INTRACELLULAR CA-2+ POTENTIATES NA+/H+ EXCHANGE AND CELL-DIFFERENTIATION INDUCED BY PHORBOL ESTER IN U937 CELLS

Eur. J. Biochem. 183, 709-714 (1989) 4" FEBS 1989

Intracellular Ca2 potentiates Na /H exchange and cell differentiation induced by phorbol ester in U937 cells +

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Javier ALVAKEZ ',Javicr GARCi A-SANCHO ', Faustino MOLLINEDO' and Ana SANCHEZ I I

Departamcnto de Bioquimica y Biologia Molecular y Fisiologia, Facultad de Medicina, Universidad de Valladolid Centro dc Investigaciones Biolbgicas, Madrid

(Rcccived February 8/April 28. 1989)

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EJB 89 0160

The human cell line U937 differentiates to monocyte macrophage-like cells in response to tumour-promoting phorbol esters. This effect is attributed to activation of protein kinase C. We show here that U937 cell differentiation induced by 12-0-tetradecanoylphorbol 13-acetate (TPA) is associated with cytoplasmic alkalinization. Ethyl-isopropyl-amiloride (EIPA), a potent inhibitor of N a + / H +exchange, blocked both cytoplasmic alkalinization and cell differentiation. Cell acidification by addition of 2 - 4 mM sodium propionate also blocked TPAinduced U937 cell differentiation. These results suggest that a sustained cell alkalinization mediated by activation of N a + / H t exchange is essential for TPA-induced differentiation in U937 cells. The increase of cytoplasmic free calcium concentration ([Ca' 'Ii) by addition of the calcium ionophore ionomycin enhanced TPA-induced alkalinization by increasing the apparent affinity of the Na+/H+ antiporter for intracellular H +. Treatment with ionornycin also potentiated differentiation of U937 cells induced by TPA. This synergism suggests that [Ca2+Ii either potentiates the activation of protein kinase C or triggers additional transducing mechanisms. The key events of this interaction occur during the first 30 min of treatment, even though cell differentiation manifests much later.

The human cell line U937 has the properties of monoblasts or immature monocytes [I]. This cell line is induced to differentiate into more mature monocyte-macrophages by 12-0tetradecanoylphorbol 13-acetate (TPA) 12, 31 as well as by other agents, such as retinoic acid [l, 41, human interferon [ 5 , 61, butyrate [7] and calcitriol ('1,25-dihydroxy-vitamin D3') [8 - 101.The molecular mechanisms underlying U937 differentiation are still unknown. Phorbol esters have been reported to induce differentiation of the promyelocytic HL-60 cell line by enhancing N a + / H + exchange activity [ll]. In addition, it has been shown recently that cell alkalinization by activation of the N a f / H t exchange system is a general response of U937 cells to several differentiating agents 112, 131. The aim of the present study is to examine further the role of the N a + / H + antiporter in TPA-induced U937 cell differentiation. Calcium ions are able to affect both protein kinase C [14] and N a + / H + exchange in some cells [15]. For this reason, the possible implications of intracellular Ca2+ in the regulation of N a + / H exchange activity and U937 cell differentiation have been examined. +

EXPERIMENTAL PROCEDURES Materials RPMI 1640 culture medium, fetal calf serum and L-glutamine were purchased from Flow Laboratories (Irvine, UK). Correspondence to J . Garcia-Sancho, Departamento Bioquimica, Facultad de Medicina, 47005-Valladolid, Spain Abbreviations. BCECF, 2',7'-bis(carboxyethyl)-5(6)-carboxy-fl uorescein; EIPA, ethyl-isopropyl-amiloride; TPA, 12-0-tetradecanoylphorbol 13-acetate; [Ca' +I,, intracellular free calcium concentration; pHi, intracellular pH.

