Biochimica et Biophysica Acta, 1091 (1991) 158-164 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0167-4889/91/$03.50 ADONIS 016748899100074R
158
BBAMCR 12859
Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards peutrophils Michael A. Baxter 1, Christopher M. Bunce 2, Janet M. Lord 2, Philip J. French 3, Robert H. Michell 3 and Geoffrey Brown 2 I Department of Medicine, Universityof Birmingham. Birmingham (U.K.), 2 Department of Immunology, University of Birmingham, Birmingham ( U,K,) and 3 Department of Biochemistry, University of Birmingham, Birmingham ( U.K.) (Received I April 1990)
Key words: Inositol transport; HL60 cell; Myeloid differentiation; Neutrophil
[aHllnositol uptake by H I ~ cells was measured during DMSO-induced differentiation towards neutrophils. The values for gm (53.2/AM) and Vm,~ (5.3 pmol/min per 10 ~ cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. lnosltol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations, lnositol transport rates changed during DMSOinduced differentiation of H I ~ cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intraceilular inositol concentration from 10.9 to 132.7 pM seen between day I and day S. After 4 days exposure to DMSO the rate of inosltoi transport fell to a value of 3.2 + 0.3 pmol/min per 10 ~ cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 ;~M. The inositol transport rate, thus, appears to closely accompany changes in the intraceUular concentration of inosltol. Inositol ~vansport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced ~ cells, but the Km for inositol transport was similar in both cell types and was unchanged during H I ~ differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositoi transporter, one consequence of which is the alteration of
intraceUular inusitul concentrations. Introduction
The central role of phosphatidylinositol 4,5-bisphosphate hydrolysis in the generation of intracellular second messengers is well established [1]. Many extracellular stimuli have been shown to promote phosphatidylinositol 4,5-bisphosphate breakdown in target cells, with the subsequent generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol [2]. The Ins(1,4,5)P3 generated in this way stimulates the mobilisation of calcium from intracellular stores [3] and diacylglycerol activates the calcium-phospholipiddependent protein kinase, protein kinase C [4]. Several workers have proposed that the subsequent covalent
Abbreviations: lns(1,4,5)P3, inositol 1,4,5-triphosphate; DMSO, dimethylsulphoside. Correspondence: M. Baxter, Department of Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
modification of key cellular proteins, by protein kinases and phosphatases, is central to the co-ordinated control of cell growth and differentiation [5]. Whilst the precise role of inositol lipids and inositol phosphates in cell growth and differentiation has not yet been clearly defined, the importance of inositol lipid hydrolysis is implied by the observation that phorbol esters which, like diacylglycerol, are potent activators of protein kinase C [6], are also capable of inducing cellular differentiation [7]. HL60 is a human bipotent promyeloid cell line which, in response to specific external stimuli, undergoes growth arrest and differentiates into one of two functionally and morphologically distinct blood cell types. Dimethylsulphoxide (DMSO) or retinoic acid promote the differentiation of HL60 towards neutrophils [8,9], whilst vitamin D-3 or phorbol esters (such as 12-O-tetradecanoylphorbol-13-acetate; TPA) induce monocyte differentiation [7,10]. HL60 calls have been used extensively as a model system to study cellular events, including changes in inositol lipid metabolism, which occur
159 during growth arrest and induction of differentiation [11,12]. Exposure of HL60 cells to either DMSO or TPA has been shown to produce marked changes in the relative amounts of several inositol poly-phosphates, notably inositol tetrakisphosphate, pentakisphosphate and hexakisphosphate. Furthermore, an increase in the concentration of intracellular inositol has also been noted in HL60 cells during DMSO-induced growth arrest and differentiation (French, P.J., unpublished data). Although the pathways of metabolism of inositol poly-phosphates within cells have not yet been fully established, it is possible that changes in the level of intracellular inositol might result from the breakdown of phosphoinositides or inositol phosphates. Alternatively, inositol could be derived from extracellular sources via stimulation of transmembrane inositol transport [13], several reports have suggested that cellular differentiation is preceded by the activation of other plasma membrane transport systems [14,15]. Inositol transport has previously been studied in a number of mammalian tissues and cell lines, including HL60. In these cells, it is an active, highly specific and sodium-dependent process [13,16]. The inositol transporter is, therefore, a potential site for the regulation of intracellular inositol concentration. This study has investigated inositol transport into HL60 cells undergoing neutrophil differentiation and has considered these findings in relation to the cells' intracellular inositol concentration, growth arrest and maturation. The role of inositol transport in the fully differentiated human peripheral blood neutrophil is also considered. Materials and Methods
Cell culture HL60 cells were maintained in long-term exponential growth in inositol free RPMI 1640 medium (Northumbria Biologicals, Northumberland, U.K.) supplemented with I mg/l myo-inositol (Gibco, Paisley, U.K.), a serum replacement (ITS + pre-mix (Universal Biologicals, London, U.K.)), L-glutamine (Gibco) and antibiotics (100 U / m l penicillin and 50 # g / m l streptomycin, Gibco). Differentiation of HL60 cells towards neutrophils when maintained in this medium was optimally induced by the addition of 0.970 DMSO. At time zero, cells were resuspended at 2.10 5 cells/nil in medium containing 0.970 DMSO and established as 200 ml cultures in 175 cm2 tissue culture flasks (Nunc, Paisley, U.K.) and cultured for up to 7 days. Aliquots of cells were taken at daily intervals to assess numbers of viable cells and to monitor the differentiation status of the culture as revealed by the cells ability to phagocytose complement-coated yeasts [17]. Both uninduced and DMSO-induced HL60 cultures were maiutained at 37 °C in a humidified atmosphere containing 570 CO2.
