JOURNAL OF CELLULAR PHYSIOLOGY 151:361-366 (1992)
Na+/H+ Antiporter Gene Expression Increases During Retinoic Acid-Induced Granulocytic Differentiation of HL60 Cells C A D I P A R T H I N. RAO,* CLAUDE SARDET, JACQUES POUYSSECUR, BRADFORD C. BERK
AND
Cardiology Division, Emory University School of Medicine, Atlanta, Georgia 30322 (G.N.R., B.C.B.1, and Centre de Biochirriie, Centre Ndtioridl de /a Recherche Srientifique, Universitt;de Nice, 06034, Nice, France (C.S., 1.P.) Uuring differentiation of human leukemic HL60 cells into granulocytes, sustained increases in intracellular pH and Na+/H+ antiporter activity have been observed. In the present study we report that retinoic acid (RA)-induced granulocytic differentiation ol' HL60 cells causes an -18-Cold increase in the steady-state rnRNA levels for the Na+/H+ antiporter. This was due to an increase in the rate of Naf/H+ antiporter gene transcription as measured by nuclear run-on analysis. Antiporter protein synthesis increased by seven-fold during RA-induced granulocytic differentiation of HL60 cells as measured by irnmunoprecipitation of j5Smethionine-labeled proteins with the RPI -c28 Na+/H+ antiporter antibody. N o increase in antiporter rnRNA was observed in response to etretinate, an analogue of retinoic acid, which did not induce differentiation. Thus, N a t / H ' antiporter gene expression is associated with RA-induced granulocytic differentiation of HL60 cells. The present findings and our previous data (Rao et al., 1991) dcmonstrate that Na ' /H ' antiporter gene expression is a generalized feature of HLhO cell differentiation. ic; I Y W WIIPY LISS, Inr.
The Naf/HC antiporter has been described by Sardet et al. (1990) as a 100 kD plasma membrane protein that is present in virtually all mammalian cells (Boron and Boulpaep, 1983; Grinstein et al., 1989; Mahnensmith and Aronson, 1985).It regulates intracellular pH (pH,) by mediating a one-to-one exchange of intracellular Hf and extracellular Nat (Boron and Boulpaep, 1983; Grinstein et al., 1989; Mahnensmith and Aronson, 1985; Sardet et al., 1990).An important role for the Naf/Hf antiporter has been shown in early signal transduction events required for growth factor-induced mitogenesis (Burns and Rozengurt, 1983; Cassel et al., 1983; Moolenaar, 1986; Paris and Pouyssegur, 1984). The antiporter also appears to be important in the control of cellular proliferation: mutant cells showing no Na+/Hf antiporter activity fail to undergo cell division at neutral and acidic pH in HCO ,-free medium (Pouyssegur et al., 19841, and transformed cells have higher pH, values than the respective nontransformed cells (Doppler et al., 1987; Hagag e t al., 1987; Ober and Pardee, 1987). However, the normal physiological role of the antiporter in the regulation of cell pH is not clear. In the presence of HCO-, there is little change in pH, in response to growth factors (Aickin and Thomas, 1977; Russell and Boron, 1976), and antiporter-deficient mutants grow well (Agarwal et al., 1986; Pouyssegur et al., 1984). We postulated that the antiporter may play a role in long-term cellular responses such as differentiation, based on the findings that Nat/Hf antiporter activity increases during differentiation of human promyelo-
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0 1992 WILEY-LISS, INC.
cytic HL60 cells (Besterman et al., 1985; Costa-Casnellie et al., 1988; Hazav et al., 1989; Ladoux et al., 1987; Restrepo et al., 1987). We chose HL60 cells for our in vitro model for cellular differentiation because they can be induced to differentiate into either granulocyte or macrophage-like cells by treatment with retinoic acid (RA) or phorbol12-myristate 13-acetate (PMA), respectively (Collins et al., 1978; Lotem and Sachs, 1979; Rovera et al., 1979). Using these cells, we have previously reported that PMA-induced monocytic differentiation of HL60 cells was associated with large increases in Na+/Hf antiporter mRNA and protein expression (Rao et al., 1991). Here we report that the steady-state levels of antiporter mRNA and protein also increase 18-fold and 7-fold7respectively, in response to RA-induced granulocytic differentiation of HL60 cells. These increases are accounted for by a n increase in the rate of antiporter gene transcription. These findings demonstrate that Na+/Hf antiporter expression is a generalized feature of HL60 cell differentiation.
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MATERIALS AND METHODS Cell culture HL60 cells (kindly provided by Dr. Diego Restrepo, Monell Chemical Senses Center, Philadelphia, PA)
Received September 16,1991; accepted December 23,1991.
*To whom reprint requests/correspondence should be addressed.
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were grown in RPMI 1640 medium supplemented with 10% (vivj heat-inactivated fetal bovine serum, 100 unitsiml penicillin, and 100 pg/ml stretomycin. The cultures were maintained in humidified 95% air-5% C 0 2 at 37°C by passage of 1-2 x lo5 cells/ml every other day. Cell viability was tested by trypan blue exclusion assay. To induce differentiation, HL60 cells were treated with retinoic acid (RA) (1pM) for various times. The extent of differentiation was measured by the ability of cells to reduce nitroblue tetrazolium salt (NBT). The NBT reduction assay was performed as described (Davies et al., 1985; Li et al., 1973). RNA blot analysis HL60 cells were treated with RA (1pM) for varying times and total cellular RNA was isolated from all cells (approximately 8 x 1O7 cells for each time point) by the guanidinium isothiocyanate-cesium chloride protocol of Chirgwin et al. (1979). Poly A' RNA was selected by passing total cellular RNA in high salt containing 20 mM Tris-HC1 buffer (pH 7.5) through a n oligo(dT1-cellulose column and eluting it with salt-free 20 mM TrisHC1 buffer (pH 7.5) as described earlier by Aviv and Leder (1972). Poly A+ RNA (10 pg) from each time point was size fractionated by electrophoresis on 1% agarose, 2% formaldehyde gel. After transfer to a Nytran membrane as described by Thomas (1980), the RNA was crosslinked to the membrane using UV irradiation (Stratalinker, Stratagene, La Jolla, CA). After 4 h prehybridization in 50% (viv) formamide, 5X SSC (1X SSC = 0.15 M NaC1, 0.015 M sodium citrate), 5X Denhardt's (1X Denhardt's = 0.02% (wiv) each of Ficoll, polyvinyl pyrolidonc and bovine serum albumin), 50 mM sodium phosphate (pH 6.51, and 250 pgiml of sheared salmon sperm DNA a t 42"C, the Nytran membrane was hybridized in the above solution containing 10% (wiv) dextran sulfate and 1 x lo6 c p d m l of cDNA probe for 16 h at 42°C. The cDNA probes-Na+/H+ antiporter cDNA (Bam H1 fragment, bp 731-2646 of human cDNA, clone c28, a s described by Sardet et al., 1989) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA (a full length rat cDNA, clone pRGAPD 13, Fort et al., 1985)-were radiolabeled using a GIBCOiBRL random primers labeling kit as per manufacturer's protocol with lw3'P1dCTP (specific activity 3,000 Ciimmole, DuPont-New England Nuclear). After hybridization, the Nytran membrane was washed three times in 2X SSC, 0.2% SDS (15 min, 2 5 T ) and twice in 0.1X SSC, 0.110 SDS (15 min, 60°C). The membrane was then exposed to Kodak X-Omat AR x-ray film with a n intensifying screen a t -70°C for 16 h. Densitometric analysis of the autoradiograms exposed in the linear range of film density was made on a Pharmacia Ultroscan XL laser densitometer. Nuclear run-on assay Nuclei from uninduced and induced HL60 cells (approximately 2 x 10' cells) were prepared by the technique of Groudine et al. (1981). Run-on transcription was carried out at 30°C for 30 min in a reaction mixture consisting of 210 pI nuclear suspension (3-5 x lo7 nuclei in 40% (viv) glycerol, 50 mM Tris-HC1, pH 8.3, 5 mM MgCl,, and 0.1 M EDTA), 60 p15X nuclear run-on buffer (12.5 mM MgCl,, 750 mM KC1, 1.25 mM each
100
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1
1
201 /d o v . 0
I
24
,
I
40
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72
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96
.
