The addition of the protein kinase C activator, PMA enhances this secretion. Preincubation with 500 n okadaic acid for 45 minutes inhibited secretion induced by the calcium ionophore A23187, in the presence or absence of PMA (Fig. 4). The extent of secretion induced by PMA plus A23187 is similar to that of antigen, and okadaic acid inhibits their levels of secretion to a similar extent. It has been shown in mast cells that okadaic acid has no effect on calcium release from intracellular stores or the influx of extracellular calcium (Takei et al., 1993; Kitani et al., 1996). Furthermore it has been established that in vitro, okadaic acid has no effect on a number of protein kinases, including protein kinase C (Hardie et al., 1991). These results suggest that okadaic acid inhibits secretion at a point in the secretory pathway downstream of the rise in intracellular Ca2+ and activation of protein kinase C.

860

Cell Biology International, Vol. 22, No. 11/12, 1998 120

70

50

60

40

Release (%)

Release (%)

50

30

20

40 30

% Release in absence of inhibitor

100 80 60 Me-OAte 40 20 OA 0

200

400

600

800

1000

Concentration (nM)

20

10 10

0

A23187

PMA + A23187

Fig. 4. Effects of okadaic acid on [3H] 5-HT release induced by the calcium ionophore A23187. RBL-2H3 cells were preincubated for 45 min with 500 n okadaic acid before addition of either 1000 n A23187 or the combination of 50 n PMA and 500 n A23187. After 30 min the activation was stopped and the supernatant assayed for [3H] 5-HT release which is expressed as a percentage of the total [3H] 5-HT present in the cells. The results represent meansSE of triplicates from four separate experiments.

Inhibition of antigen-induced secretion by methyl okadaate Methyl okadaate, an analogue of okadaic acid was also capable of inhibiting antigen-induced secretion when cells were preincubated for 45 minutes (Fig. 5). As can be seen in the inset, when compared directly, methyl okadaate was less effective than the same concentration of okadaic acid, but similarly to okadaic acid, inhibition was both time and concentration dependent (data not shown). In vitro, methyl okadaate has little inhibitory effect on PP1 or PP2A, so it is most likely that methyl okadaate is converted to the active acidic form by intracellular enzymes, as has been suggested (Holmes et al., 1990; Peachell and Munday, 1993). Effects of okadaic acid on protein phosphorylation The intracellular effect of phosphatase inhibition should be to increase the phosphorylation level of specific proteins upon which the kinases and phosphatases which interact with them are active (Haystead et al., 1989). We therefore examined changes in protein phosphorylation in the RBL2H3 cells in the presence of okadaic acid and also in the presence of methyl okadaate to determine whether it had a similar effect to that of okadaic acid. For these experiments a newer form of okadaic acid was used, the ammonium salt of

0

1000 500 1500 Methyl okadaate (nM)

2000

Fig. 5. Inhibition of DNP-BSA-induced [3H] 5-HT release by methyl okadaate. RBL-2H3 cells were preincubated with increasing concentrations of methyl okadaate for 45 min before addition of 25 ng/ml of DNP-BSA for 30 min. Results are meansSE of triplicates from at least three experiments. The inset shows that when compared directly, methyl okadaate (Me-OAte) was a less effective inhibitor than okadaic acid (OA).

okadaic acid (okadaic acid-salt; OA-s) as it was soluble in buffer and had a longer storage life. Although this okadaic acid-salt form inhibited secretion to the same extent as that of the free acid, the time course of inhibition was delayed. When inhibition is compared directly, preincubation for 30 min with the non-salt form gives 67% inhibition of secretion whilst 45 min preincubation with the okadaic acid-salt is required to give a similar level of inhibition (62%). Both forms of okadaic acid achieve greater than 80% inhibition of secretion after 60 min preincubation. Cells preincubated with 32 P-orthophosphoric acid, were incubated with 1000 n okadaic acid-salt or methyl okadaate for 30–60 minutes. The cells were lysed and proteins separated by SDS-PAGE as outlined in Methods. Figure 6 shows that the addition of okadaic acidsalt, time-dependently increases the phosphorylation of a number of proteins. The predominant proteins are of molecular weights 55 and 18 kDa. The addition of methyl okadaate increases the phosphorylation of the same two proteins, confirming that it is acting in the same manner as okadaic acid-salt. In order to examine the effects of okadaic acid on protein phosphorylation in more detail, the cells were lysed and triton-insoluble cytoskeletal proteins and triton-soluble proteins separated by SDS-PAGE as outlined in Methods. Following