Isolation of human blood neutrophi/s For inositol transport studies normal mature neutrophils were prepared using density sedimentation as previously described [18]. Briefly, 14 ml of heparinised peripheral blood were mixed with 2 ml of Hespan (DuPont, Stevenage, U.K.) and left to stand in universal containers (Sterilin, Paisley, U.K.) at 37 °C for 40 rain. The leucocyte-enriched supernate was removed and the cells washed twice in phosphate-buffered saline (once at 1500 rpm, 10 rain, 4 ° C; and twice at 1000 rpm, 10 rain, 4°C; MSE 3000, MSE, Crawley, U.K.). In control transport studies HL60 cells were incubated in 87.570 human AB serum, 0.12570 Hespan for 40 min at 37°C prior to inositol transport assays as described below. Inositol transport assays HL60 cells were harvested from the culture medium (10 min, 1000 rpm, MSE3000) and washed twice in phosphate-buffered saline (PBS). Cells were resuspended in PBS at (2-9). 107 cells/ml and a 600 #! aliquot was subsequently mixed with 0.1-1/~Ci [14C]inulin (9.42 mCi/mmol, Amersham International, Amersham, U.K.). In a separate tube, 500 #l PBS containing [3H]inositol (Amersham International) at a specific activity of 0.2 pCi/nmol, inositol (0-150/~M) and glucose (0-20 mM) was simultaneously incubated for 10 min at 37 ° C. Inositol uptake was initiated by the addition of 500/Ll of the cell/inulin mixture. 200-600 pl aliquots were taken at time intervals up to 15 min and the reaction terminated by dilution in ice-cold PBS (400/tl). Cells were recovered by spinning rapidly (30 s, microfuge B, Beckman, High Wycombe, U.K.) through dibutyl phthalate oil (Sigma, Poole, U.K.), snap freezing in liquid nitrogen and excision of the tip of the microfuge tube with a hot scalpel blade. Tips were transferred to a scintillation vial containing 400/~l distilled water, 50 pl Optisolve (FSA Laboratory Supplies, Loughborough, U.K.) and 4 ml scintillation fluid. 14C and aH radioactivity was measured using a dual label programme (LKB Mk3, LKB-Pharmacia, Milton Keynes, U.K.). Labelling and extraction of [SH]inositol HL60 cells were incubated for 6 days in modified RPMI 1640 medium as described above and containing 1/~Ci//~g [3H]inositol as previously described [19], prior to treatment with 0.970 DMSO for up to 7 days. Inositol and inositol phosphates were extracted from control and DMSO-treated cells essentially by the method of Berridge et al. [20]. Briefly, 10 7 cells were harvested, washed once in inositol free RPMI 1640 medium and lysed by the addition of 500 ~1 of 2070 TCA containing 250 /~g/ml phytic acid and 0.570 phytate hydrolysate [19]. Inositol lipids were sedimented (1 rain, 6500 rpm, MSE microcentaur, MSE) and the soluble inositol and
160 inositol phosphates transferred to a 5 ml polypropylene tube. The lipid pellet was washed once with 500/tl of 2~ TCA, the supernatants pooled and washed four times with 4 ml of diethyl ether. Following the last diethyl ether wash 100 ttl of 50 mM EDTA (pH 7) was added to the inositol extract which was stored at - 20 ° C prior to fractionation by HPLC. The inositol extract was applied to a 25 cm partisil P10 SAX HPLC column (Thames Chromatography, Maidenhead, U.K.) and inositol separated from inositol phosphates, which were retained by the column and subsequently removed with 1 M ammonium phosphate. 0.5 ml fractions were collected into 38~ methanol prior to the addition of liquid scintillant and liquid scintillation spectrometry. The distribution of [3H]inositol within HL60 cells after 15 min incubation was determined as follows. HL60 cells (0.64.10'~/ml) were incubated with 15/~M inositol and 0.4 ~tCi/nmol [3H]inositol in 1 ml of PBS at 37 o C. After 15 rain, 600 ~tl aliquots were placed onto a silicone oil cushion (62~ Siliconil APl00; 38~$ Siliconil AR20, Wacker-Chemic GMBH, Munich), which was layered over 500/~1 of 20~ TCA. The incubation was terminated by centrifugation of the cells through the oil into the TCA (MSE Microcentaur, 13000 rpm, 1 rain, Anderson, K. personal communication). Buffer and oil layers were discarded, leaving the TCA and cell pellet. The TCA layer was placed into a 5 ml polypropylene tube and 0.5 ml of distilled water added before washing four times with 4 ml diethyl ether. Following the last diethyl ether wash 100 ~tl of 50 mM EDTA (pH 7) was added and the volume of the sample made up to 10 ml with distilled water. This extract was loaded onto a Dowex AG1-X8 (formate form, Bio-Rad Laboratories,
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Fig. 1. Effect of extracellular inositol concentration on inositol uptake into HL60 cells. HL60 cells ((0.2-1)- 107/ml) were incubated at 37 o C in PBS containin8 5 (e e), 10 (0 0), 15 (ra ra) or 50 FM (U I ) inositol and [3Hl-inositol (0.2 FCi/nmol). Aliquots were taken at the time points shown and cells were collected by spinning through dibutyl phthalate oil, as described in Materials and Methods. Cell pellets were counted for 3H radioactivity by fiquid scintillation spectrometry.
Hemel Hempstead, U.K.) 2.5 cm column. Any remaining unbound material was eluted using 5 ml of distilled water, combined with the column eluate and taken as the 'free inositol fraction'. Inositol phosphates were recovered by elution of the column with 2 M ammonium formate/0.1 M formic acid. A 2 ml aliquot of each extract was suspended in 20 ml of scintillant (15 g PPO; 0.375 g POPOP in 2.5 I xylene; 1 : 1 mixture with Triton X-100) and the radioactivity determined by liquid scintillation counting. The pellet was resuspended in 0.3 ml distilled water and 1.125 ml of a chloroform/methanol/concentrated HCI mixture (100: 200: 1) added. This mixture was left overnight at room temperature to allow extraction of the inositol phospholipids from the particulate cellular fraction. Each extract was mixed with 0.375/tl of 0.3 M HCI containing 1 mM inositol and 2 M KCI before centrifugation (2000 rpm, 5 rain, MSE 3000). The lower phase was collected and air dried in a 5 ml polypropylene tube and dried under a stream of nitrogen. After addition of 5 ml of scintillant, radioactivity was determined by liquid scintillation counting. Results
Previous studies in this laboratory (McConnell, F.M., unpublished data) have shown that HL60 cells do not synthesise inositol de novo and must obtain 95% or more of their inositol requirement from the extracellular growth medium as preformed inositol. In the inositol transport assays reported in this study, total 3H counts were derived from both intracellular [3H]inositol and extracellular [3H]inositol trapped in the cell pellet. The volume of extracellular fluid estimated using [14C]inulin was small (0.06 + 0.06/zl/10 z cells), particularly when compared with an intracellular volume for HL60 of 1 /tl/106 cells. However, the specific activity of [3H]inositol in the medium was high (0.2/tCi/nmol) and even a small extracellular contamination could produce large errors in the recorded counts. A correction for extracellular [3H]inositol contamination was therefore made to all data.