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120
.
144
Time, hr Fig. 1. Time course for RA-induced granulocytic differentiation of HL60 cells. HL60 cells were treated with 1FM RA for indicated times, and differentiation was measured as percentage of cells positive for NBT assay.
ATP, GTP and CTP, 25 mM Tris-HC1 (pH 8.0)), and 50 pl [a-32PlUTP (500 pCi, specific activity 3,000 Cii mmole, DuPont-New England Nuclear). Reactions were terminated by the addition of 8 pl of 11 mgiml RNase-free DNase I and incubation a t 30°C for 5 min. "P-labeled RNA was purified a s described by Linial et al. (1985). Plasmids containing cDNAs for the Na+/H+antiporter and GAPDH were linearized, denatured (0.2 M NaOH for 30 min a t 25"C), neutralized with 6X SSC (10 volumes) and applied to a Nytran membrane (10 pgislot) using a slot blot apparatus. After prehybridization for 3 h in 100 mM TES-HCI buffer (pH 7.41, 0.2% SDS, 10 mM EDTA, 0.3 M NaC1, 1X Denhardt's, and 250 pg/ml Escherichia coZi tRNA, the membrane was hybridized in the above solution containing 1 x lo7 cpmiml of 32P-labeled nuclear RNA transcripts for 48 h at 65°C. Membranes were washed twice in 2X SSC, 0.1% SDS (15 min, 25"C), and twice in 0.1X SSC, 0.1% SDS (60 min, 60°C).
Immunoprecipitation analysis HL60 cells were metabolically labeled for 24 h using ["Slmethionine (200 pCi/ml) -+ 1 pM RA (total treatment time = 72 h) and membranes were isolated as described by Sardet et al. (1990). Equal amounts of membrane proteins from uninduced and induced HL60 cells were immunoprecipitated using affinity-purified antiNaf/H+ antiporter antibodies (RPl-c28).The immunoprecipitated proteins were separated on SDS-7.5% PAGE under reducing conditions described by Sardet et al. (1990) and Laemmli (1970) and subjected to autoradiography.
RESULTS Retinoic acid caused a time-dependent differentiation of HL60 cells into granulocytes (Fig. l ) . About 2-3% of untreated cells were NBT positive, a marker for granulocytic differentiation. This number did not vary significantly in cells treated with RA for up to 12 h. Treatment for 24 h, however, caused differentiation
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Na ' iH+ ANTIPORTER AND HMO CELL DIFFERENTIATION
+GAPDH Fig. 2. Steady-state levels of Na'lH' antiporter and GAPDH mRNA during RA-induced granulocyt,ic differentiation of HL60 cells. Equal amounts of poly A t RNA (10pg/lane)from RA-treated and untreated HL60 cells were analyzed by Northern blotting for steady-state levels of Na'iH ' antiporter and GAPDH mRNA using the respective "P-laheled cDNA probes.
in - 14%of the cells. Following 6 days of RA treatment, a maximum of - 90% of cells had differentiated. In differentiated granulocytic HL60 cells (Ladoux et al., 1987; Restrepo et a]., 1987) and normal circulating granulocytes (Grinstein and Furuya, 19861, the Na+/H+ antiporter plays a critical role in the recovery of pH, following acidosis and cell activation (PMA, fMLP). To understand further the role of the Na+/H+ antiporter during HL60 cell differentiation at the molecular level, cells were treated with RA, and poly A + RNA was isolated a t varying times following induction. Equal amounts of poly A t RNA (10 pg) at each time point were analyzed by Northern blotting using a cloned 32P-labeled human Na+/H+ antiporter cDNA. No significant change was observed in Na'lH+ antiporter mRNA levels after 3 h treatment with RA (Fig. 2 upper panel; Table 1). However, after 6 h RA treatment, the steady-state mRNA levels for the antiporter increased by eightfold. Na+/H+ antiporter mRNA levels steadily increased, reaching a maximum after 24 h RA treatment (- 18-fold increase). Treatment with RA for up to 72 h did not produce any further increases in the Na'/H+ antiporter mRNA levels. To prove that induction of Na+/Ht antiporter mRNA by RA was not due t o a global increase in the RNA synthesis, two experiments were performed. First, total RNA
TABLE 1. Na+/H+ antiporter steady-state mRNA levels and transcriptional rates in HMO cells during retinoic acid-induced granulocytic differentiation Treatment (Hours) 3 6 24 72
Fold increase in mRNA levels
Fold increase in transcriptional rate
0.4
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8.0 17.6 18.4
8.3
synthesis, as measured by 3H-uridine uptake into trichloroacetic acid-precipitable material decreased by 20% in RA-treated cells (60,800 2 5,200 vs 49,000 2 3,050 cpm). Second, the levels of GAPDH mRNA, a glycolytic enzyme that is expressed constitutively in most cells, were not significantly different in RAtreated and untreated cells (Fig. 2 lower panel). Together these findings indicate that the induction of Na+/H+ antiporter mRNA by RA in HL60 cells is specific. To prove that the induction of Na'/H' antiporter RNA was due to induction of differentiation, rather than to a direct effect of retinoids on the antiporter gene, HL60 cells were treated with etretinate, a synthetic analogue of RA that does not induce differentiation (Ladoux et al., 1987).In contrast to RA, etretinate
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+ Vector + GAPDH Na/H
Fig. 4. Transcription rates of Na ' /H antiporter and GAPDH genes during RA-induced granulocytic differentiation of HL60 cells. '"Plabeled RNA transcripts were isolated from nuclei of RA-treated and untreated HL60 cells and hybridized to filter-immobillzed Na /Hi antiporter cDNA and GAPDH cDNA.