Cell Biology International, Vol. 22, No. 11/12, 1998

Fig. 6. Increased protein phosphorylation induced by okadaic acid and methyl okadaate. RBL-2H3 cells were preincubated with 32Pi for 2 hours in phosphate-free buffer before a further preincubation with 1000 n of the ammonium salt of okadaic acid (okadaic acid-salt; OA-s) or methyl okadaate (Me-OAte) for 30, 45 or 60 min. The okadaic acid-salt inhibited secretion to the same extent as the non-salt form but inhibition was delayed in onset. Preincubation for 30 min with the non-salt form inhibits secretion to a similar level as 45 min preincubation with the okadaic acid-salt form. Cells were also left for 45 min without inhibitors (C). After the required times, supernatants were removed and cells lysed with Laemmli sample buffer and proteins separated on a 12.5% gel by SDS-PAGE. The molecular weights of the two major proteins (55 kDa and 18 kDa) exhibiting increases in phosphorylation were determined from molecular weight markers run alongside the samples.

incubation with 1000 n okadaic acid-salt for 45 minutes, a number of cytoskeletal and soluble proteins exhibit increased phosphorylation. Proteins were separated on gels of varying polyacrylamide concentration and autoradiograms were exposed for various times to highlight different proteins. In the cytoskeletal pellet, the proteins which exhibited the greatest increase in phosphorylation were those of 55, 18 and 16 kDa, as is apparent in Figure 7A. Smaller increases were seen in proteins of 168 and 107 kDa. In the triton-soluble fraction, okadaic acid-salt-induced increases in phosphorylation were also seen in proteins of 100, 55, 49 and 20 kDa. We identified the 55 kDa protein as the cytoskeletal intermediate filament protein vimentin, by Western immunoblotting with a monoclonal antivimentin antibody (Fig. 7B). In unstimulated cells vimentin is predominantly in the triton-insoluble or cytoskeletal fraction. Okadaic acid-salt causes the partial translocation of vimentin to the triton-

861

Fig. 7. Effects of okadaic acid on specific protein phosphorylation and translocation. (A) Cells were preincubated with 32Pi for 2 hours before a further incubation for 45 min with 1000 n okadaic acid ammonium salt (OA-s) or buffer. The cells were then lysed as described in Materials and Methods and the triton-insoluble (pellet) and soluble (supernatant) fractions separated. An autoradiogram of phosphorylated proteins in a 12.5% gel is shown. Increases in phosphorylation are seen mainly in proteins of 55, 18 and 16 kDa. The autoradiogram is representative of four separate experiments. (B) Cells without preincubation with 32Pi but incubated with OA-s and proteins separated as above were transferred to PVDF membranes and incubated with a monoclonal antivimentin antibody as described in Materials and Methods. A representative Western immunoblot showing all the bands detected by chemiluminescence is shown.

soluble fraction (Fig. 7B). The same results were seen when vimentin was detected in the immunoblot with the method of 4-chloro-1-naphthol from 32 Pi labelled samples, clearly identifying this protein as vimentin. Cell lysates were also separated by twodimensional gel electrophoresis (Ludowyke et al., 1989, 1996) and the pIs of the 18 and 16 kDa proteins were found to be very basic (data not shown). These proteins appeared to be abundant by Coomassie Blue staining, and after determining the amino acid composition and searches using the