Antibiotics were from Laboratorios Llorente (Madrid, Spain). TPA was purchased from Sigma (St Louis, MO, USA). TPA was dissolved in dimethylsulfoxide at a concentration of 1 mM and stored in aliquots at - 20" C. Ionomycin was purchased from Calbiochem-Behring (San Diego, CA, USA). Nigericin was a gift of Lilly Indiana de Espaiia (Madrid, Spain). Acetoxymethyl esters of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and fura-2 were purchased from Molecular Probes (Eugene, OR, USA). Ethylisopropyl-amiloride (EIPA) was a generous gift of Drs T. Friedrich and B. Burchardt (Max-Planck-Institut fur Biophysik, Frankfurt, FRG). The anti-Mol a subunit (CD1lb) Bear1 monoclonal antibody [16] was a generous gift of Dr J. E. De Vries (Unicet, Dardilly, France). P3X63 myeloma culture supernatant was used as negative control. Cell culture

U937 cells were grown at 37°C in a humidified atmosphere containing 5% COz in RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 50 pg/ml gentamycin. Cell differentiation was induced by adding 100 nM TPA and assessed from cell cluster formation, adhesion to culture plates [17] and Mol antigen expression [18]. pHi and [CaZ+]imeasurements

Intracellular pH was measured fluorometrically using BCECF [19]. Cells were loaded with BCECF by incubating 1-2 x lo6 cells/ml with 5 pM BCECF-acetoxymethyl ester for 45 min at 37°C. The incubation medium was a saline solution containing 140mM NaC1, 1 mM MgC12, 0.2mM

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l n f r a c e l l u l a r pH

Fig. 1 . Effects of' TPA and ionomycin on Na'IH' exchunge activity of U9.17 cel1.y. BCECF-loaded cells were acidified by addition of 15 mM sodium propionatc and the ensuing recovery of the intracellular pH towards the initial lcvel was followed during 10 min. The alkalinization rate, calculated from the slope of pHi recovery and mcasured as ApH/min, is plotted against pHi. Values obtained in control cells (a) and in cells treated with either 2 pM ionomycin (b), 100 nM TPA (c) or ionomycin + TPA (d) are shown. Ionophore or TPA were added 1 min before propionate. In parallel experiments with fura-2-loaded cclls TPA did not modify [Ca2'Ii (resting level about 100 nM) whereas ionomycin produced a sustained increase of [Ca''li to about 900 nM. The initial valuc of pHi was 7.2- 7.3 and the addition of propionate lowered it by about 0.4 within less than 5 s. The results are representative of live similar experiments

CaCI,, 10 mM glucose, 10 mM K/Hepes pH 7.4. BCECFloaded cells were pelleted by centrifugation for 7 min at 300 g and resuspended at 1 - 2 x lo6 cells/ml in the same medium. Intracellular pH was estimated from fluorescence measuremcnts (500-nm excitation; 530-nm emission) in aliquots of the cell suspension incubated at 37 "C under magnetic stirring in a Hitachi 650-10s spectrofluorimeter. The signal was calibrated with nigericin in high-K' medium [20] and corrected for the leak of the dye. Intracellular Ca" concentration was measured using fura-2 as described previously [21, 221. Immun~fluorescenceJlowcytometry A suspension of cells (100 pl at 3 x 106/ml) in the incubation medium described above was mixed with 60 pl of Bear-1 anti-Mol a subunit monoclonal antibody and incubated for 30 min at 4°C. Then, cells were washed twice with phosphate-buffered saline and resuspended in 100 pl fluorescein-isothiocyanate-conjugated goat anti-(mouse IgC) (Kallestad, Austin, TX), previously diluted 1 :40 in phosphate-buffered saline. Subsequently, cells were washed three times and subjected to immunofluorescence flow cytometry in an EPICS-C cytofluorometer (Coulter Scientific, Harpeanden, UK).