To analyze whether increases in Na ' /H antiporter mRNA levels in RA-treated HL60 cells were also reflected a t the protein level, cells were metabolically labeled with [35Slmethionine and membranes were isolated. Equal amounts of membrane proteins from untreated and RA-treated HL60 cells were immunoprecipitated with affinity-purified anti-Na+/H antiporter antibodies \RPl-c28) (Sardet et al., 1990), the immunoprecipitates were separated by SDS-PAGE under reducing conditions, and proteins were visualized by autoradiography. The Na+/H+antiporter can be identified by its apparent molecular size (- 100 kD) and phosphorylation as described earlier (Rao et al., 1991). sevenfold increase in the As shown in Figure 5 , a intensity of the Na+/H+ antiporter band was observed in RA-treated (72 h) HL60 cells as compared to untreated cells. This finding is consistent with the steadystate levels of antiporter mRNA. +
+GAPDH Fig. 3. Effect of etretinate on the steady-state levels of Na-IH' antiporter and GAPDH mRNA. HL60 cells were treated with either RA (1 p,M) or etretinate (1 +M), and poly A+ RNA was isolated. Equal amounts of poly A+ RNA from agonist-treated and untreated HL60 cells were analyzed for Na ' /H ' antiporter and GAPDH mRNA by Northern blotting as described in Figure 2.
did not increase Na+/H ' antiporter mRNA levels in HL60 cells (Fig. 3). This result indicates that the induction of Na+/H+antiporter mRNA by RA in HL60 cells is closely associated with differentiation. Further, since near maximal increases in the steady-state mRNA levels for the Na+iH+ antiporter (- 18-fold at 24 h) were reached before a significant population of cells had differentiated (- 14% a t 24 h), induction of the Na'/H+ antiporter gene precedes the HL60 cell differentiation. To determine whether RA-induced increases in antiporter mRNA levels were due to increased gene transcription, we next performed nuclear run-on assays in HL60 cell nuclei treated with RA. As shown in Figure 4 and Table 1, treatment with RA for 72 h caused a n eightfold increase in Na+/H+ antiporter transcription. In contrast, the rate of GAPDH transcription showed a small decrease in RA-treated cells consistent with the generalized decrease in total RNA synthesis. Although a n eightfold increase in the Na+/H+ antiporter transcription rate may account for the 18-fold increase in its steady-state mRNA levels over time, posttranscriptional mechanisms (such as message stabilization) cannot be ruled out. Overall, nuclear run-on results clearly show that during RA-induced granulocytic differentiation of HL60 cells, transcription of Na+/H+ antiporter gene increases significantly.
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DISCUSSION Ladoux et al. (1987) have reported increases in pH, and Na+/H+ antiporter activity in HL60 cells during RA-induced granulocytic differentiation. The increase in Na+/H+ antiporter activity preceded the onset of differentiation and persisted throughout the course of differentiation. The present findings strongly suggest that these increases in activity are due to induction of antiporter gene expression. The fact that etretinate, a retinoid that did not cause differentiation, failed to induce antiporter gene expression indicates that induction of the antiporter gene is tightly associated with differentiation of HL60 cells. We now have strong evidence from the present study and our previous work with PMA-induced monocytic differentiation that induction of the N a t / H ' antiporter is closely associated with HL60 cell myeloid differentiation. These data are: (1)the increase in antiporter expression temporally preceded differentiation induced by either RA (present study) or PMA (Rao et al., 1991), (2) agents that mimic RA and PMA (etretinate and l-oleolyl-2-acetylglycerol,respectively) in activating certain signal transduction pathways but that fail to induce differentiation, also do not induce antiporter expression, and (3) inhibition of differentiation by cyclo-
Na ' /H ' ANTIPORTER AND HL60 CELL DIFFERENTIATION
i i
110 kD Na/H
47 kD
Fig. 5. Na ' /H antiporter protein synthesis during RA-induced granulocytic differentiation of HL60 cells. The molecular size markers were phosphorylase b ( 1 10 kDat and ovalbumin 147 kDa).
heximide prevented antiporter induction (Rao et al., 1991). Since induction of the Na +/H+antiporter gene was observed during both PMA-induced monocytic and RA-induced granulocytic differentiation of HL60 cells, it appears that induction of the Na+/H+antiporter gene is a common feature of myeloid differentiation. Future studies with other model systems for differentiation (e.g., PC12 cell line) will address the question of whether induction of the Na+/H+ antiporter gene is a common feature of the differentiation process or restricted to myeloid differentiation. The marked increases in Na+/H+ antiporter expression during differentiation of HL60 cells into monocytes or granulocytes suggest a n important physiological function. The most compelling reason for leukocytes to express more antiporter is that during the respiratory burst activated by bacteria or chemotactic peptides, there is a n intense cyctoplasmic acidification (Grinstein and Furuya, 1984, 1986). In response to PMA stimulation of the respiratory burst, both neutrophils (Grinstein and Furuya, 1984, 1986) and DMSOdifferentiated HL60 cells (Restrepo et al., 1987) exhibit a n amiloride-sensitive acid recovery mediated by the Na '/H+ antiporter. Despite agreement on the role of the antiporter in pH, recovery in response to PMA, there are conflicting data regarding the role of pH, in HL60 cell differentiation and the mechanisms by which antiporter activity is regulated. Ladoux et al. (1987, 1989) reported a n increase in both pH, and Na+/H+ antiporter activity (measured in the absence of HCO-,) during DMSO and RA-induced granulocytic differentiation of HL60 cells. This group observed a 1.7-fold in-
365
crease in antiporter V,, in RA-differentiated HL60 cells (Ladoux et al., 1987). In contrast, studies of DMSO-differentiated HL60 cells have shown no change in pHi when measured in the presence of HCOp3 (Restrepo et al., 1988) and no increase in the antiporter V,, (Costa-Casnellie et al., 1988). The only difference in antiporter regulation observed by the latter group in DMSO-differentiated HL60 cells was a decrease in K, for Na'. However, Restrepo et al. (1987) observed a ninefold increase in the rate of amiloride-sensitive H' efflux following PMA stimulation in DMSO-differentiated HL60 cells compared to undifferentiated cells. In contrast, Ladoux et al. (1989) and our preliminary results (Rao and Berk, unpublished observations) show no PMA-induced pH, response in RA-induced HL60 cells. These data suggest that there are important differences in the effects of PMA on antiporter activity in RA-differentiated as compared to DMSO-differentiated HL60 cells. The data in the present study suggest that the values do not accurately for pH, and Na + /H+ antiporter V,, reflect the level of antiporter protein expression. For example, Ladoux et al. (1987) observed a 1.7-fold increase in antiporter V,,. However, we found at least a sevenfold increase in antiporter protein expression and a n 18-fold increase in its mRNA levels. We propose four possible mechanisms to account for this disparity in findings. First, i t is possible that there are "spare" antiporters that are not physically associated with the plasma membrane but are available for insertion during cellular acidification. Second, maximal activation of the antiporters may not occur in response to acid loading alone as employed by Ladoux et al. (1987). This could occur if, e.g., activation occurred by several mechanisms such as phosphorylation (Sardet et al., 1990) and binding of a regulatory protein (Morel1et al., 1990), or if inactivation (e.g., dephosphorylation) was more rapid in response to acid loading compared to the oxidative burst. The findings of the ninefold increase in Na+/H+antiporter activity in differentiated HL60 cells in response to PMA-induced oxidative burst (Restrepo et al., 1987) would support such a mechanism. Third, antiporters may be present in the cell but functionally inactive due to association with a negative regulatory protein. Fourth, if HL60 cells are capable of expressing more than one Na+/H+ antiporter isoform (Sardet et al., 19911, it is possible that the increase in mRNA and protein may be due in part t o a n isoform that has a relatively low V,,. Future experiments will be required to characterize isoform expression, posttranslational regulatory mechanisms, and the functional role of antiporter induction in HL60 cell differentiation.
LITERATURE CITED Agarwal, N., Haggerty, H., Adelherg, E. and Slayman, C. (1986) Isolation and characterization of a Na-H antiporter-deficient mutant of LLC-PKl Cells. Am. J. Physiol., 251tC825-C830. Aickin, C.C., and Thomas, R.C. (1977) An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibres. J. Physiol. (Land), 273:295-316. Aviv, H., and Leder, P. (1972) Purification of biologically active globin messenger KNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA, 69:1408-1412.