Characterisation of the HL60 inositol transporter Fig. 1 shows that the accumulation of intracellular inositol into uninduced HL60 cells at a variety of extracellular inositol concentrations was linear for up to 15 min at 37°C. We have determined the mean cell volume of HL60 cells in the uninduced state and also during differentiation towards neutrophils (data not shown). Using this information, data from Fig. 1 and the intracellular labelling experiments outlined below, we have calculated that after 15 min incubation at 37 o C, the concentration of intracellular inositol exceeds that of extracellular inositol. At this point inositol is being transported against a concentration gradient, a
161 property characteristic of an active process. The linearity of inositol accumulation during the first 15 rain of incubation at 37°C strongly suggests that the time courses shown represent initial rates of inositol accumulation. Although such uptake kinetics are consistent with all previous studies of inositol transport I13,18,21], it is in marked contrast to the transport kinetics of hexose sugars, such as glucose [22]. This may indicate that in HL60 cells the inositol transport rate is relatively slow in both directions, but that influx is much more rapid than efflux. This has been confirmed in other cell types [23,24]. Analysis of cell extracts from HL60 cells labelled with [3H]inositol for 15 min revealed that 76.7 + 2.13~ (n ffi 5) of the counts were present as free inositol with only 0.8 :k 0.06~ (n ffi 5) in the form of inositol phosphates. There was, however,, a rapid sequestration of [3H]inositol into phospholipids (23.3 + 2.13~ (n = 5)) and this may partially explain the observed maintenance of the initial uptake rates. Inositol uptake rates were measured over a range of physiological concentrations of inositol (5-150 pM). A plot of inositol concentration against inositol transport rates (Fig. 2), demonstrates kinetics typical of a highly specific, carrier mediated system. Failure to achieve complete saturation of inositol uptake probably reflects the fact that HL60 cells, in addition to a high affinity transport system, also possesses a non-specific, passive, low affinity carrier system. Such a mechanism is well characterised in other cell systems, including myeloid
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Concentration of inositolgmolar (S)
Fig. 2. Rate of inositol uptake into HL60 cells versus inositol concentration. HL60 cells ((0.2-1)-107/ml) were incubated for 10 rain at Y/°C in PBS containing inositol (5-150 FM) and [3H]inositol (0.2 pCi/nmol). Aliquots were taken for the estimation of inositol uptake
and cells were collected by spinning through dibutyl phthalate oil, as described in materialsand methods. Cell pellets were counted for 3H radioactivity by liquid scintillation spectrometry. Values shown are mean+ S.E. of five determinations. Insert: Reciprocal values for the above data are plotted as a Lineweaver-Burkeplot. The intercept on the x-axis= - 1 / K m, the intercept on the y-axis= 1/Vma~.
TABLE I
Effect of extraceilular glucose concentration on the kinetics of inositol uptake into HL60 cells HL60 cells (2.106/ml) were incubated at 3 7 ° C in PBS containing inositol (5-150 ~M), [3H]inositol and D-glucose (0-20 raM). Cell samples were taken after 10 min for estimation of inositol uptake as described in Materials and Methods. K m and Vmax values were determined from a double-reciprocal plot of the inositol uptake data, numbers in parentheses indicate the number of replicates used to
determine each mean value Glucose concentration (raM) 0 5 10
20
Km
Vraax
(~M)
(pmol/min per 106cells)
53.2+ 3.9 (5) 53.0+ 2.0 (4) 56.8± 4.0 (4) 55.5 + 4.4 (4)
5.3 + 0.53 (5) 5.3 + 0.2 (4) 5.5 + 0.45 (4) 4.1 + 0.4 (4)
cells, derived from human tissues [13,18]. The reported K m value for the low affinity transporter is approx. 400 /~M, whilst the normal serum concentration of inositol is in the range 50-100 ~M [13], suggesting that this transporter has an insignificant physiological role. Similarly, the low affinity uptake of inositol would be expected to have little effect on the kinetics of the specific, high affinity inositol transport, which was determined using low inositol concentrations (5-150/~M). This is supported by the linear double-r~c,.p,'cca! plot (insert (Fig. 2; r values 0.96-1.00) from which the K m and Vma~ values for the high affinity transporter were calculated. Calculation of these parameters using a S vs. S ~ V plot gave comparable results (data not shown). In undifferentiated HL60 cells the K m and i ma~ values for inositol transport were 53.2 + 3.9 ~M (n = 5) and 5.3 :t: 0.5 pmol/min per 106 cells (n = 5), respectively, and are in good agreement with published values for this cell line [13]. Furthermore, Table I shows that, even at high concentrations of glucose (20 raM), no significant effect was seen on either the Km or Vma~ for inositol transport. Taken together, the above data suggest that the transport of inositol into HL60 cells is via an active, specific, carrier mechanism which is distinct from the glucose transporter. Inositol transport rate and accumulation during H L 6 0 cell differentiation The time-course of the effect of DMSO on the growth and differentiation of HL60 cells is shown in Fig. 3A. Following a 2-3 day phase of exponential growth, there is a dramatic arrest of cell growth which is accompanied by a rapid induction of phagocytic ability. After 7 days of culture in 0.9~ DMSO, 82% of the cells exhibit functional and morphological characteristics of mature neutrophils. Fig. 3B shows the concommitant changes in Vma~ for inositol transport seen in HL60 cells when
162 exposed to 0.9~ DMSO. After 2 days of DMSO treatment the rate of inositol transport increased from 5.3 40.5 pmol/min per 106 cells (n = 5) to 10.44-0.4 pmol/min per 10 6 cells (n = 3). The level of inositol transport then remained elevated for 3 days, peaking at 12.4 + 0.4 pmol/min per 106 cells (n = 3) at day 4, before returning to a lower rate of 3.2 4- 0.38 pmol/min per 106 ceils (n = 3) after 7 days when the cells had optimally differentiated towards neutrophils (Fig. 3A). In contrast to the rate of inositol transport, the Km values did not vary significantly throughout the 7 days of treatment with DMSO (legend to Fig. 3). The observed changes in inositol transport were accompanied by changes in intracellular inositol concentration. As shown in Fig. 3B, uninduced HL60 cells had an intracellular inositol concentration of 12.6 /~M which was unchanged (10.9 aM) after 1 day of exposure to DM$O. However, by day 2 intracellular inositol had increased to 28.9/~M and inositol levels continued to rise in the differentiating cells to a value of 132.7/~M after 5 days exposure to DMSO. This value represented the peak of inositol accumulation within the cells and from day 5 to day 7 inositol concentrations fell slowly, but progressively to a lower value of 112.0 #M. The data shown in Fig. 3 suggest that the increased intracellular free inositol seen during the differentiation of HL60 cells towards neutrophils is associated with, and at least in
part produced by, an increased capacity of the cells to transport inositol from the extracellular medium. Inositol transport in neutrophils
It is of interest that in terminally differentiated HL60 cells, after 7 days of DMSO exposure, the rate of inositol transport (Vmax) fell to a rate which was below that of the undifferentiated cells. This suggests that, although, inositol transport has an important role in precursor and differentiating myeloid cells, the requirement for inositol uptake is reduced in the fully differentiated neutrophils. In an attempt to address this question we have measured the rate of inositol transport in mature human blood neutrophils. Although the K m of the neutrophil system for inositol transport (64 + 6.0 /~M, n = 8) was similar to that of HL60 cells, the rate of inositol transport was very much slower. As shown in Fig. 3B, the Vmax for inositol transport in neutrophils was estimated at 0.3 4-0.07 pmol/min per 106 cells (n - 8) compared with 5.3 4- 0.53 (n - 5) and 3.2 4- 0.38 pmol/min per 106 cells (n = 3) for uninduced and day 7 DMSO-treated HL60 cells, respectively. These findings are supported in a recent report by Simmons and coworkers [18], who also demonstrated relatively slow rates of inositol transport in human peripheral blood neutrophils. However, to rule out the possibility that the isolation of neutrophils with Hespan (see Materials and
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FiB. 3. Chanees in inositol transport and intracellular inositol concentration during HL60 cell differentiation towards neutrophils. HL60 cells were cultured for 7 days in medium containing 1 mg/i inositol and 0.9% DMSO v/v. (A) Cells were removed daily for enumeration of viable cells (O [J) and assessment of the percentage of cells able to phagocytose complement coated yeasts (e e). (B) Human peripheral blood neutrophils (N) and, on appropriate days HL60 cells, were resuspended at (2-9). 106/ml in PBS for determination of inositol uptake rates and Vm~, values ( I i), as described in Materials and Methods. K m values for inositol transport were unchanged by exposure of HL60 cells to 0.9% DMSO and were identical for HL60 cells and human neutrophils (range 53.2+3.9/tM to 67.6+5.2 /~M ( n = 5 ) and 64.0+6.0/~M ( n = 8 ) , respectively), in parallel experiments (n = 2), in which cells were labelled to equilibrium with [aH]inositol prior to exposure to DMSO, intracellular inositol concentrations (o ~ o) were determined in tripficate by the method of Berridge et el. [20], as described in Materials and Methods.