862

ExPasy protein database, they were identified as likely to be histones, which are very basic and known to increase in phosphorylation with okadaic acid (Ajiro et al., 1996). DISCUSSION In the present study we have established that inhibitors of serine/threonine protein phosphatases inhibited granule mediator secretion from the RBL-2H3 mucosal mast cell line when activated with antigen or the calcium ionophore A23187. Furthermore, we have identified vimentin as a major substrate of the phosphatases. In vitro studies show that calyculin A is equipotent against PP1 and PP2A but that PP2A is approximately 10-fold more sensitive to okadaic acid than PP1. Our results may then suggest that the major phosphatase involved in the inhibitory process is PP2A. However, the relative intracellular concentrations of PP1 and PP2A and their accessibility to the inhibitors needs to be considered. Nevertheless, as inhibition of PP1 and/or PP2A inhibits secretion, these phosphatases appear to have an integral part in the pathway leading to secretion. At which point or points within the secretory pathway the phosphatases are acting then becomes an important question. With regard to this question, it is of great importance that okadaic acid inhibited secretion induced by both antigen and Ca2+ ionophore. IgE receptor aggregation by specific antigen leads to the activation of a pathway of well defined intracellular biochemical signals including tyrosine kinases, phospholipases, specific protein kinase C isozymes and an increase of intracellular calcium (Ali et al., 1989; Cunha-Melo et al., 1989; Ozawa et al., 1993; Lee and Oliver, 1995; Beaven and Baumgartner, 1996). Protein kinase C activation and a rise in intracellular calcium are critical elements required for secretion (Cunha-Melo et al., 1989; Ozawa et al., 1993). The addition of a calcium ionophore like A23187 which directly increases intracellular calcium, bypasses the strict requirement for tyrosine kinase and phospholipase activity to induce secretion. However we have recently shown that secretion induced by A23187 was strongly correlated with protein kinase C activation (Ludowyke et al., 1996). Previous work had suggested that okadaic acid only slightly suppressed A23187-induced secretion from RBL2H3 cells (Kitani et al., 1996), yet in our hands okadaic acid inhibited secretion induced by A23187. PMA, which activates protein kinase C

Cell Biology International, Vol. 22, No. 11/12, 1998

maximally (Cunha-Melo et al., 1989; Ludowyke et al., 1996), enhances secretion induced by A23187 to a level similar to that of antigen, yet this secretion is also inhibited by okadaic acid. This suggests that the point of inhibition is downstream of the rise in intracellular calcium and activation of protein kinase C. Alterations in cell morphology induced by okadaic acid have been documented in neutrophils (Kreienbuhl et al., 1992) and mast cells (Kitani et al., 1996). Our present results on the morphological effects of okadaic acid on RBL-2H3 cells concur with the rounding of the cells in the presence of okadaic acid, but differ in a number of other aspects which have implications for the secretory process. Even at low concentrations of okadaic acid the cells became rounder, but significantly, even at 1000 n they did not become detached from the substratum in contrast to other reports (Kitani et al., 1996). This is an important point as it is known that attachment is required for optimal antigen-induced secretion to occur (Hamawy et al., 1992) and that during the secretory process the cells spread out on the substratum, almost doubling their surface area and decrease in height by about half (Ludowyke et al., 1994; Kawasugi et al., 1995). We have noted that the time course of rounding of the cells correlated with the time course of inhibition, and that the round cells did not spread out when activated with antigen, although whether the rounding is part of the inhibitory process is unclear. Therefore as calyculin A caused the cells to become detached, these events could not occur and it was then difficult to interpret the role of phosphatases in secretion. For this reason, and also for the induction of extensive plasma membrane blebbing, calyculin A was not used in the later experiments. In total cell lysates a number of proteins exhibited an increased phosphorylation following addition of okadaic acid, with the predominant ones being of 55 kDa and 18 kDa. The phosphorylation of these proteins increased with time after addition of okadaic acid. Importantly, the okadaic acid analogue, methyl okadaate, which in vitro has little effect upon PP1 or PP2A, also increased the phosphorylation of these same proteins, but at a slower rate, which is correlated with its slower rate of inhibition of secretion (Fig. 5). These data strengthen the reasoning that intracellular enzymes can cleave this analogue, yielding the free okadaic acid, which can then inhibit the phosphatases (Holmes et al., 1990; Peachell and Munday, 1993). A more detailed analysis of the phosphorylated proteins in triton-soluble and insoluble fractions,