RESULTS Activation of Na+/H' exchange by TPA and ionomycin N a + / H + exchange activity was estimated as the rate of cytoplasmic alkalinization of BCECF-loaded cells after instantaneous acidification by addition of 15 mM sodium propionate to the incubation medium [23]. Treatment of the U937 cells with either ionomycin or TPA increased the rate of cytoplasmic alkalinization, and the effect was larger when both drugs were added together (Fig. 1). The alkalinization was

inhibited by more than 90% by removal of extracellular Na' or by addition of 40 pM EIPA, an inhibitor of the Na'/ H' exchange activity [24]. These results indicate that both ionomycin and TPA are able to increase the affinity for internal H + of the Na'/H+ antiporter allowing a faster rate of H + extrusion at a given pHi value. This would result in a shift of the cytoplasmic pH towards more alkaline values. A similar mechanism has been reported for a variety of stimuli in other cells [15]. The fact that the effects of ionomycin and TPA are additive suggests that the pathway for N a + / H + exchange activation may differ for both drugs. Phorbol esters have been shown to increase the activity of the N a f / H + exchanger in several cell lines [25-271. This effect is related to phosphorylation of membrane proteins mediated by protein kinase C [15]. The effects of [Ca'+], on N a + / H Cexchange are unclear [28, 291. In lymphocytes the increase of [Ca'+Ii has been reported to stimulate Na+/H exchange, but this effect was secondary to cell shrinkage due to KC1 loss on activation of Ca2+-dependent K + and CIchannels. Shrinkage would then initiate stimulation of protein kinase(s), followed by activation of Na '/H' exchange and cytoplasmic alkalinization [30]. We tested the possible operation of this mechanism in U937 cells. However, addition of ionomycin did not produce any change of cell volume in U937 cells. This is consistent with a recent report showing that U937 cells lack Ca'+-dependent K + channels (311. I-urthermore, osmotic cell shrinkage induced by the addition of concentrated NaCl solutions did not produce any activation of N a + / H' exchange in U937 cells (data not shown). +

Eflects of T P A and ionomycin on U937 intracellular p H , cell dijfkreri t ia t ion mid M o I antigen exprcssion

Fig. 2A shows the effects of incubating U937 cells in serum-free saline solution with either TPA or ionomycin on intracellular pH. Intracellular pH was monitored for 30 min after the start of the experiment. Both trcatmcnts produced an initial transient cell acidification followed by a more sustained alkalinization. Initial acidification on stimulation has been reported in 'other cells [29, 32, 331, where it was attributed to a stimulation of cell metabolism. The long-lasting cell alkalinization found in U937 cells was to be expected from the effects of ionomycin and TPA on N a + / H +exchange described above (Fig. 1). EIPA inhibited this alkalinization, confirming the involvement of N a + / H + exchange (trace 3 in Fig. 2A). When both drugs were added together, ionomycin potentiated the intracellular alkalinization induced by TPA (cf. traces 1 and 2 in Fig. 2A). This observation is consistent with the effects on N a + / H +exchange described above (Fig. 1). If cells first treated with TPA in serum-free solution during 30 min were then sedimented by centrifugation and resuspended in serum-containing culture medium, cell differentiation, monitored by formation of cell clusters and by cell adhesion to culture plates, was observed after 12- 14 h of incubation at 37°C (cf. a and b in Fig. 2B). This is not surprising since it had been reported previously that the effect of added TPA is not reversed by washing [34]. Interestingly, addition of ionomycin during the 30-min pretreatment with TPA potentiated differentiation of U937 cells after 12 - 14 h of incubation in serum-containing culture medium (cf. b and c in Fig. 2B). Measurements of adherent cells in four experiments gave means (+ SD) of 20 f 4% in cells incubated with TPA alone and 39 4 % in cells incubated with both TPA and ionomycin. The enhancement of TPA-induced cell differentiation by ionomycin was a most surprising outcome since the

