phosphodiesterase and exocytotic secretion from HL60 cells ... The non-differentiated HL60 cell can be stimulated to secrete when Ca2" and guanosine ...
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Biochem. J. (1988) 250, 375-382 (Printed in Great Britain
Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin 0 Jane STUTCHFIELD and Shamshad COCKCROFT Department of Experimental Pathology, School of Medicine, University College London, University Street, London WCIE 6JJ, U.K.
The non-differentiated HL60 cell can be stimulated to secrete when Ca2" and guanosine 5'-[y-thio]triphosphate (GTPyS) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentrationdependence on Ca2" between pCa 8 and pCa 5. The half-maximal requirements for Ca2" for PPI-pde activation and secretion are pCa 6.4 + 0.1 and pCa 6.2 + 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTPyS > guanosine 5'-[/y-imido]triphosphate > guanosine 5'-[y-methylene]triphosphate > XTP ITP, but the maximal response achieved by each compound compared with GTPyS is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTPyS-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTPyS in stimulating secretion, suggesting that the effect of GTPyS cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2" and GTPyS requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (Gp) and the other for activating exocytosis (GE), acting in series. -
INTRODUCTION Guanine nucelotide regulatory proteins are intermediary molecules that transduce information between incoming signals (e.g. hormone receptor) and target proteins (such as adenylate cyclase) [1,2]. One such system involves the activation of polyphosphoinositide phosphodiesterase (PPI-pde) which is responsible for hydrolysing PIP2 (phosphatidylinositol bisphosphate) and possibly PIP (phosphatidylinositol phosphate) [3,4]. The products of hydrolysis, IP3 (inositol 1,4,5-trisphosphate) and DG (diacylglycerol), are intracellular signals involved in mobilizing cytosolic Ca2" and activating protein kinase C respectively [5,6]. PPI-pde activation is coupled to receptors by a putative G protein, Gp [3,4]. Evidence for this includes activation of PPI-pde by stable GTP analogues and their ability to potentiate the effects of receptor-directed agonists in plasma membranes or permeabilized cell preparations [3,4,7-101. A role for guanine nucleotides other than their role in activating PPI-pde has been described recently in the process of exocytosis in a variety of permeabilized cells [1 1-14]. Exocytosis of secretory granules is one important example of cell activation where Ca2+ and DG are
already proposed as the effectors for initiating secretion [15,16]. In neutrophils [11], adrenal chromaffin cells (12] and RINm5F cells [13], it has been shown that GTPyS can stimulate Ca2l-independent secretion, conditions which would not stimulate PPI-pde [11,12,17]. In mast cells, secretion triggered by Ca2" plus GTPyS is resistant to inhibition by neomycin [14], an inhibitor of PPI-pde. We have designated this putative G-protein GE [11]. To differentiate further the effects of guanine nucleotides between PPI-pde activation and exocytosis, we have used the clonal line, HL60 cells. These cells are a human promyelocytic cell line which can be terminally differentiated either into a neutrophil-like or a macrophage-like cell depending on the inducer [18-20]. We demonstrate here that the undifferentiated cell has the intracellular machinery to undergo exocytosis and PPI-pde activation although cell surface receptors for agonists such as f MetLeu-Phe are absent. GTP and its stable analogues and Ca2" buffers were introduced directly into the cytosol by permeabilizing the plasma membrane with streptolysin 0, a bacterial cytolysin [14,21,22]. We are able to demonstrate a dual role for guanine nucleotides, one for PPI-pde activation and the other involved in exocytosis.
Abbreviations used: PPI-pde, polyphosphoinositide phosphodiesterase; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; IP1, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; GTPyS, guanosine 5'-[y-thiojtriphosphate; GppNHp, guanosine 5'-[fly-imido]triphosphate; GppCH2p, guanosine 5'-[fly-methylene]triphosphate; G protein, guanine nucleotide regulatory protein; DG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate.