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Besterman, J.M., May J r , W.S., LeVine 111, H., Cragoe Jr, E.J., and Cuatrecasas, P. (1985) Amiloride inhibits phorbol ester-stimulated Na+iH+ exchange and protein kinase C. J. Biol. Chem., 260:11551159. Boron, W.F., and Boulpaep, E.L. 119831 Intracellular pH regulation in the renal proximal tuhule of the salamander. Na-H exchange. J. Gen. Physiol., 81:29-52. Burns, C.P., and Rozengurt, E. (1983)Serum, platelet-derived growth factor, vasopressin and phorbol esters increase intracellular pH in Swiss 3T3 cells. Biochem. Biophys. Res. Commun. 1l6:931-938. Cassel, D., Rothenberg, P., Zhuang, Y.X., Deuel, T.F., and Glaser, L. (1983)Platelet-derived a o w t h factor stimulates Na+iH+ exchange and induces cytoplasmmc alkalinization in NR6 cells. Proc. Nail. Acad. Sci. USA, 80t6224-6228. Chirrwin, J.M.. Prubvla, A.E., MacDonald. R.J., and Rutter. W.J. (1679) Isolation ofhi”ological1yactive ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18:5294-5299. Collins, S.J., Ruscetti, F.W., Gallagher, R.E., and Gallo, R.C. (1978) Terminal differentiation of human Dromvelocvtic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc. Natl. Acad. Sci. USA, 752458-2462. Costa-Casnellie, M.R.. Segel, G.B.. and Lichtman. M.A. (1988) The NaT:H+ exchanger in ymmature and mature granulocytic HL-60 cells. Property changes induced by intracellular acidification and cell maturation. J. Biol. Chem., 263t11851-11855. Davies, P.J., Murtaugh, M.P., Moore dr., W.T., Johnson, G.S., and Lucas, D. (1985)Retinoic acid-induced expression oftissue transglutaminase in human promyelocytic leukemia (HL-60) cells. J . Biol. Chem., 2605166-5174. Doppler, W., Jaggi, R., and Groner, B. (1987) Induction of v-mos and activated Ha-ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression. Gene, 54r747-153. Fort, Ph., Marty, L., Piechaczyk, M., el-Sabrouty, S., Dani, C., Jeauteur, P., and Blanchard, J.M. (1985) Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3phosphate-dehydrogenase multigenic family. Nucleic Acids Res., 13t1431-1442. Grinstein, S.. and Furuva, W. (1984) Amiloride-sensitive Na+/H+ exchange in human neutrophils: mechanism of activation by chemotactic factors. Biochem. Biophys. Res. Commun., 122:755-762. Grinstein, S., and Furuya, W. (1986) Cytoplasmic pH regulation in phorbol ester-activated human neutrophils. Am. J. Physiol., 251: C55-65. Grinstein, S., Rotin, D., and Mason, M.J. (1989) Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim. Biophys. Acta, 988:73-97. Groudine, M., Peretz, M., and Weintraub, H. (1981)Transcriptional regulation of hemoglobin switching in chicken emhryos. Mol. Cell Biol., 1281-288. Hagag, N., Lacal, J.C., Graber, M., Aaronson, S., and Viola, M.V. (19871Microinjection ofras p21 induces a rapid rise in intracellular pH. Mol. Cell Biol. 7:1984-1988. Hazav, P., Shany, S., Moran, A,, and Levy, R. (1989) Involvement of intracellularpH elevation in the effect ofl,25-dihydroxyvitamin D3 on HTA0 cells. Cancer Res., 49t72-75. Ladoux, A,, Cragoe Jr., E.J., Geny, B., Ahita, J.P., and Frelin, C. (1987)Differentiation of human promyelocytic HL 60 cells by retinoic acid is accompanied by an increase in the intracellular pH. The role of the Na+/H+ exchange system. J. Biol. Chem.,262:811-816. Ladoux, A., Krawice, I., Damais, C., and Frelin, C. (1989) Phorbol
esters and chemotactic factors induce distinct changes in cvtodasmic Ca2 and pH in granulocytic-like HL60 cells. Biichim. Bioihys. Acta. ~~~. > - 10 - - - 1.?:55-59 ~-
Laenimli, U.K. (1970) Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature, 227,680-685. Li, C.Y., Lam, K.W., and Yam, L.T. (1973) Esterases in human leukocytes. J. Histochem. Cytochem., 21:1-12. Linial, M., Gunderson, N., and Groudine, M. (198.5)Enhanced transcription of c-myc in bursa1 lymphoma cells requires continuous protein synthesis. Science, 230:1126-1132. Lotem, J., and Sachs, 1,. (1979) Regulation of normal differentiation in niouse and human niyeloid leukemic cells by phorbol esters and the mechanism of tumor promotion. Proc. Natl. Acad. Sci. USA, 76: 5158-5162. Mahnensniith, R.L., and Aronson, P.S. (19853The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ. Res. 56t773-788. Moolenaar, W.H. (1986)Effects of growth factors on intracellular pH regulation. Annu. Rev. Physiol., 48t363-376. Morel], G . , Stepock, D., Shenolikar, S., and Weinman, E.J. (19901 Identification of a putative Na’iH exchanger regulatory cofactor in rabbit renal BBM. Am. J. Physiol., 259:F867-F871. Ober, S.S.,and Pardee, A.B. (19873Intracellular pH is increased after transformation of Chinese hamster embryo fibroblasts. Proc. Natl. Acad. Sci. USA, 84.2766-2770. Paris, S., and Pouyssegur, J . (1984) Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H + . J. Biol. Chem., 259:10989-10994. Pouyssegur, J., Sardet, C., Franchi, A,, L’Allemain, G., and Paris, S. (1984) A specific mutation abolishing N a i ~ i H +antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Natl. Acad. Sci. USA, 81 t48334837. Rao, G.N., deRoux, N., Sardet, C., Pouyssegur, J., and Berk, B.C. (1991) N a ’ /H ’ antiporter gene expression during monocytic differentiation ofHL60 cells. J . Biol. Chem., 26613485-13488. Restrepo, I]., Kozody, D.J., and Knauf, P.A. (1987) Changes in Na ’ /H ’ exchange regulation upon granulocytic differentiation of HL60 cells. Am. J . Physiol., 253:C619-C624. Restrepo, D., Kozody, D.J., Spinelli, L.J., and Knauf, P.A. (1988) pH homeostasis in promyelocytic leukemic HL60 cells. J. Gen. Physiol, 92t489-507. Rovera, G., Santoli, D., and Damsky, C. (1979) Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc. Natl. Acad. Sci. USA, 76: 2779-2783. Russell, J.M., and Boron, W.F. (1976) Role of choloride transport in regulation of intracellular pH. Nature, 264:73-74. Sardet, C., Franchi, A., and Pouyssegur, J. (1989)Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+iH+ antiporter. Cell, !ifi:271-280. Sardet, C., Counillon, L., Franchi, A ,, and Pouyssegur, J. (1990) Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD. Science, 247t723-726. Sardet, C., Fafournoux, P., and Pouyssegur, J . (1991) a-thrombin, epidermal growth factor and okadaic acid activate the Na ‘/H’ cxchancer. NHE-1. hv uhosuhorvlatine a set of common sites. J . Biol. C h e i . , 266:19166~19171: Thomas, P.S. (1980)Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA. 773201-5205. +
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Na+/H+ Antiporter Gene Expression Increases During Retinoic Acid-Induced Granulocytic Differentiation of HL60 Cells C A D I P A R T H I N. RAO,* CLAUDE SARDET, JACQUES POUYSSECUR, BRADFORD C. BERK
AND
Cardiology Division, Emory University School of Medicine, Atlanta, Georgia 30322 (G.N.R., B.C.B.1, and Centre de Biochirriie, Centre Ndtioridl de /a Recherche Srientifique, Universitt;de Nice, 06034, Nice, France (C.S., 1.P.) Uuring differentiation of human leukemic HL60 cells into granulocytes, sustained increases in intracellular pH and Na+/H+ antiporter activity have been observed. In the present study we report that retinoic acid (RA)-induced granulocytic differentiation ol' HL60 cells causes an -18-Cold increase in the steady-state rnRNA levels for the Na+/H+ antiporter. This was due to an increase in the rate of Naf/H+ antiporter gene transcription as measured by nuclear run-on analysis. Antiporter protein synthesis increased by seven-fold during RA-induced granulocytic differentiation of HL60 cells as measured by irnmunoprecipitation of j5Smethionine-labeled proteins with the RPI -c28 Na+/H+ antiporter antibody. N o increase in antiporter rnRNA was observed in response to etretinate, an analogue of retinoic acid, which did not induce differentiation. Thus, N a t / H ' antiporter gene expression is associated with RA-induced granulocytic differentiation of HL60 cells. The present findings and our previous data (Rao et al., 1991) dcmonstrate that Na ' /H ' antiporter gene expression is a generalized feature of HLhO cell differentiation. ic; I Y W WIIPY LISS, Inr.