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TIME IN MINUTES

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B

CONTROL

T PA

I O N 0 + TPA

Fig. 2.effects of ionornycin and TPA on intracellular p H and cell dgferentiation. (A) Cells were incubated in serum-free saline medium with the following additions: (1) 100 nM TPA; (2) 2 pM ionomycin followed by 100 nM TPA; (3) as 2 except 40 pM EIPA was added 2 min before ionomycin. Intracellular pH was followed for 30 min and the value at this time is given ( 0 ) .(B) After the 30-min treatment described above, the cells were sedimented, resuspended in culture medium containing 10% serum and incubated at 37°C in a humidified atmosphere with 5% COz. Photomicrographs were taken 12 h later. (a) Control cells with n o additions during the 30-min pretreatment; (b) TPA-pretreated cells; (c) cells pretreated with TPA and ionomycin

ionophore is lost on resuspension in the serum-containing medium by binding to the plasma proteins [35, 361. The ionophoric effects are then restricted in time to the first 30 min of incubation. In fact, ionomycin did not potentiate TPAinduced cell differentiation when added directly to the serumcontaining culture medium. This implies that the interactions between TPA and Ca2+ during this early period are responsible for the effects on cell differentiation observed 12- 14 h later. U937 cell differentiation was also monitored by the appearance of the Mol antigen in the plasma membrane. This antigen has been reported to function both as a C3bi receptor (CR3) [37] and a general adhesion molecule [38-411. It is present in mature blood monocytes and granulocytes and its expression is largely increased during phorbol-ester-induced

U937 cell differentiation [18]. The 30-min pretreatment with ionomycin and TPA described above did not increase the expression of Mol antigen as compared with the effect produced by TPA alone. The proportion of Mol-positive cells (mean f SD, n = 4 ) was 3.5+ 1.7% in control cells, 21.4 2.2% in TPA-treated cells and 15.1 0.5% in cells treated with TPA and ionomycin. These results indicate a dissociation between Mol expression and cell adhesiveness and aggregation and suggest that the effects of Ca2+and TPA are not entirely superimposable. This is consistent with the recent observation that anti-Mol antibodies do not effectively inhibit U937 cell aggregation [18]. Values of pHi after a 12-72-h incubation in serum-containing culture medium with 100 nM TPA were 0.26-0.70 higher than those obtained in control cells, not treated with

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T PA + E I PA J -1

w

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LL 0 (L

w m

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3

z w

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a

B

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RELATIVE FLUORESCENCE INTENSITY (log scale)

Fig. 3. ,5ffic.is qf EIPA (40 p M ) on the dq'erentiation of UY37 cells induced by TPA (100 n M j . Cells were incubated for 24 h at 37 'C in serumcontaining culture medium with no other additions (a), 100 nM TPA (b) or 100 n M TPA + 40 pM ETPA (c). Then photomicrographs were taken (A) or Mol antigen expression was examined by immunofluorescence flow cytometry as described in Experimental procedures (B). The dashed line corresponds to staining with the negative control myeloma P3X63 IgG,

TPA (resting pHi, 7.34 0.02; data from six different experiments, not shown). This observation confirms previous reports [12]. Estimations of pHi were performed in these experiments by loading the cells with BCECF and resuspending them in nominally bicarbonate-free saline solution for measurements of fluorescence. This manoeuvre would largely eliminate possible contributions of bicarbonate-dependent systems to the setting of pHi. However, it has been shown previously that pHi values measured under these conditions are representative of the values reached by the U937 cells in the bicarbonate-containing culture medium [I21 (see Discussion). Cell differentiation, monitored either by cell aggregation and adhesion (Fig. 3A) or by Mol antigen expression (Fig. 3B), was inhibited by EIPA. Measurements of the adherent cells in three experiments gave means ( & SD) of 48 f 6% in cells incubated for 24 h with TPA alone and 15 f 2% in cells incubated with TPA and EIPA. These results suggest that the activation of the N a + / H + exchange is closely related to differentiation in U937 cells. Differentiation was also inhibited by cytoplasmic acidification achieved by the addition of 2-4 mM sodium propionate to the culture medium. This

inhibition took place when propionate was added either at the same time as TPA, or 5 h later, or even 9 h later. Similar time courses of inhibition of differentiation were observed when 40 pM EIPA was added to TPA-stimulated cells (not shown). These results suggest that a sustained alkalinization above a threshold pHi is required for differentiation of U937 cells.