163 Methods) impairs inositol transport, HL60 cells were similarly treated prior to inositol transport assays. In these experiments, neither the K m (45 #M) nor the Vm~, (4.6 pmol/min per 10 6 cells) for inositol transport were significantly altered when compared to non-Hespantreated HL60 cells. Therefore, the reduced rate of inositol transport observed in terminally differentiated HL60 cells in vitro, is likely to be a reflection of a reduced dependence on extracellular inositol in normal peripheral blood neutrophils. Discussion
Although the role of inositol phospholipids in the regulation of cell function is well established, their precise role in cellular differentiation is less well understood. The observation that a number of growth factors stimulate phosphatidylinositol 4,5-bisphosphate breakdown [2] and that phorbol esters, powerful promoters of cell differentiation, appear to exert their effects via protein kinase C activation [7] has been taken as evidence that inositol phospholipid hydrolysis has a role in regulating cell growth and differentiation. In addition, increases in the levels of intracellular InsP~ and InsP6, which are probably not derived from lipids, have been reported in HL60 cells stimulated to differentiate towards neutrophils [12]. The physiological significance of these observations remains unclear. It has also been noted (French, P.J., unpublished data) and confirmed in this study that there is a large increase in intraceUular free inositol when HL60 cells are stimulated to differentiate towards neutrophils by exposure to DMSO. The source of this inositol was previously unknown but the overall rise in intracellular inositol phosphates during HL60 neutrophil differentiation [12] suggested that it was unlikely to be solely derived from metabolism of the higher inositol phosphates. The most likely explanation now seems to be enhanced entry from the extracellular medium mediated by an increase in the rate of transmembrane transport. Inositol is transported into many mammalian cells and tissues by a specific, active and sodium-dependent mechanism characteristic of a system mediated by a specific protein transporter [13,18,21] and modulation of intracellular inositol concentrations via inositol transport has been proposed previously [25,26]. For example, tissues affected by the complications of uncontrolled acute and chronic diabetes have been shown to be inositol-depleted [26]: these tissues also have a reduced rate of inositol transport [25,26]. Furthermore, reactivation of inositol transport in experimental model systems of diabetes has been claimed to restore intracellular inositol concentrations to normal [26] and in some circumstances to reverse the clinical course of diabetic complications [27]. This study has investigated the role of inositol transport as a regulator of intracellular inositol concentra-
tion. The data presented here suggest that inositol uptake in HL60 cells is mediated by a specific and active process with K m and Vmax values similar to those reported in other systems. DMSO-induced growth arrest and cellular differentiation of HL60 was accompanied by an activation of inositol transport via an increase in Vmax, leading to a large rise (13-fold) in the intracellular inositol concentration. Interestingly, however, the changes in intracdlular inositol concentration and Vmax were not parallel over the 7 day treatment period. Inositol levels rose between days 1 and 5 of DMSO treatment, the period leading up to the cells growth arrest. The increase in limax preceded the rise in inositol concentration. Similarly, the decline in Vm~x from day 3 occurred 1 day prior to the decline in inositol concentration which continued gradually up to day 7. Thus, the apparent increase in the rate of inositol transport and the increased intracellular inositol concentration occur in association with growth arrest: they precede the acquisition of the differentiated phenotype, which is accompanied by a slight fall in intracellular inositol levels and a substantially reduced Vma~ for inositol transport. Cellular differentiation appears to be ~sociated with the activation of a number of cell membrane enzymes and transporters, in particular large increases in the flux of Na + ions have been noted [14,15,28]. Moreover, it has recently been suggested that Na+/K+-ATPase is a target enzyme for a number of the putative second messenger systems which are thought to play a role in cell regulation [14]. Inositol transport is sodium-dependent [16] and it may be that these apparently separate phenomena are mechanistically linked. Alternatively, their simultaneous occurrence may simply serve to highlight the importance of regulation of membrane function in the transduction of intracellular messages into altered cell function, growth or differentiation status. The exact role of regulated inositol transport in the control of intraceUular inositol during growth arrest and the induction of differentiation is unknown. However, the observation that inositol transport rates decrease as HL60 cells become terminally differentiated, has an interesting parallel in the slow rates of inositol transport in mature blood neutrophils. It is possible that the reduced rate of inositol transport seen in neutrophils and to a lesser extent in DMSO-induced HL60 cells, is the consequence of either direct inhibition of the transporters' intrinsic activity or reduced expression of the transporter protein which occurs as the precursor cells become committed to their fully differentiated forms. Thus, inositol transport, although important in sustaining the inositol supply in growing and differentiating cells, is less important in mature cells, possibly because mature, non-dividing cells have a reduced inositol requirement. The decreased inositol transport capacity of periph-
164 eral blood neutrophils and DMSO-induced HL60 cells may be caused either by interconversion of transporters between active and inactiw=-states or by alteration of the rate of synthesis of the transporters and their expression at the plasma membrane. Both types of process are involved in the superficially similar regulation of intracenular glucose levels by the glucose transporter [29]. The regulation of inositol transport in HL60 cells reported here, may provide an appropriate experimental system in which to answer these questions.