Cell Biology International, Vol. 22, No. 11/12, 1998

reveals a relatively small number of other proteins which are affected by the addition of okadaic acid. Of the three major phosphorylated proteins in the triton-insoluble fraction, we have identified the 55 kDa protein as the intermediate filament protein, vimentin by Western immunoblotting. This concurs with reports in other cell types that phosphatase inhibitors increase vimentin phosphorylation (Eriksson et al., 1992; Lee et al., 1992; Gutierrez et al., 1995). While this study was in progress, others reported that okadaic acid increased the phosphorylation of two proteins of 18 kDa and 68 kDa in RBL-2H3 cells (Kitani et al., 1996). We did not see changes in a 68 kDa protein, but only in a 55 kDa protein. We have also shown here that okadaic acid leads to increased association of vimentin with the triton-soluble fraction. A function if any, for vimentin in the secretory process in RBL-2H3 cells is unknown, but other evidence such as the translocation of PKC- to colocalize with vimentin filaments upon antigen activation (Spudich et al., 1992), and the phosphorylation of vimentin in permeabilized rat peritoneal mast cells (Tasaka, 1994) suggests a role may exist. We have also observed that okadaic acid increases the phosphorylation of another cytoskeletal protein, myosin, (unpublished observations), consistent with results seen in macrophages (Wilson et al., 1991). Within the mast cell population there are two distinct subtypes which differentiate due to the tissue microenvironment (Beaven and Baumgartner, 1996). Mast cells are commonly described as connective tissue or serosal mast cells (CTMC; which generally contain the granular enzymes tryptase and chymase-MCTC) and mucosal mast cells (MMC; which generally contain tryptase-MCT) (Schulman, 1993). Both subtypes respond to crosslinking of their high affinity IgE receptors and to calcium ionophores, but only CTMCs respond to neuropeptides, polyamines and morphine analogues (Beaven and Baumgartner, 1996; Schulman, 1993). It was shown in human lung mast cells (Peachell and Munday, 1993), which are of the MMC subtype, and we have shown here in RBL-2H3 cells which are homologous to MMCs (Seldin et al., 1985; Beaven and Baumgartner, 1996), that okadaic acid inhibits both IgE-based and non IgE-based secretion. Evidence in the literature suggests that in rat peritoneal mast cells (which are of the CTMC subtype), okadaic acid inhibits IgE-based secretion but has little or no effect on non-IgE based activators (Takei et al., 1993; Alfonso et al., 1994; Estevez et al., 1994; Kitani et al., 1996). This

863

suggests that the requirement for phosphatases in the secretory process may vary between the different mast cell subtypes. However, it must be taken into consideration that within these experiments, preincubation times varied and the level of secretion induced by IgE and non IgE-based activators were not matched, which makes this interpretation more difficult. Our data lead us to conclude that phosphatases are required in the RBL-2H3 cell secretory pathway, at a point distal to the rise in intracellular calcium and activation of protein kinase C. We have identified vimentin as a substrate of the phosphatases, but clearly, before a mechanism of the inhibitory process initiated by okadaic acid may be defined, the identification of the other proteins exhibiting alterations in phosphorylation with okadaic acid must be completed.

ACKNOWLEDGEMENTS R.I.L. is supported by the Sternberg Research Fellowship. We are very thankful for the technical assistance of Ms P. Keyhani. We are grateful for financial support from The Clive and Vera Ramaciotti Foundation, The Asthma Foundation of NSW, Community Health and AntiTuberculosis Association and St. Vincent’s Hospital. We thank Dr P.W. French and Associate Prof. W. Sewell for critical reading of the manuscript. REFERENCES A K, Y K, U K, N Y, 1996. Alteration of cell cycle-dependent histone phosphorylations by okadaic acid. Induction of mitosis-specific H3 phosphorylation and chromatin condensation in mammalian interphase cells. J Biol Chem 271: 13,197–13,201. A A, B MA, V MR, L MC, B LM, 1994. Effect of signal transduction pathways on the action of thapsigargin on rat mast cells—crosstalks between cellular signalling and cytosolic pH. Biochem Pharmacol 47: 1813–1820. A H, C- DM, B MA, 1989. The rise in concentration of free Ca2+ and of pH provides sequential, synergistic signals for secretion in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells. J Immunol 143: 2626–2633. B MA, B RA, 1996. Downstream signals initiated in mast cells by FC-epsilon-RI and other receptors. Curr Opin Immunol 8: 766–772. B C, G N, 1995. Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated

864

neutrophils—studies with selective inhibitors Ro 31-8220 and calyculin A. Cell Signal 7: 721–732. B C, T A, 1988. Inhibitory effect of a marinesponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J 256: 283–290. B LM, A A, B MA, V MR, L MC, C AG, 1992. Influence of protein kinase C, cAMP and phosphatase activity on histamine release produced by compound 48/80 and sodium fluoride on rat mast cells. Agents Actions 37: 1–7. C P, 1989. The structure and regulation of protein phosphatases. Ann Rev Biochem 58: 453–508. C P, C PT, 1989. Protein phosphatases come of age. J Biol Chem 264: 21,435–21,438. C-M JR, G HM, A H, H FL, H KP, B MA, 1989. Studies of protein kinase C in the rat basophilic leukemia (RBL-2H3) cell reveal that antigeninduced signals are not mimicked by the actions of phorbol myristate acetate and Ca2+ ionophore. J Immunol 143: 2617–2625. D B, C C, P E, H J, P A, 1995. Contrasting effects of calyculin A and okadaic acid on the respiratory burst of human neutrophils. Eur J Pharmacol 288: 193–200. E JE, B DL, V R, O J, F H, G RD, 1992. Cytoskeletal integrity in interphase cells requires protein phosphatase activity. Proc Natl Acad Sci USA 89: 11,093–11,097. E MD, V MR, L MC, B LM, 1994. Effect of okadaic acid on immunologic and nonimmunologic histamine release in rat mast cells. Biochem Pharmacol 47: 591–593. G LM, Q JL, R J, V S, R JA, 1995. The protein phosphatase inhibitor calyculin-A affects catecholamine secretion and granular distribution in cultured adrenomedullary chromaffin cells. Eur J Cell Biol 68: 88–95. H W, 1994. Cell proliferation status, cytokine action and protein tyrosine phosphorylation modulate leukotriene biosynthesis in a basophil leukaemia and a mastocytoma cell line. Biochem J 299: 467–472. H MM, O C, M SE, S RP, 1992. Adherence of rat basophilic leukemia (RBL-2H3) cells to fibronectin-coated surfaces enhances secretion. J Immunol 149: 615–621. H DG, H TA, S AT, 1991. Use of okadaic acid to inhibit protein phosphatases in intact cells. Methods Enzymol 201: 469–476. H TAJ, S ATR, C D, H RC, T Y, C P, H DG, 1989. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature 337: 78–81 H CF, L HA, C F, S FJ, 1990. Inhibition of protein phosphatases-1 and -2A with acanthifolicin. Comparison with diarrhetic shellfish toxins and identification of a region on okadaic acid important for phosphatase inhibition. FEBS Lett 270: 216–218. K K, F PW, P R, L RI, 1995. Focal adhesion formation is associated with secretion of allergic mediators. Cell Motility Cytoskel 31: 215–224. K S, T R, N Y, M Y, I K, 1996. The effect of okadaic acid on histamine release, cell morphology and phosphorylation in rat basophilic leukemia (RBL-2H3) cells, human basophils and rat peritoneal mast cells. Int Arch Allergy Immunol 110: 339–347.