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METHODS AND MATERIALS Materials Streptolysin 0 was obtained from Wellcome Diagnostics (Dartford, Kent, U.K.). RPMI-1640, Medium 199, glutamine, penicillin and streptomycin were all obtained from Flow Laboratories. Foetal calf serum was obtained from Imperial Laboratories. [3H]Inositol was obtained from either NEN or Amersham. All nucleotides were obtained from Boehringer Mannheim except XTP and dGDP which were obtained from Sigma.
to Medium 199 for the last 18-24 h, which varied from 24 to 36 % [mean 27 % ± 7 (S.D.), n = 20]. In later experiments we supplemented the Medium 199 with bovine serum albumin (3.5 mg/ml) (as a substitute for the foetal calf serum) and we found that secretion was no longer impaired. The formation of inositol phosphates was unaffected by the change in protocol. For the time-course experiments, 100 ,1 aliquots were removed at the indicated times into 1 ml of ice-cold 0.15 M-NaCl buffered at pH 7 with 10 mM-potassium phosphate and the cells processed as indicated above.
Methods Culturing of HL60 cells. HL60 cells were grown in RPMI-1640 supplemented with 15 % heat-inactivated foetal calf serum, L-glutamine (2 mM), streptomycin (50 ,ug/ml) and penicillin (50 i.u./ml). The cells were passaged at starting densities of (0.2-0.3) x 106 cells/ml and maintained in suspension culture in an air/CO2 (19: 1) humidified atmosphere at 37 'C. The cell cultures were diluted every 2-3 days so that the cell density was maintained at (1-2) x 106 cells/ml.
Calcium buffers. Ca2l was buffered with 3 mM-EGTA at concentrations between pCa 8 and pCa 5 and free Mg2+ was maintained at 2 mm. CaEGTA buffers were prepared as described previously [25]. For nominally zero Ca2+, 3 mM-EGTA was used. The maximum error due to varying the concentration of ATP in the range 0-10 mm was GppNHp > GppCH2p > XTP ITP. GTP and dGTP are inactive. From Figs. 3(a) and 3(b), it is clear that the ability of the different nucleotides to stimulate maximal secretion is greater than their ability to stimulate IP3 production. For example, if the maximal secretory response due to GTPyS is expressed as 100 %, then the maximal secretory response to GppNHp is 75% ± 6 (n = 5). In contrast, if IP3 production due to GTPyS is expressed as 100 00, then the maximal secretory response due to GppNHp is only 22 % ± 2 (n = 3). This difference in efficacy is true for all the nucleotides tested. When XTP and ITP were used between 0.01 and 10 mm, secretion was increased in a concentration-dependent manner (Fig. 3a) whilst IP3 increased only up to 3 mM. At 10 mM-XTP and -ITP (and GTP), a decrease in IP3 (and 'P2 and IP1) back to zero-time values was evident (Fig. 3b). We tested whether GDP or dGDP could selectively inhibit secretion or PPI-pde activation. Fig. 4 illustrates the effect of 1 mM-GDP or -dGDP on the concentrationdependence of GTPyS for secretion. Both compounds shift the requirement of secretion for GTPyS to higher concentrations. Fig. 5(b) illustrates the concentrationdependence for dGDP to inhibit secretion induced by two concentrations of GTPyS. The effect of dGDP on IP3 production due to GTPyS is similar to secretion (Fig. Sa). Similar results were obtained for GDP (results not shown). Both dGDP and GDP act as competitive inhibitors of GTPyS-induced IP3 production and secretion.
0.8 F 0.6 [ 0.4 0
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Fig. 3. Effect of GTP and its analogues on (a) /I-glucuronidase secretion and (b) IP3 production from streptolysin-Opermeabilized HL60 cells HL60 cells were incubated in the presence of 1 mMMgATP, 0.4 i.u. of streptolysin 0/ml and pCa 5. *, GTPyS; *, GppNHp; A, GppCH2p; 0, XTP; O, ITP; A, GTP; V, dGTP.