Acknowledgements This study was supported by grants from the Leukaemia Research Fund, the British Diabetic Association and the Medical Research Council. P.J.F. is the recipient of a SERC-CASE studentship and J.M.L. is a Royal Society 1983 University Research Fellow. References 1 Miehell, R,H,, Drummond, A. and Downes, C.P., eds. (1989) Inositol Lipids in Cell Signalling. Academic Press, London. 2 Berridge, M.J. (1984) Biochem. J. 220, 345-360. 3 Berridge, MJ. and h'vine, R.F. (1984) Nature 312, 315-321. 4 Nishizuka, Y.N. (1988) Nature 334, 661-665. 5 Lord, J.M., Bunce, C.M. and Brown, G. (1988) Br. J. Cancer 58, 549-555. 6 Kraft, A.S. and Anderson, W.B. (1983) Nature 303, 621-623. 7 Huberman, E. and Callaham, M.J. (1979) Proc. Natl. Acad. Sci. USA 76, 1293-1297. 8 Collins, S J , Ruscetti, F.W., Gallagher, R.E. and Galio. R.C. (1978) Pro¢, Natl. Acad. Sci, USA 75, 2458-2562. 9 Hemmi, H. and Breitman, T. (1987) Blood 69, 501-507.
10 Tanaka, H., Abe, E., Miyaura, C., Kuribayashi, T., Konno, K., Nishii, Y. and Suda, T. (1982) Biochem. J. 204, 713-719. 11 Collins, S.J. (1987) Blood 70, 1233-1244. 12 Michell, R.H., King, C.E., Piper, C.E., Stephens, L.R., Bunce, C.M., Guy, G.R. and Brown, G. (1989) in Cell Physiology of Blood. Vol. 43 Society of General Physiologists series (Gunn, R.D. and Parker, J.C., eds.), pp. 345-355. 13 Yorek, M.A., Dunlop, J.A. and Ginsberg, B. (1986) Arch. Biochem. Biophys. 246, 801-807. 14 Oishi, K., Zhengs, B. and Kuo, J.F. (1990) J. Biol. Chem. 265, 70-75. 15 Rozengurt, E. (1986) Science 234, 161-166. 16 Greene, D.A., Lattimer, S.A. and Sima, A.A.F. (1987) N. EngI.J. Med. 316, 599-606. 17 Toksoz, D., Bunce, C.M., Stone, P.C.W., Michell, R.H. and Brown, G. (1982) Leuk. Res. 6, 491-498. 18 Simmons, D., Bonford, J. and Ng, L.L. (1990) Clin. Sci. 78, 335-341. 19 French, PJ., Bunce, C.M., Brown, G., Creba, J.A. and Michell, R.H. (1988) Biochem. Soc. Trans. 16, 985-986. 20 Berridge, M.J., Dawson, R.M.C, Downes, C.P., Heslop, J.P. and Irvine, R.F. (1983) Biochem. J. 212, 473-482. 21 Greene, D.A. and Lattimer, S.A. (1982) J. Clin. Invest. 70, 10091018. 22 Joost, H.G., Weber, T.M. and Cushman, S.W. (1988) Biochem. J. 249, 155-161. 23 Cotlier, E. (1970) Invest. Opthalmol. 9, 681-691. 24 Prpic, V., Blackmore, P.F. and Exton, J.H. (1982) J. Biol. Ch~m. 257, 11315-11322. 25 Gillon, K.R.W. and Hawthorne, J.N. (1983) Biochem. J. 210, 775-787. 26 Greene, D.A. and Lattimer, S.A. (1984) Diabetes 33, 712-716. 27 Mayer, J.H. and Thomlinson, D.R. (1983) Diabetologia 25, 433438. 28 Vallance, S., Downes, C.P., Cragoe, E.J. and Whetton, A.D. (1990) Biochem. J. 26, 359-364. 29 Joost, H.G. and Weber, T.M. (1989) Diabetologia 32, 831-838.
158
BBAMCR 12859
Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards peutrophils Michael A. Baxter 1, Christopher M. Bunce 2, Janet M. Lord 2, Philip J. French 3, Robert H. Michell 3 and Geoffrey Brown 2 I Department of Medicine, Universityof Birmingham. Birmingham (U.K.), 2 Department of Immunology, University of Birmingham, Birmingham ( U,K,) and 3 Department of Biochemistry, University of Birmingham, Birmingham ( U.K.) (Received I April 1990)
Key words: Inositol transport; HL60 cell; Myeloid differentiation; Neutrophil
[aHllnositol uptake by H I ~ cells was measured during DMSO-induced differentiation towards neutrophils. The values for gm (53.2/AM) and Vm,~ (5.3 pmol/min per 10 ~ cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. lnosltol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations, lnositol transport rates changed during DMSOinduced differentiation of H I ~ cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intraceilular inositol concentration from 10.9 to 132.7 pM seen between day I and day S. After 4 days exposure to DMSO the rate of inosltoi transport fell to a value of 3.2 + 0.3 pmol/min per 10 ~ cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 ;~M. The inositol transport rate, thus, appears to closely accompany changes in the intraceUular concentration of inosltol. Inositol ~vansport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced ~ cells, but the Km for inositol transport was similar in both cell types and was unchanged during H I ~ differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositoi transporter, one consequence of which is the alteration of
intraceUular inusitul concentrations. Introduction
The central role of phosphatidylinositol 4,5-bisphosphate hydrolysis in the generation of intracellular second messengers is well established [1]. Many extracellular stimuli have been shown to promote phosphatidylinositol 4,5-bisphosphate breakdown in target cells, with the subsequent generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol [2]. The Ins(1,4,5)P3 generated in this way stimulates the mobilisation of calcium from intracellular stores [3] and diacylglycerol activates the calcium-phospholipiddependent protein kinase, protein kinase C [4]. Several workers have proposed that the subsequent covalent
Abbreviations: lns(1,4,5)P3, inositol 1,4,5-triphosphate; DMSO, dimethylsulphoside. Correspondence: M. Baxter, Department of Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
modification of key cellular proteins, by protein kinases and phosphatases, is central to the co-ordinated control of cell growth and differentiation [5]. Whilst the precise role of inositol lipids and inositol phosphates in cell growth and differentiation has not yet been clearly defined, the importance of inositol lipid hydrolysis is implied by the observation that phorbol esters which, like diacylglycerol, are potent activators of protein kinase C [6], are also capable of inducing cellular differentiation [7]. HL60 is a human bipotent promyeloid cell line which, in response to specific external stimuli, undergoes growth arrest and differentiates into one of two functionally and morphologically distinct blood cell types. Dimethylsulphoxide (DMSO) or retinoic acid promote the differentiation of HL60 towards neutrophils [8,9], whilst vitamin D-3 or phorbol esters (such as 12-O-tetradecanoylphorbol-13-acetate; TPA) induce monocyte differentiation [7,10]. HL60 calls have been used extensively as a model system to study cellular events, including changes in inositol lipid metabolism, which occur