The Naf/HC antiporter has been described by Sardet et al. (1990) as a 100 kD plasma membrane protein that is present in virtually all mammalian cells (Boron and Boulpaep, 1983; Grinstein et al., 1989; Mahnensmith and Aronson, 1985).It regulates intracellular pH (pH,) by mediating a one-to-one exchange of intracellular Hf and extracellular Nat (Boron and Boulpaep, 1983; Grinstein et al., 1989; Mahnensmith and Aronson, 1985; Sardet et al., 1990).An important role for the Naf/Hf antiporter has been shown in early signal transduction events required for growth factor-induced mitogenesis (Burns and Rozengurt, 1983; Cassel et al., 1983; Moolenaar, 1986; Paris and Pouyssegur, 1984). The antiporter also appears to be important in the control of cellular proliferation: mutant cells showing no Na+/Hf antiporter activity fail to undergo cell division at neutral and acidic pH in HCO ,-free medium (Pouyssegur et al., 19841, and transformed cells have higher pH, values than the respective nontransformed cells (Doppler et al., 1987; Hagag e t al., 1987; Ober and Pardee, 1987). However, the normal physiological role of the antiporter in the regulation of cell pH is not clear. In the presence of HCO-, there is little change in pH, in response to growth factors (Aickin and Thomas, 1977; Russell and Boron, 1976), and antiporter-deficient mutants grow well (Agarwal et al., 1986; Pouyssegur et al., 1984). We postulated that the antiporter may play a role in long-term cellular responses such as differentiation, based on the findings that Nat/Hf antiporter activity increases during differentiation of human promyelo-
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0 1992 WILEY-LISS, INC.
cytic HL60 cells (Besterman et al., 1985; Costa-Casnellie et al., 1988; Hazav et al., 1989; Ladoux et al., 1987; Restrepo et al., 1987). We chose HL60 cells for our in vitro model for cellular differentiation because they can be induced to differentiate into either granulocyte or macrophage-like cells by treatment with retinoic acid (RA) or phorbol12-myristate 13-acetate (PMA), respectively (Collins et al., 1978; Lotem and Sachs, 1979; Rovera et al., 1979). Using these cells, we have previously reported that PMA-induced monocytic differentiation of HL60 cells was associated with large increases in Na+/Hf antiporter mRNA and protein expression (Rao et al., 1991). Here we report that the steady-state levels of antiporter mRNA and protein also increase 18-fold and 7-fold7respectively, in response to RA-induced granulocytic differentiation of HL60 cells. These increases are accounted for by a n increase in the rate of antiporter gene transcription. These findings demonstrate that Na+/Hf antiporter expression is a generalized feature of HL60 cell differentiation.
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MATERIALS AND METHODS Cell culture HL60 cells (kindly provided by Dr. Diego Restrepo, Monell Chemical Senses Center, Philadelphia, PA)
Received September 16,1991; accepted December 23,1991.
*To whom reprint requests/correspondence should be addressed.
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were grown in RPMI 1640 medium supplemented with 10% (vivj heat-inactivated fetal bovine serum, 100 unitsiml penicillin, and 100 pg/ml stretomycin. The cultures were maintained in humidified 95% air-5% C 0 2 at 37°C by passage of 1-2 x lo5 cells/ml every other day. Cell viability was tested by trypan blue exclusion assay. To induce differentiation, HL60 cells were treated with retinoic acid (RA) (1pM) for various times. The extent of differentiation was measured by the ability of cells to reduce nitroblue tetrazolium salt (NBT). The NBT reduction assay was performed as described (Davies et al., 1985; Li et al., 1973). RNA blot analysis HL60 cells were treated with RA (1pM) for varying times and total cellular RNA was isolated from all cells (approximately 8 x 1O7 cells for each time point) by the guanidinium isothiocyanate-cesium chloride protocol of Chirgwin et al. (1979). Poly A' RNA was selected by passing total cellular RNA in high salt containing 20 mM Tris-HC1 buffer (pH 7.5) through a n oligo(dT1-cellulose column and eluting it with salt-free 20 mM TrisHC1 buffer (pH 7.5) as described earlier by Aviv and Leder (1972). Poly A+ RNA (10 pg) from each time point was size fractionated by electrophoresis on 1% agarose, 2% formaldehyde gel. After transfer to a Nytran membrane as described by Thomas (1980), the RNA was crosslinked to the membrane using UV irradiation (Stratalinker, Stratagene, La Jolla, CA). After 4 h prehybridization in 50% (viv) formamide, 5X SSC (1X SSC = 0.15 M NaC1, 0.015 M sodium citrate), 5X Denhardt's (1X Denhardt's = 0.02% (wiv) each of Ficoll, polyvinyl pyrolidonc and bovine serum albumin), 50 mM sodium phosphate (pH 6.51, and 250 pgiml of sheared salmon sperm DNA a t 42"C, the Nytran membrane was hybridized in the above solution containing 10% (wiv) dextran sulfate and 1 x lo6 c p d m l of cDNA probe for 16 h at 42°C. The cDNA probes-Na+/H+ antiporter cDNA (Bam H1 fragment, bp 731-2646 of human cDNA, clone c28, a s described by Sardet et al., 1989) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA (a full length rat cDNA, clone pRGAPD 13, Fort et al., 1985)-were radiolabeled using a GIBCOiBRL random primers labeling kit as per manufacturer's protocol with lw3'P1dCTP (specific activity 3,000 Ciimmole, DuPont-New England Nuclear). After hybridization, the Nytran membrane was washed three times in 2X SSC, 0.2% SDS (15 min, 2 5 T ) and twice in 0.1X SSC, 0.110 SDS (15 min, 60°C). The membrane was then exposed to Kodak X-Omat AR x-ray film with a n intensifying screen a t -70°C for 16 h. Densitometric analysis of the autoradiograms exposed in the linear range of film density was made on a Pharmacia Ultroscan XL laser densitometer. Nuclear run-on assay Nuclei from uninduced and induced HL60 cells (approximately 2 x 10' cells) were prepared by the technique of Groudine et al. (1981). Run-on transcription was carried out at 30°C for 30 min in a reaction mixture consisting of 210 pI nuclear suspension (3-5 x lo7 nuclei in 40% (viv) glycerol, 50 mM Tris-HC1, pH 8.3, 5 mM MgCl,, and 0.1 M EDTA), 60 p15X nuclear run-on buffer (12.5 mM MgCl,, 750 mM KC1, 1.25 mM each
100
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40
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72
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96
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120
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144
Time, hr Fig. 1. Time course for RA-induced granulocytic differentiation of HL60 cells. HL60 cells were treated with 1FM RA for indicated times, and differentiation was measured as percentage of cells positive for NBT assay.