DISCUSSION We report here evidence for the involvement of N a + / H C exchange-mediated intracellular alkalinization in differentiation of U937 cells induced by TPA. Both U937 cell alkalinization and differentiation were blocked by amiloride derivatives. It has been shown recently that the presence or the absence of bicarbonate in the incubation medium can make large differences in the pHi responses of renal mesangial cells to growth factors [42]. It could then be argued that, in the present work, pHi measurements were performed in nominally bicarbonate-free medium whereas differentiation

71 3 took place in bicarbonate-containing culture medium. However, it has been shown previously that the presence or absence of bicarbonate does not produce a major modification of the intracellular pH changes obtained in U937 cells and that N a + dependent Cl-/HCOj exchange [43], which exists in these cells, does not play a major role in their differentiation process [12]. It has also been argued that a concentration of EIPA sufficient for blocking N a + / H +exchange (10 pM) was unable to stop differentiation of U937 cells when added to serumcontaining culture medium [13]. Since EIPA is a highly apolar molecule, it could be bound to plasma proteins when added to serum-containing medium. As a consequence concentrations higher than those required in serum-free saline solutions would be needed to inhibit N a f / H exchange. This phenomenon has been shown to happen with several ionophores [35, 361 and it could also be responsible for the failure of ionomycin to potentiate TPA-induced differentiation of U937 cells when added directly to serum-containing medium. On the other hand, we find that an alternative acidifying maneouvre, the addition of sodium propionate, is also able to stop differentiation. According to our results, intracellular alkalinization should be maintained for many hours to allow differentiation, since, even when the addition of propionate or EIPA was delayed 9 h with respect to TPA, differentiation was stopped. I t has, finally, been argued that alkalinization is not necessary for U937 cell differentiation, since very low concentrations of TPA (0.1 nM) were able to produce some differentiation without measurable changes of pHi [13]. These results should be regarded with caution since, given the small fraction of differentiated cells obtained under these conditions, the expected variation of the total fluorescent signal reporting pHi might well fall within the experimental error. The activation of protein kinase C and the subsequent stimulation of the N a + / H + antiporter have been implicated in differentiation of several cell types [ I I , 26, 27, 441. We report here a synergism between C a 2 +and phorbol esters that leads to a stronger activation of N a c / H + exchange as well as to a faster and more effective differentiation of U937 cells. Synergistic effects between [Ca2+Iiand protein kinase C activation, leading to transferrin receptor phosphorylation and down-regulation, have also been reported in HL-60 leukemic cells [45]. Further evidence indicates that the increase of [Ca2+Ii enhances the binding of protein kinase C to the plasma membrane and increases the affinity of phorbol esters for the enzyme, leading to a synergistic and faster activation [46, 471. This mechanism might also explain the synergism between [Ca2'Ii and TPA reported here for U937 cells. Recent evidence indicates that induction of differentiation by TPA may involve different ways of activation of protein kinase C and/or other molecular mechanisms [48 - 511. In this context, the synergistic effect of Ca2 on TPA-induced U937 cell differentiation might be related to one of these alternative pathways. The inability of ionomycin to potentiate TPA-induced expression of Mol antigen parallel to the increase of cell differentiation, as judged from adhesion to culture plates and aggregation, might point in this direction. In any case, the critical interactions between intracellular Ca2+ and TPA took place during the first 30 min of incubation, even though the active state of the N a + / H +exchanger and the cytoplasmic alkalinization are required for at least 9 h and full expression of differentiation requires at least 10 12 h. This observation points out that phenomena happening at early stages, well separated in time from cell differentiation, play a prominent role in predetermining the later pathways of these processes. +

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This work was supported by grants from the Direccion General de Investigacidn Cientifica y Tecnica (PB86-0312) and the Fondo de lnvestigaciones Sanitarias de la Seguridad Social (88jl748).

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