159 during growth arrest and induction of differentiation [11,12]. Exposure of HL60 cells to either DMSO or TPA has been shown to produce marked changes in the relative amounts of several inositol poly-phosphates, notably inositol tetrakisphosphate, pentakisphosphate and hexakisphosphate. Furthermore, an increase in the concentration of intracellular inositol has also been noted in HL60 cells during DMSO-induced growth arrest and differentiation (French, P.J., unpublished data). Although the pathways of metabolism of inositol poly-phosphates within cells have not yet been fully established, it is possible that changes in the level of intracellular inositol might result from the breakdown of phosphoinositides or inositol phosphates. Alternatively, inositol could be derived from extracellular sources via stimulation of transmembrane inositol transport [13], several reports have suggested that cellular differentiation is preceded by the activation of other plasma membrane transport systems [14,15]. Inositol transport has previously been studied in a number of mammalian tissues and cell lines, including HL60. In these cells, it is an active, highly specific and sodium-dependent process [13,16]. The inositol transporter is, therefore, a potential site for the regulation of intracellular inositol concentration. This study has investigated inositol transport into HL60 cells undergoing neutrophil differentiation and has considered these findings in relation to the cells' intracellular inositol concentration, growth arrest and maturation. The role of inositol transport in the fully differentiated human peripheral blood neutrophil is also considered. Materials and Methods
Cell culture HL60 cells were maintained in long-term exponential growth in inositol free RPMI 1640 medium (Northumbria Biologicals, Northumberland, U.K.) supplemented with I mg/l myo-inositol (Gibco, Paisley, U.K.), a serum replacement (ITS + pre-mix (Universal Biologicals, London, U.K.)), L-glutamine (Gibco) and antibiotics (100 U / m l penicillin and 50 # g / m l streptomycin, Gibco). Differentiation of HL60 cells towards neutrophils when maintained in this medium was optimally induced by the addition of 0.970 DMSO. At time zero, cells were resuspended at 2.10 5 cells/nil in medium containing 0.970 DMSO and established as 200 ml cultures in 175 cm2 tissue culture flasks (Nunc, Paisley, U.K.) and cultured for up to 7 days. Aliquots of cells were taken at daily intervals to assess numbers of viable cells and to monitor the differentiation status of the culture as revealed by the cells ability to phagocytose complement-coated yeasts [17]. Both uninduced and DMSO-induced HL60 cultures were maiutained at 37 °C in a humidified atmosphere containing 570 CO2.
Isolation of human blood neutrophi/s For inositol transport studies normal mature neutrophils were prepared using density sedimentation as previously described [18]. Briefly, 14 ml of heparinised peripheral blood were mixed with 2 ml of Hespan (DuPont, Stevenage, U.K.) and left to stand in universal containers (Sterilin, Paisley, U.K.) at 37 °C for 40 rain. The leucocyte-enriched supernate was removed and the cells washed twice in phosphate-buffered saline (once at 1500 rpm, 10 rain, 4 ° C; and twice at 1000 rpm, 10 rain, 4°C; MSE 3000, MSE, Crawley, U.K.). In control transport studies HL60 cells were incubated in 87.570 human AB serum, 0.12570 Hespan for 40 min at 37°C prior to inositol transport assays as described below. Inositol transport assays HL60 cells were harvested from the culture medium (10 min, 1000 rpm, MSE3000) and washed twice in phosphate-buffered saline (PBS). Cells were resuspended in PBS at (2-9). 107 cells/ml and a 600 #! aliquot was subsequently mixed with 0.1-1/~Ci [14C]inulin (9.42 mCi/mmol, Amersham International, Amersham, U.K.). In a separate tube, 500 #l PBS containing [3H]inositol (Amersham International) at a specific activity of 0.2 pCi/nmol, inositol (0-150/~M) and glucose (0-20 mM) was simultaneously incubated for 10 min at 37 ° C. Inositol uptake was initiated by the addition of 500/Ll of the cell/inulin mixture. 200-600 pl aliquots were taken at time intervals up to 15 min and the reaction terminated by dilution in ice-cold PBS (400/tl). Cells were recovered by spinning rapidly (30 s, microfuge B, Beckman, High Wycombe, U.K.) through dibutyl phthalate oil (Sigma, Poole, U.K.), snap freezing in liquid nitrogen and excision of the tip of the microfuge tube with a hot scalpel blade. Tips were transferred to a scintillation vial containing 400/~l distilled water, 50 pl Optisolve (FSA Laboratory Supplies, Loughborough, U.K.) and 4 ml scintillation fluid. 14C and aH radioactivity was measured using a dual label programme (LKB Mk3, LKB-Pharmacia, Milton Keynes, U.K.). Labelling and extraction of [SH]inositol HL60 cells were incubated for 6 days in modified RPMI 1640 medium as described above and containing 1/~Ci//~g [3H]inositol as previously described [19], prior to treatment with 0.970 DMSO for up to 7 days. Inositol and inositol phosphates were extracted from control and DMSO-treated cells essentially by the method of Berridge et al. [20]. Briefly, 10 7 cells were harvested, washed once in inositol free RPMI 1640 medium and lysed by the addition of 500 ~1 of 2070 TCA containing 250 /~g/ml phytic acid and 0.570 phytate hydrolysate [19]. Inositol lipids were sedimented (1 rain, 6500 rpm, MSE microcentaur, MSE) and the soluble inositol and
160 inositol phosphates transferred to a 5 ml polypropylene tube. The lipid pellet was washed once with 500/tl of 2~ TCA, the supernatants pooled and washed four times with 4 ml of diethyl ether. Following the last diethyl ether wash 100 ttl of 50 mM EDTA (pH 7) was added to the inositol extract which was stored at - 20 ° C prior to fractionation by HPLC. The inositol extract was applied to a 25 cm partisil P10 SAX HPLC column (Thames Chromatography, Maidenhead, U.K.) and inositol separated from inositol phosphates, which were retained by the column and subsequently removed with 1 M ammonium phosphate. 0.5 ml fractions were collected into 38~ methanol prior to the addition of liquid scintillant and liquid scintillation spectrometry. The distribution of [3H]inositol within HL60 cells after 15 min incubation was determined as follows. HL60 cells (0.64.10'~/ml) were incubated with 15/~M inositol and 0.4 ~tCi/nmol [3H]inositol in 1 ml of PBS at 37 o C. After 15 rain, 600 ~tl aliquots were placed onto a silicone oil cushion (62~ Siliconil APl00; 38~$ Siliconil AR20, Wacker-Chemic GMBH, Munich), which was layered over 500/~1 of 20~ TCA. The incubation was terminated by centrifugation of the cells through the oil into the TCA (MSE Microcentaur, 13000 rpm, 1 rain, Anderson, K. personal communication). Buffer and oil layers were discarded, leaving the TCA and cell pellet. The TCA layer was placed into a 5 ml polypropylene tube and 0.5 ml of distilled water added before washing four times with 4 ml diethyl ether. Following the last diethyl ether wash 100 ~tl of 50 mM EDTA (pH 7) was added and the volume of the sample made up to 10 ml with distilled water. This extract was loaded onto a Dowex AG1-X8 (formate form, Bio-Rad Laboratories,
!