ATP, GTP and CTP, 25 mM Tris-HC1 (pH 8.0)), and 50 pl [a-32PlUTP (500 pCi, specific activity 3,000 Cii mmole, DuPont-New England Nuclear). Reactions were terminated by the addition of 8 pl of 11 mgiml RNase-free DNase I and incubation a t 30°C for 5 min. "P-labeled RNA was purified a s described by Linial et al. (1985). Plasmids containing cDNAs for the Na+/H+antiporter and GAPDH were linearized, denatured (0.2 M NaOH for 30 min a t 25"C), neutralized with 6X SSC (10 volumes) and applied to a Nytran membrane (10 pgislot) using a slot blot apparatus. After prehybridization for 3 h in 100 mM TES-HCI buffer (pH 7.41, 0.2% SDS, 10 mM EDTA, 0.3 M NaC1, 1X Denhardt's, and 250 pg/ml Escherichia coZi tRNA, the membrane was hybridized in the above solution containing 1 x lo7 cpmiml of 32P-labeled nuclear RNA transcripts for 48 h at 65°C. Membranes were washed twice in 2X SSC, 0.1% SDS (15 min, 25"C), and twice in 0.1X SSC, 0.1% SDS (60 min, 60°C).
Immunoprecipitation analysis HL60 cells were metabolically labeled for 24 h using ["Slmethionine (200 pCi/ml) -+ 1 pM RA (total treatment time = 72 h) and membranes were isolated as described by Sardet et al. (1990). Equal amounts of membrane proteins from uninduced and induced HL60 cells were immunoprecipitated using affinity-purified antiNaf/H+ antiporter antibodies (RPl-c28).The immunoprecipitated proteins were separated on SDS-7.5% PAGE under reducing conditions described by Sardet et al. (1990) and Laemmli (1970) and subjected to autoradiography.
RESULTS Retinoic acid caused a time-dependent differentiation of HL60 cells into granulocytes (Fig. l ) . About 2-3% of untreated cells were NBT positive, a marker for granulocytic differentiation. This number did not vary significantly in cells treated with RA for up to 12 h. Treatment for 24 h, however, caused differentiation
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Na ' iH+ ANTIPORTER AND HMO CELL DIFFERENTIATION
+GAPDH Fig. 2. Steady-state levels of Na'lH' antiporter and GAPDH mRNA during RA-induced granulocyt,ic differentiation of HL60 cells. Equal amounts of poly A t RNA (10pg/lane)from RA-treated and untreated HL60 cells were analyzed by Northern blotting for steady-state levels of Na'iH ' antiporter and GAPDH mRNA using the respective "P-laheled cDNA probes.
in - 14%of the cells. Following 6 days of RA treatment, a maximum of - 90% of cells had differentiated. In differentiated granulocytic HL60 cells (Ladoux et al., 1987; Restrepo et a]., 1987) and normal circulating granulocytes (Grinstein and Furuya, 19861, the Na+/H+ antiporter plays a critical role in the recovery of pH, following acidosis and cell activation (PMA, fMLP). To understand further the role of the Na+/H+ antiporter during HL60 cell differentiation at the molecular level, cells were treated with RA, and poly A + RNA was isolated a t varying times following induction. Equal amounts of poly A t RNA (10 pg) at each time point were analyzed by Northern blotting using a cloned 32P-labeled human Na+/H+ antiporter cDNA. No significant change was observed in Na'lH+ antiporter mRNA levels after 3 h treatment with RA (Fig. 2 upper panel; Table 1). However, after 6 h RA treatment, the steady-state mRNA levels for the antiporter increased by eightfold. Na+/H+ antiporter mRNA levels steadily increased, reaching a maximum after 24 h RA treatment (- 18-fold increase). Treatment with RA for up to 72 h did not produce any further increases in the Na'/H+ antiporter mRNA levels. To prove that induction of Na+/Ht antiporter mRNA by RA was not due t o a global increase in the RNA synthesis, two experiments were performed. First, total RNA
TABLE 1. Na+/H+ antiporter steady-state mRNA levels and transcriptional rates in HMO cells during retinoic acid-induced granulocytic differentiation Treatment (Hours) 3 6 24 72
Fold increase in mRNA levels
Fold increase in transcriptional rate
0.4
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8.0 17.6 18.4
8.3
synthesis, as measured by 3H-uridine uptake into trichloroacetic acid-precipitable material decreased by 20% in RA-treated cells (60,800 2 5,200 vs 49,000 2 3,050 cpm). Second, the levels of GAPDH mRNA, a glycolytic enzyme that is expressed constitutively in most cells, were not significantly different in RAtreated and untreated cells (Fig. 2 lower panel). Together these findings indicate that the induction of Na+/H+ antiporter mRNA by RA in HL60 cells is specific. To prove that the induction of Na'/H' antiporter RNA was due to induction of differentiation, rather than to a direct effect of retinoids on the antiporter gene, HL60 cells were treated with etretinate, a synthetic analogue of RA that does not induce differentiation (Ladoux et al., 1987).In contrast to RA, etretinate
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+ Vector + GAPDH Na/H
Fig. 4. Transcription rates of Na ' /H antiporter and GAPDH genes during RA-induced granulocytic differentiation of HL60 cells. '"Plabeled RNA transcripts were isolated from nuclei of RA-treated and untreated HL60 cells and hybridized to filter-immobillzed Na /Hi antiporter cDNA and GAPDH cDNA.