Cell Biology International, Vol. 22, No. 11/12, 1998

K P, K H, N V, 1992. Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulusdependent increases in cytoskeletal actin of human neutrophils. Blood 80: 2911–2919. L RJ, O JM, 1995. Roles for Ca2+ stores release and two Ca2+ influx pathways in the Fc epsilon R1-activated Ca2+ responses of RBL-2H3 mast cells. Mol Biol Cell 6: 825–839. L WC, Y JS, Y SD, L YK, 1992. Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells. J Cell Biochem 49: 378–393. L KM, 1992. Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca2+ transients in human platelets. Role of a cdc2-related protein kinase. Biochemistry 31: 6553–6561. L RI, K K, F PW, 1994. ATPGamma-S induces actin and myosin rearrangement during histamine secretion in a rat basophilic leukemia cell line (RBL-2H3). Eur J Cell Biol 64: 357–367. L RI, S LL, 1994. Calcium-independent secretion by ATP-gamma-S from a permeabilized rat basophilic leukaemia cell line (RBL-2H3). Cell Signal 6: 223–231. L RI, P I, B MA, A RS, 1989. Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J Biol Chem 264: 12,492–12,501. L RI, S LL, MN CM, 1996. Calcium ionophore-induced secretion from mast cells correlates with myosin light chain phosphorylation by protein kinase C. J Immunol 157: 5130–5138. M K, H RJ, A H, C-M JR, B MA, 1988. Assessment of IgE-receptor function through measurement of hydrolysis of membrane inositol phospholipids. New insights on the phenomena of biphasic antigen concentration-response curves and desensitization. J Immunol 140: 3919–3927. M K, S M, K J, Y M, A H, S E, K T, K J, M T, 1992. The effects of okadaic acid and calyculin A on thrombin induced platelet reaction. Biochem Int 26: 327–334. N M, T H, S M, M K, T I, D K, K T, S H, N M, S S, 1994. Calyculin A and okadaic acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A. Cell Signal 6: 59–71. O K, S Z, K MG, B PM, M H, M JF, B MA, 1993. Ca2+ dependent and Ca2+ -independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells. J Biol Chem 268: 1749–1756. P PT, M MR, 1993. Regulation of human lung mast cell function by phosphatase inhibitors. J Immunol 151: 3808–3816. S N, S H, T C, 1994. Protein phosphatase inhibitors induce the release of serotonin from rat basophilic leukemia cells (RBL-2H3). Biochim Biophys Acta 1221: 291–296. S ES, 1993. The role of mast cells in inflammatory responses in the lung. Crit Rev Immunol 13: 35–70. S DC, A S, A KF, S RL, H A, C JP, W RG, 1985. Homology of the rat

Cell Biology International, Vol. 22, No. 11/12, 1998

basophilic leukemia cell and the rat mucosal mast cell. Proc Natl Acad Sci USA 82: 3871–3875. S A, M T, S L, 1992. Association of the beta isoform of protein kinase C with vimentin filaments. Cell Motil Cytoskel 22: 250–256. T M, M H, E K, 1993. Effect of okadaic acid on histamine release from rat peritoneal mast cells activated by anti-igE. J Pharm Pharmacol 45: 750–752. T K, 1994. Development of the research in the field of histamine release. J Pharm Soc Jpn 114: 147–159. W PD, V ND, 1990. Regulation of norepinephrine secretion in permeabilized PC12 cells by Ca2+ -stimulated phosphorylation. Effects of protein phosphatases and phosphatase inhibitors. J Biol Chem 265: 10,352–10,357. W TR, W SP, 1992. Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C. Br J Pharmacol 105: 627–31.

865

W AK, T A, R JC, D L P, 1991. Okadaic acid, a phosphatase inhibitor, decreases macrophage motility. Am J Physiol 260: L105–L112. W YN, W PD, 1991. Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells. Biochim Biophys Acta 1092: 384–390. Y N, T Y, K Y, W A, I F, 1991. Inhibitory effect of okadaic acid on carbachol-evoked secretion of catecholamines in cultured bovine adrenal medullary cells. Biochem Biophys Res Commun 174: 77–83. Y Y, S M, K J, K T, S T, T K, Y F, S N, 1995. Cytoskeletal reorganization of human platelets induced by the protein phosphatase 1/2A inhibitors okadaic acid and calyculin A. Biochem J 307: 439–449.