80"
40
i
30
20
_e
i
lO
O, 0 Time (rain)
Fig. 1. Effect of extracellular inositol concentration on inositol uptake into HL60 cells. HL60 cells ((0.2-1)- 107/ml) were incubated at 37 o C in PBS containin8 5 (e e), 10 (0 0), 15 (ra ra) or 50 FM (U I ) inositol and [3Hl-inositol (0.2 FCi/nmol). Aliquots were taken at the time points shown and cells were collected by spinning through dibutyl phthalate oil, as described in Materials and Methods. Cell pellets were counted for 3H radioactivity by fiquid scintillation spectrometry.
Hemel Hempstead, U.K.) 2.5 cm column. Any remaining unbound material was eluted using 5 ml of distilled water, combined with the column eluate and taken as the 'free inositol fraction'. Inositol phosphates were recovered by elution of the column with 2 M ammonium formate/0.1 M formic acid. A 2 ml aliquot of each extract was suspended in 20 ml of scintillant (15 g PPO; 0.375 g POPOP in 2.5 I xylene; 1 : 1 mixture with Triton X-100) and the radioactivity determined by liquid scintillation counting. The pellet was resuspended in 0.3 ml distilled water and 1.125 ml of a chloroform/methanol/concentrated HCI mixture (100: 200: 1) added. This mixture was left overnight at room temperature to allow extraction of the inositol phospholipids from the particulate cellular fraction. Each extract was mixed with 0.375/tl of 0.3 M HCI containing 1 mM inositol and 2 M KCI before centrifugation (2000 rpm, 5 rain, MSE 3000). The lower phase was collected and air dried in a 5 ml polypropylene tube and dried under a stream of nitrogen. After addition of 5 ml of scintillant, radioactivity was determined by liquid scintillation counting. Results
Previous studies in this laboratory (McConnell, F.M., unpublished data) have shown that HL60 cells do not synthesise inositol de novo and must obtain 95% or more of their inositol requirement from the extracellular growth medium as preformed inositol. In the inositol transport assays reported in this study, total 3H counts were derived from both intracellular [3H]inositol and extracellular [3H]inositol trapped in the cell pellet. The volume of extracellular fluid estimated using [14C]inulin was small (0.06 + 0.06/zl/10 z cells), particularly when compared with an intracellular volume for HL60 of 1 /tl/106 cells. However, the specific activity of [3H]inositol in the medium was high (0.2/tCi/nmol) and even a small extracellular contamination could produce large errors in the recorded counts. A correction for extracellular [3H]inositol contamination was therefore made to all data.
Characterisation of the HL60 inositol transporter Fig. 1 shows that the accumulation of intracellular inositol into uninduced HL60 cells at a variety of extracellular inositol concentrations was linear for up to 15 min at 37°C. We have determined the mean cell volume of HL60 cells in the uninduced state and also during differentiation towards neutrophils (data not shown). Using this information, data from Fig. 1 and the intracellular labelling experiments outlined below, we have calculated that after 15 min incubation at 37 o C, the concentration of intracellular inositol exceeds that of extracellular inositol. At this point inositol is being transported against a concentration gradient, a
161 property characteristic of an active process. The linearity of inositol accumulation during the first 15 rain of incubation at 37°C strongly suggests that the time courses shown represent initial rates of inositol accumulation. Although such uptake kinetics are consistent with all previous studies of inositol transport I13,18,21], it is in marked contrast to the transport kinetics of hexose sugars, such as glucose [22]. This may indicate that in HL60 cells the inositol transport rate is relatively slow in both directions, but that influx is much more rapid than efflux. This has been confirmed in other cell types [23,24]. Analysis of cell extracts from HL60 cells labelled with [3H]inositol for 15 min revealed that 76.7 + 2.13~ (n ffi 5) of the counts were present as free inositol with only 0.8 :k 0.06~ (n ffi 5) in the form of inositol phosphates. There was, however,, a rapid sequestration of [3H]inositol into phospholipids (23.3 + 2.13~ (n = 5)) and this may partially explain the observed maintenance of the initial uptake rates. Inositol uptake rates were measured over a range of physiological concentrations of inositol (5-150 pM). A plot of inositol concentration against inositol transport rates (Fig. 2), demonstrates kinetics typical of a highly specific, carrier mediated system. Failure to achieve complete saturation of inositol uptake probably reflects the fact that HL60 cells, in addition to a high affinity transport system, also possesses a non-specific, passive, low affinity carrier system. Such a mechanism is well characterised in other cell systems, including myeloid
$
3
S
2
1
0:l
'1
0
20
40
60
80
100
0'.2 120
140
Concentration of inositolgmolar (S)
Fig. 2. Rate of inositol uptake into HL60 cells versus inositol concentration. HL60 cells ((0.2-1)-107/ml) were incubated for 10 rain at Y/°C in PBS containing inositol (5-150 FM) and [3H]inositol (0.2 pCi/nmol). Aliquots were taken for the estimation of inositol uptake
and cells were collected by spinning through dibutyl phthalate oil, as described in materialsand methods. Cell pellets were counted for 3H radioactivity by liquid scintillation spectrometry. Values shown are mean+ S.E. of five determinations. Insert: Reciprocal values for the above data are plotted as a Lineweaver-Burkeplot. The intercept on the x-axis= - 1 / K m, the intercept on the y-axis= 1/Vma~.