4010
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show a similar dependence on the presence of Ca2l; halfmaximal for secretion is pCa 6.2 + 0.2 (S.D.) (n = 14) and for IP3 production is pCa 6.4+0.1 (S.D.) (n = 6). The ability of a given nucleotide to support secretion was compared with its ability to stimulate PPI-pde activity (Figs. 3a and 3b). In these experiments MgATP was present at 1 mm and Ca2+ at pCa 5. Fig. 3(a) illustrates the concentration-dependence of GTPyS, GppNHp, GppCH2p, XTP, ITP, GTP and dGTP to stimulate secretion. Fig. 3(b) illustrates the concentrationdependence of these compounds to stimulate lP3 formation. dGTP was inactive and the data are not included in Fig. 3(b) for clarity. UTP and CTP were also tested for their ability to stimulate secretion and 1P3 production,
10 0
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t Zero
-8
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Fig. 4. Inhibition by GDP and dGDP of secretion induced by GTPyS HL60 cells were incubated in the presence of 1 mmMgATP, streptolysin 0 and pCa 5. *, control; *, 1 mmGDP; A, 1 mM-dGDP. 1988
Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytosis 3
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Fig. 6. Effect of PMA and PMA plus GTPyS on Ca2l-dependent secretion HL60 cells were permeabilized in the presence of MgATP (1 mM) by streptolysin 0. 0, Control; El, PMA (100 nM); 0, GTPyS (25,UM); *, PMA (100 nM) plus GTPyS (25 /LM).
201A 0
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a
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Fig. 5. Effect of increasing concentrations of dGDP
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formation and (b) secretion HL60 cells were incubated in the presence of MgATP, streptolysin 0 and pCa 5. A, control; *, GTPyS; *, 25 uM-GTPyS.
(a) IP3
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The effect of GTPyS on secretion could be explained by its ability to generate IP3 and diacylglycerol. The function of IP3 to mobilize Ca2" can be disregarded in the present experimental conditions, i.e. in the presence of buffered Ca2". In contrast, DG would function as an activator of protein kinase C. To test this possibility, we compared the effect of PMA (a potent and stable activator of protein kinase C) and GTPyS on secretion (Fig. 6). PMA alone is able to promote some secretion in the presence of Ca2" but it is clearly less effective than GTPyS. When GTPyS and PMA are presented simultaneously, the dependence on Ca2" for secretion is shifted to lower concentrations. Substantial secretion is now evident even in the presence of 3 mM-EGTA, i.e. nominally Ca2"-free (Fig. 6). The effect of PMA on secretion is dependent on the presence of MgATP, which is consistent with PMA acting via protein kinase C activation (results not shown). To exclude further the role of GTPyS as simply being the promoter of DG formation, we studied the requirement for MgATP since it is essential for protein phosphorylation by protein kinase C. Figs. 7(a) and 7(b) illustrate the MgATP dependence for both IP3 production and secretion. The cells were metabolically inhibited to suppress the endogenous ATP pool. In the absence of MgATP, an increase in IP3 due to GTPyS is observed from 280 d.p.m. to 880 d.p.m. at pCa 5. However, Vol. 250
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Fig. 7. Effect of MgATP on (a) IP3 formation and (b) secretion HL60 cells, pretreated with metabolic inhibitors, were permeabilized in a glucose-free medium in the presence of different concentrations of MgATP. *, pCa 7; *, pCa 5; El, pCa 7 plus GTPyS (25 sM); 0, pCa 5 plus GTPyS (25 /uM).
380
J. Stutchfield and S. Cockcroft 60 r
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Fig. 8. Effect of omitting MgATP of pCa 5
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(a) Ca2+-dependence stimulated with GTPyS and (b) GTPyS-dependence in the
presence
(a) HL60 cells, pretreated with metabolic inhibitors, were permeabilized with streptolysin 0 in the presence (closed symbols) or absence (open symbols) of 1 mM-MgATP in glucose-free medium. El, no MgATP; *, 1 mM-MgATP; 0, no MgATP plus GTPyS (25 uM); *, 1 mm-MgATP plus GTPyS (25 /tM). (b) HL60 cells, pretreated with metabolic inhibitors, were permeabilized with streptolysin 0 in the presence (@) or absence (0) of 1 mM-MgATP in a glucose-free medium containing pCa 5 and GTPyS at the concentrations indicated.