To analyze whether increases in Na ' /H antiporter mRNA levels in RA-treated HL60 cells were also reflected a t the protein level, cells were metabolically labeled with [35Slmethionine and membranes were isolated. Equal amounts of membrane proteins from untreated and RA-treated HL60 cells were immunoprecipitated with affinity-purified anti-Na+/H antiporter antibodies \RPl-c28) (Sardet et al., 1990), the immunoprecipitates were separated by SDS-PAGE under reducing conditions, and proteins were visualized by autoradiography. The Na+/H+antiporter can be identified by its apparent molecular size (- 100 kD) and phosphorylation as described earlier (Rao et al., 1991). sevenfold increase in the As shown in Figure 5 , a intensity of the Na+/H+ antiporter band was observed in RA-treated (72 h) HL60 cells as compared to untreated cells. This finding is consistent with the steadystate levels of antiporter mRNA. +
+GAPDH Fig. 3. Effect of etretinate on the steady-state levels of Na-IH' antiporter and GAPDH mRNA. HL60 cells were treated with either RA (1 p,M) or etretinate (1 +M), and poly A+ RNA was isolated. Equal amounts of poly A+ RNA from agonist-treated and untreated HL60 cells were analyzed for Na ' /H ' antiporter and GAPDH mRNA by Northern blotting as described in Figure 2.
did not increase Na+/H ' antiporter mRNA levels in HL60 cells (Fig. 3). This result indicates that the induction of Na+/H+antiporter mRNA by RA in HL60 cells is closely associated with differentiation. Further, since near maximal increases in the steady-state mRNA levels for the Na+iH+ antiporter (- 18-fold at 24 h) were reached before a significant population of cells had differentiated (- 14% a t 24 h), induction of the Na'/H+ antiporter gene precedes the HL60 cell differentiation. To determine whether RA-induced increases in antiporter mRNA levels were due to increased gene transcription, we next performed nuclear run-on assays in HL60 cell nuclei treated with RA. As shown in Figure 4 and Table 1, treatment with RA for 72 h caused a n eightfold increase in Na+/H+ antiporter transcription. In contrast, the rate of GAPDH transcription showed a small decrease in RA-treated cells consistent with the generalized decrease in total RNA synthesis. Although a n eightfold increase in the Na+/H+ antiporter transcription rate may account for the 18-fold increase in its steady-state mRNA levels over time, posttranscriptional mechanisms (such as message stabilization) cannot be ruled out. Overall, nuclear run-on results clearly show that during RA-induced granulocytic differentiation of HL60 cells, transcription of Na+/H+ antiporter gene increases significantly.
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DISCUSSION Ladoux et al. (1987) have reported increases in pH, and Na+/H+ antiporter activity in HL60 cells during RA-induced granulocytic differentiation. The increase in Na+/H+ antiporter activity preceded the onset of differentiation and persisted throughout the course of differentiation. The present findings strongly suggest that these increases in activity are due to induction of antiporter gene expression. The fact that etretinate, a retinoid that did not cause differentiation, failed to induce antiporter gene expression indicates that induction of the antiporter gene is tightly associated with differentiation of HL60 cells. We now have strong evidence from the present study and our previous work with PMA-induced monocytic differentiation that induction of the N a t / H ' antiporter is closely associated with HL60 cell myeloid differentiation. These data are: (1)the increase in antiporter expression temporally preceded differentiation induced by either RA (present study) or PMA (Rao et al., 1991), (2) agents that mimic RA and PMA (etretinate and l-oleolyl-2-acetylglycerol,respectively) in activating certain signal transduction pathways but that fail to induce differentiation, also do not induce antiporter expression, and (3) inhibition of differentiation by cyclo-
Na ' /H ' ANTIPORTER AND HL60 CELL DIFFERENTIATION
i i
110 kD Na/H
47 kD
Fig. 5. Na ' /H antiporter protein synthesis during RA-induced granulocytic differentiation of HL60 cells. The molecular size markers were phosphorylase b ( 1 10 kDat and ovalbumin 147 kDa).
heximide prevented antiporter induction (Rao et al., 1991). Since induction of the Na +/H+antiporter gene was observed during both PMA-induced monocytic and RA-induced granulocytic differentiation of HL60 cells, it appears that induction of the Na+/H+antiporter gene is a common feature of myeloid differentiation. Future studies with other model systems for differentiation (e.g., PC12 cell line) will address the question of whether induction of the Na+/H+ antiporter gene is a common feature of the differentiation process or restricted to myeloid differentiation. The marked increases in Na+/H+ antiporter expression during differentiation of HL60 cells into monocytes or granulocytes suggest a n important physiological function. The most compelling reason for leukocytes to express more antiporter is that during the respiratory burst activated by bacteria or chemotactic peptides, there is a n intense cyctoplasmic acidification (Grinstein and Furuya, 1984, 1986). In response to PMA stimulation of the respiratory burst, both neutrophils (Grinstein and Furuya, 1984, 1986) and DMSOdifferentiated HL60 cells (Restrepo et al., 1987) exhibit a n amiloride-sensitive acid recovery mediated by the Na '/H+ antiporter. Despite agreement on the role of the antiporter in pH, recovery in response to PMA, there are conflicting data regarding the role of pH, in HL60 cell differentiation and the mechanisms by which antiporter activity is regulated. Ladoux et al. (1987, 1989) reported a n increase in both pH, and Na+/H+ antiporter activity (measured in the absence of HCO-,) during DMSO and RA-induced granulocytic differentiation of HL60 cells. This group observed a 1.7-fold in-
365
crease in antiporter V,, in RA-differentiated HL60 cells (Ladoux et al., 1987). In contrast, studies of DMSO-differentiated HL60 cells have shown no change in pHi when measured in the presence of HCOp3 (Restrepo et al., 1988) and no increase in the antiporter V,, (Costa-Casnellie et al., 1988). The only difference in antiporter regulation observed by the latter group in DMSO-differentiated HL60 cells was a decrease in K, for Na'. However, Restrepo et al. (1987) observed a ninefold increase in the rate of amiloride-sensitive H' efflux following PMA stimulation in DMSO-differentiated HL60 cells compared to undifferentiated cells. In contrast, Ladoux et al. (1989) and our preliminary results (Rao and Berk, unpublished observations) show no PMA-induced pH, response in RA-induced HL60 cells. These data suggest that there are important differences in the effects of PMA on antiporter activity in RA-differentiated as compared to DMSO-differentiated HL60 cells. The data in the present study suggest that the values do not accurately for pH, and Na + /H+ antiporter V,, reflect the level of antiporter protein expression. For example, Ladoux et al. (1987) observed a 1.7-fold increase in antiporter V,,. However, we found at least a sevenfold increase in antiporter protein expression and a n 18-fold increase in its mRNA levels. We propose four possible mechanisms to account for this disparity in findings. First, i t is possible that there are "spare" antiporters that are not physically associated with the plasma membrane but are available for insertion during cellular acidification. Second, maximal activation of the antiporters may not occur in response to acid loading alone as employed by Ladoux et al. (1987). This could occur if, e.g., activation occurred by several mechanisms such as phosphorylation (Sardet et al., 1990) and binding of a regulatory protein (Morel1et al., 1990), or if inactivation (e.g., dephosphorylation) was more rapid in response to acid loading compared to the oxidative burst. The findings of the ninefold increase in Na+/H+antiporter activity in differentiated HL60 cells in response to PMA-induced oxidative burst (Restrepo et al., 1987) would support such a mechanism. Third, antiporters may be present in the cell but functionally inactive due to association with a negative regulatory protein. Fourth, if HL60 cells are capable of expressing more than one Na+/H+ antiporter isoform (Sardet et al., 19911, it is possible that the increase in mRNA and protein may be due in part t o a n isoform that has a relatively low V,,. Future experiments will be required to characterize isoform expression, posttranslational regulatory mechanisms, and the functional role of antiporter induction in HL60 cell differentiation.
LITERATURE CITED Agarwal, N., Haggerty, H., Adelherg, E. and Slayman, C. (1986) Isolation and characterization of a Na-H antiporter-deficient mutant of LLC-PKl Cells. Am. J. Physiol., 251tC825-C830. Aickin, C.C., and Thomas, R.C. (1977) An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibres. J. Physiol. (Land), 273:295-316. Aviv, H., and Leder, P. (1972) Purification of biologically active globin messenger KNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA, 69:1408-1412.