addition of MgATP, in a concentration-dependent greatly enhances the production of IP3 (Fig. 7a). The effect of MgATP is most likely due to its ability to maintain the level of PIP and PIP2, but this does not appreciably increase the basal activity. IP3 increases from 220 d.p.m. at no MgATP to 320 d.p.m. at pCa 7 with 10 mM-MgATP. A substantial level of secretion is still observed in the absence of MgATP and this is enhanced by its presence (Fig. 7b). ATP-independent secretion is dependent on both Ca2" and GTPyS (Figs. 8a and 8b). manner,
DISCUSSION The HL60 cell-line, established from the peripheral blood leukocytes of a patient with acute promyelocytic leukaemia, consists predominantly of promyelocytes which can be induced to undergo myeloid differentiation to granulocytes [36,37]. On differentiation with a variety of stimuli such as dimethyl sulnhoxide, retinoic acid or dibutyryl cyclic AMP, these cells acquire most known functions of the neutrophil such as chemotaxis, lysosomal enzyme secretion, phagocytosis, respiratory burst activity and bacterial killing [18,19,34,35,38]. This is accompanied by expression of membrane receptors for the chemotactic peptide fMet-Leu-Phe [18] and also the development of the superoxide-generating system [38]. Although it is known that the undifferentiated cell contains azurophil granules [34], no membrane receptors are present for fMet-Leu-Phe to test the possibility that the HL60 cell may contain an intact secretory mechanism. In this study we report the novel finding that the azurophil granules can be induced to secrete in the undifferentiated HL60 cells when calcium and guanine nucleotides are intro-
duced into the permeabilized cells. This is accompanied by activation of PPI-pde which is known to be regulated by a putative guanine nucleotide binding protein, Gp [3,4]. In order to understand the role of PPI-pde activation and the consequent generation of its products, IP3 and DG, in exocytosis, we have studied the effect of guanine nucleotides on both processes by permeabilizing HL60 cells with streptolysin 0. This agent permits the rapid efflux of soluble proteins without release of secretory material. Maximal secretion only occurs when GTPyS and Ca2l (pCa 5) are provided (Figs. la and lb). Streptolysin 0 has been previously used to permeabilize mast cells [14,21,22] where similar characteristics of lactate dehydrogenase release and histamine secretion have been observed. There is a requirement for Ca2l in GTPyS-stimulated PPI-pde activation and secretion. The half-maximal requirement for secretion and PPI-pde activation are similar; pCa 6.2 and 6.4 respectively. We have previously reported that the Ca2" requirement for PPI-pde in human neutrophils and mast cells is optimally fulfilled at pCa 7 [14,17]. In both those studies, MgATP was omitted and this is known to deplete the levels of PIP and PIP2 [28]. In the present experiments, where MgATP was included, we found that the increase in Ca21 from pCa 8 to pCa 5 led to an increase in IP3 and IP2 by GTPyS. Such calcium-dependency has also been reported for the RINm5F cells [13]. However, it should be noted that, even at pCa 8, there is a response to GTPyS (see Fig. 2) which is only totally suppressed when FGTA is raised to 10 mm (results not shown). In the intact cell, it is possible that the rise in cytosol Ca21 may further potentiate IP3 production to sustain a biphasic response. 1988
Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytosis
It could be argued that activation of PPI-pde is responsible for the secretory response in these cells. It can be inferred that the role of PPI-pde in raising the cytosolic Ca2l must be essential. However, in these experiments Ca2' has been provided in the form of Ca2+ buffers and therefore the role of IP3 can be dismissed. The second product, DG, would be active. The main argument in support of the secretory response being a product of PPI-pde activation is that they show some similar characteristics. They are: (1) a similar Ca2+ requirement, (2) similar rank order of potency of the GTP analogues, (3) identical effects of both GDP and dGDP on GTPyS-induced lP3 production and secretion. However, three separate observations suggest that the effect of GTPyS on secretion cannot be completely explained by PPI-pde activation. These are discussed below. The first hint comes from comparing the nucleotide specificity and efficacy for secretion with that for 1P3 formation. Although the nucleotide specificity for both parameters is identical, it is clear