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Besterman, J.M., May J r , W.S., LeVine 111, H., Cragoe Jr, E.J., and Cuatrecasas, P. (1985) Amiloride inhibits phorbol ester-stimulated Na+iH+ exchange and protein kinase C. J. Biol. Chem., 260:11551159. Boron, W.F., and Boulpaep, E.L. 119831 Intracellular pH regulation in the renal proximal tuhule of the salamander. Na-H exchange. J. Gen. Physiol., 81:29-52. Burns, C.P., and Rozengurt, E. (1983)Serum, platelet-derived growth factor, vasopressin and phorbol esters increase intracellular pH in Swiss 3T3 cells. Biochem. Biophys. Res. Commun. 1l6:931-938. Cassel, D., Rothenberg, P., Zhuang, Y.X., Deuel, T.F., and Glaser, L. (1983)Platelet-derived a o w t h factor stimulates Na+iH+ exchange and induces cytoplasmmc alkalinization in NR6 cells. Proc. Nail. Acad. Sci. USA, 80t6224-6228. Chirrwin, J.M.. Prubvla, A.E., MacDonald. R.J., and Rutter. W.J. (1679) Isolation ofhi”ological1yactive ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18:5294-5299. Collins, S.J., Ruscetti, F.W., Gallagher, R.E., and Gallo, R.C. (1978) Terminal differentiation of human Dromvelocvtic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc. Natl. Acad. Sci. USA, 752458-2462. Costa-Casnellie, M.R.. Segel, G.B.. and Lichtman. M.A. (1988) The NaT:H+ exchanger in ymmature and mature granulocytic HL-60 cells. Property changes induced by intracellular acidification and cell maturation. J. Biol. Chem., 263t11851-11855. Davies, P.J., Murtaugh, M.P., Moore dr., W.T., Johnson, G.S., and Lucas, D. (1985)Retinoic acid-induced expression oftissue transglutaminase in human promyelocytic leukemia (HL-60) cells. J . Biol. Chem., 2605166-5174. Doppler, W., Jaggi, R., and Groner, B. (1987) Induction of v-mos and activated Ha-ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression. Gene, 54r747-153. Fort, Ph., Marty, L., Piechaczyk, M., el-Sabrouty, S., Dani, C., Jeauteur, P., and Blanchard, J.M. (1985) Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3phosphate-dehydrogenase multigenic family. Nucleic Acids Res., 13t1431-1442. Grinstein, S.. and Furuva, W. (1984) Amiloride-sensitive Na+/H+ exchange in human neutrophils: mechanism of activation by chemotactic factors. Biochem. Biophys. Res. Commun., 122:755-762. Grinstein, S., and Furuya, W. (1986) Cytoplasmic pH regulation in phorbol ester-activated human neutrophils. Am. J. Physiol., 251: C55-65. Grinstein, S., Rotin, D., and Mason, M.J. (1989) Na+/H+ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim. Biophys. Acta, 988:73-97. Groudine, M., Peretz, M., and Weintraub, H. (1981)Transcriptional regulation of hemoglobin switching in chicken emhryos. Mol. Cell Biol., 1281-288. Hagag, N., Lacal, J.C., Graber, M., Aaronson, S., and Viola, M.V. (19871Microinjection ofras p21 induces a rapid rise in intracellular pH. Mol. Cell Biol. 7:1984-1988. Hazav, P., Shany, S., Moran, A,, and Levy, R. (1989) Involvement of intracellularpH elevation in the effect ofl,25-dihydroxyvitamin D3 on HTA0 cells. Cancer Res., 49t72-75. Ladoux, A,, Cragoe Jr., E.J., Geny, B., Ahita, J.P., and Frelin, C. (1987)Differentiation of human promyelocytic HL 60 cells by retinoic acid is accompanied by an increase in the intracellular pH. The role of the Na+/H+ exchange system. J. Biol. Chem.,262:811-816. Ladoux, A., Krawice, I., Damais, C., and Frelin, C. (1989) Phorbol
esters and chemotactic factors induce distinct changes in cvtodasmic Ca2 and pH in granulocytic-like HL60 cells. Biichim. Bioihys. Acta. ~~~. > - 10 - - - 1.?:55-59 ~-
Laenimli, U.K. (1970) Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature, 227,680-685. Li, C.Y., Lam, K.W., and Yam, L.T. (1973) Esterases in human leukocytes. J. Histochem. Cytochem., 21:1-12. Linial, M., Gunderson, N., and Groudine, M. (198.5)Enhanced transcription of c-myc in bursa1 lymphoma cells requires continuous protein synthesis. Science, 230:1126-1132. Lotem, J., and Sachs, 1,. (1979) Regulation of normal differentiation in niouse and human niyeloid leukemic cells by phorbol esters and the mechanism of tumor promotion. Proc. Natl. Acad. Sci. USA, 76: 5158-5162. Mahnensniith, R.L., and Aronson, P.S. (19853The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ. Res. 56t773-788. Moolenaar, W.H. (1986)Effects of growth factors on intracellular pH regulation. Annu. Rev. Physiol., 48t363-376. Morel], G . , Stepock, D., Shenolikar, S., and Weinman, E.J. (19901 Identification of a putative Na’iH exchanger regulatory cofactor in rabbit renal BBM. Am. J. Physiol., 259:F867-F871. Ober, S.S.,and Pardee, A.B. (19873Intracellular pH is increased after transformation of Chinese hamster embryo fibroblasts. Proc. Natl. Acad. Sci. USA, 84.2766-2770. Paris, S., and Pouyssegur, J . (1984) Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H + . J. Biol. Chem., 259:10989-10994. Pouyssegur, J., Sardet, C., Franchi, A,, L’Allemain, G., and Paris, S. (1984) A specific mutation abolishing N a i ~ i H +antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Natl. Acad. Sci. USA, 81 t48334837. Rao, G.N., deRoux, N., Sardet, C., Pouyssegur, J., and Berk, B.C. (1991) N a ’ /H ’ antiporter gene expression during monocytic differentiation ofHL60 cells. J . Biol. Chem., 26613485-13488. Restrepo, I]., Kozody, D.J., and Knauf, P.A. (1987) Changes in Na ’ /H ’ exchange regulation upon granulocytic differentiation of HL60 cells. Am. J . Physiol., 253:C619-C624. Restrepo, D., Kozody, D.J., Spinelli, L.J., and Knauf, P.A. (1988) pH homeostasis in promyelocytic leukemic HL60 cells. J. Gen. Physiol, 92t489-507. Rovera, G., Santoli, D., and Damsky, C. (1979) Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc. Natl. Acad. Sci. USA, 76: 2779-2783. Russell, J.M., and Boron, W.F. (1976) Role of choloride transport in regulation of intracellular pH. Nature, 264:73-74. Sardet, C., Franchi, A., and Pouyssegur, J. (1989)Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+iH+ antiporter. Cell, !ifi:271-280. Sardet, C., Counillon, L., Franchi, A ,, and Pouyssegur, J. (1990) Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD. Science, 247t723-726. Sardet, C., Fafournoux, P., and Pouyssegur, J . (1991) a-thrombin, epidermal growth factor and okadaic acid activate the Na ‘/H’ cxchancer. NHE-1. hv uhosuhorvlatine a set of common sites. J . Biol. C h e i . , 266:19166~19171: Thomas, P.S. (1980)Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA. 773201-5205. +
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