that the efficacy of a given nucleotide is different for the two processes. For example, GppNHp is a better agonist for secretion than for PPI-pde activation. This difference in efficacy is even more apparent when one compares 1P3 production at nucleotide concentrations producing 20 % secretion. Then it is clear that there is no linear relationship between secretion and IP3 production. For example, 20% secretion induced by GppCH2p is hardly accompanied by IP3 production, whilst 20 % secretion induced by GTPyS is accompanied by a doubling of 1P3 production. Such differences in efficacy have also been noted in RINm5F cells [13] and adrenal chromaffin cells [12]. With XTP and ITP, a total dissociation of PPI-pde activation and secretion occurs. At high concentrations of these nucleotides (10 mM) it was found the IP3 production was impaired whilst secretion was unaffected. In separate experiments, Mg salts of XTP and ITP were used and this gave identical results for secretion and 1P3 production (results not shown). This would suggest that the decrease in IP3is due to the interference by XTP and ITP (and GTP) with maintaining the levels of PIP and PIP2 rather than due to changes in Mg2+ concentrations. The second argument that supports the role of a second G-protein in exocytosis is the observation that PMA cannot substitute for GTPyS. Although PMA alone can sustain some secretion (Fig. 6), it cannot mimic it. Thus GTPyS must be doing something more than increasing DG and hence protein kinase C activity. The final argument pertains to the role of MgATP in sustaining PPI-pde activity and secretion. The requirement for MgATP for PPI-pde activation and secretion is not essential. Figs. 7 and 8 demonstrate the effect of MgATP on HL60 cells which had been metabolically inhibited. Under these conditions, the response to fMetLeu-Phe (tested on differentiated cells) is completely abolished (results not shown). The observation that secretion still occurs in the absence of MgATP suggests that a direct role for protein kinase C in controlling secretion is not essential. The role of MgATP is modulatory. It not only enhances secretion but it also decreases the GTPyS and Ca2" requirement to lower concentrations (Figs. 8a and 8b). This kind of modulation by MgATP on the GTPyS and Ca2+ requirement has also been noted for mast cells [14]. Vol. 250
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The reasoning presented above allows us to suggest that in these cells, two G proteins are involved in the stimulus-secretion coupling mechanisms. The first G protein is the putative GP which is the activator of PPIpde and its main function is to generate IP3 and DG. A second G protein, which we have designated GE, must be recruited either as a consequence of receptor activation or by changes that occur after Ca2l and DG signals have been generated. In HL60 cells, secretion is a result of a network of signals which act in co-operation. In Fig. 6, it is clear that a minimum of two signals is sufficient to promote some response. Thus PMA plus pCa 5, GTPyS plus PMA, or GTPyS plus pCa 5 can promote secretion. Only in the case of GTPyS plus pCa 5 does maximal secretion occur, conditions where DG, Ca2" and another unidentified signal can be generated. GTPyS, hence G protein(s), are obviously involved in generating the Ca2l and DG signal and the challenge is to identify the third signal which is clearly under the control of a G protein as well. What does GE control? One possible candidate is a phospholipase A2. GTPyS has been reported to stimulate phospholipase A2 in FRTL-5 cells [29] and rod outer segments of bovine retina [30]. Moreover, phospholipase A2 activation in neutrophils can be stimulated by fluoride and is subject to inhibition by pertussis toxin [31,32]. In none of these studies could the possibility that activation of phospholipase A2 was stimulated independently of PPI-pde be ruled out, since both fluoride and GTPyS are potent activators of the PPI-pde [26]. Arachidonic acid metabolites have recently been suggested to be second messengers in Aplysia neurons [33]. Thus GE may not be confined to secretory systems but may be an integral part of the signalling system for cellular activation. We would like to thank Dr. Blandine Geny for introducing to us the HL60 cells. This work was financed by a grant from the MRC and the Lister Institute. S.C. is a Lister Institute
Fellow.
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Received 31 July 1987/21 September 1987; accepted 28 October 1987
1988