TABLE I
Effect of extraceilular glucose concentration on the kinetics of inositol uptake into HL60 cells HL60 cells (2.106/ml) were incubated at 3 7 ° C in PBS containing inositol (5-150 ~M), [3H]inositol and D-glucose (0-20 raM). Cell samples were taken after 10 min for estimation of inositol uptake as described in Materials and Methods. K m and Vmax values were determined from a double-reciprocal plot of the inositol uptake data, numbers in parentheses indicate the number of replicates used to
determine each mean value Glucose concentration (raM) 0 5 10
20
Km
Vraax
(~M)
(pmol/min per 106cells)
53.2+ 3.9 (5) 53.0+ 2.0 (4) 56.8± 4.0 (4) 55.5 + 4.4 (4)
5.3 + 0.53 (5) 5.3 + 0.2 (4) 5.5 + 0.45 (4) 4.1 + 0.4 (4)
cells, derived from human tissues [13,18]. The reported K m value for the low affinity transporter is approx. 400 /~M, whilst the normal serum concentration of inositol is in the range 50-100 ~M [13], suggesting that this transporter has an insignificant physiological role. Similarly, the low affinity uptake of inositol would be expected to have little effect on the kinetics of the specific, high affinity inositol transport, which was determined using low inositol concentrations (5-150/~M). This is supported by the linear double-r~c,.p,'cca! plot (insert (Fig. 2; r values 0.96-1.00) from which the K m and Vma~ values for the high affinity transporter were calculated. Calculation of these parameters using a S vs. S ~ V plot gave comparable results (data not shown). In undifferentiated HL60 cells the K m and i ma~ values for inositol transport were 53.2 + 3.9 ~M (n = 5) and 5.3 :t: 0.5 pmol/min per 106 cells (n = 5), respectively, and are in good agreement with published values for this cell line [13]. Furthermore, Table I shows that, even at high concentrations of glucose (20 raM), no significant effect was seen on either the Km or Vma~ for inositol transport. Taken together, the above data suggest that the transport of inositol into HL60 cells is via an active, specific, carrier mechanism which is distinct from the glucose transporter. Inositol transport rate and accumulation during H L 6 0 cell differentiation The time-course of the effect of DMSO on the growth and differentiation of HL60 cells is shown in Fig. 3A. Following a 2-3 day phase of exponential growth, there is a dramatic arrest of cell growth which is accompanied by a rapid induction of phagocytic ability. After 7 days of culture in 0.9~ DMSO, 82% of the cells exhibit functional and morphological characteristics of mature neutrophils. Fig. 3B shows the concommitant changes in Vma~ for inositol transport seen in HL60 cells when
162 exposed to 0.9~ DMSO. After 2 days of DMSO treatment the rate of inositol transport increased from 5.3 40.5 pmol/min per 106 cells (n = 5) to 10.44-0.4 pmol/min per 10 6 cells (n = 3). The level of inositol transport then remained elevated for 3 days, peaking at 12.4 + 0.4 pmol/min per 106 cells (n = 3) at day 4, before returning to a lower rate of 3.2 4- 0.38 pmol/min per 106 ceils (n = 3) after 7 days when the cells had optimally differentiated towards neutrophils (Fig. 3A). In contrast to the rate of inositol transport, the Km values did not vary significantly throughout the 7 days of treatment with DMSO (legend to Fig. 3). The observed changes in inositol transport were accompanied by changes in intracellular inositol concentration. As shown in Fig. 3B, uninduced HL60 cells had an intracellular inositol concentration of 12.6 /~M which was unchanged (10.9 aM) after 1 day of exposure to DM$O. However, by day 2 intracellular inositol had increased to 28.9/~M and inositol levels continued to rise in the differentiating cells to a value of 132.7/~M after 5 days exposure to DMSO. This value represented the peak of inositol accumulation within the cells and from day 5 to day 7 inositol concentrations fell slowly, but progressively to a lower value of 112.0 #M. The data shown in Fig. 3 suggest that the increased intracellular free inositol seen during the differentiation of HL60 cells towards neutrophils is associated with, and at least in
part produced by, an increased capacity of the cells to transport inositol from the extracellular medium. Inositol transport in neutrophils
It is of interest that in terminally differentiated HL60 cells, after 7 days of DMSO exposure, the rate of inositol transport (Vmax) fell to a rate which was below that of the undifferentiated cells. This suggests that, although, inositol transport has an important role in precursor and differentiating myeloid cells, the requirement for inositol uptake is reduced in the fully differentiated neutrophils. In an attempt to address this question we have measured the rate of inositol transport in mature human blood neutrophils. Although the K m of the neutrophil system for inositol transport (64 + 6.0 /~M, n = 8) was similar to that of HL60 cells, the rate of inositol transport was very much slower. As shown in Fig. 3B, the Vmax for inositol transport in neutrophils was estimated at 0.3 4-0.07 pmol/min per 106 cells (n - 8) compared with 5.3 4- 0.53 (n - 5) and 3.2 4- 0.38 pmol/min per 106 cells (n = 3) for uninduced and day 7 DMSO-treated HL60 cells, respectively. These findings are supported in a recent report by Simmons and coworkers [18], who also demonstrated relatively slow rates of inositol transport in human peripheral blood neutrophils. However, to rule out the possibility that the isolation of neutrophils with Hespan (see Materials and
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FiB. 3. Chanees in inositol transport and intracellular inositol concentration during HL60 cell differentiation towards neutrophils. HL60 cells were cultured for 7 days in medium containing 1 mg/i inositol and 0.9% DMSO v/v. (A) Cells were removed daily for enumeration of viable cells (O [J) and assessment of the percentage of cells able to phagocytose complement coated yeasts (e e). (B) Human peripheral blood neutrophils (N) and, on appropriate days HL60 cells, were resuspended at (2-9). 106/ml in PBS for determination of inositol uptake rates and Vm~, values ( I i), as described in Materials and Methods. K m values for inositol transport were unchanged by exposure of HL60 cells to 0.9% DMSO and were identical for HL60 cells and human neutrophils (range 53.2+3.9/tM to 67.6+5.2 /~M ( n = 5 ) and 64.0+6.0/~M ( n = 8 ) , respectively), in parallel experiments (n = 2), in which cells were labelled to equilibrium with [aH]inositol prior to exposure to DMSO, intracellular inositol concentrations (o ~ o) were determined in tripficate by the method of Berridge et el. [20], as described in Materials and Methods.
163 Methods) impairs inositol transport, HL60 cells were similarly treated prior to inositol transport assays. In these experiments, neither the K m (45 #M) nor the Vm~, (4.6 pmol/min per 10 6 cells) for inositol transport were significantly altered when compared to non-Hespantreated HL60 cells. Therefore, the reduced rate of inositol transport observed in terminally differentiated HL60 cells in vitro, is likely to be a reflection of a reduced dependence on extracellular inositol in normal peripheral blood neutrophils. Discussion
Although the role of inositol phospholipids in the regulation of cell function is well established, their precise role in cellular differentiation is less well understood. The observation that a number of growth factors stimulate phosphatidylinositol 4,5-bisphosphate breakdown [2] and that phorbol esters, powerful promoters of cell differentiation, appear to exert their effects via protein kinase C activation [7] has been taken as evidence that inositol phospholipid hydrolysis has a role in regulating cell growth and differentiation. In addition, increases in the levels of intracellular InsP~ and InsP6, which are probably not derived from lipids, have been reported in HL60 cells stimulated to differentiate towards neutrophils [12]. The physiological significance of these observations remains unclear. It has also been noted (French, P.J., unpublished data) and confirmed in this study that there is a large increase in intraceUular free inositol when HL60 cells are stimulated to differentiate towards neutrophils by exposure to DMSO. The source of this inositol was previously unknown but the overall rise in intracellular inositol phosphates during HL60 neutrophil differentiation [12] suggested that it was unlikely to be solely derived from metabolism of the higher inositol phosphates. The most likely explanation now seems to be enhanced entry from the extracellular medium mediated by an increase in the rate of transmembrane transport. Inositol is transported into many mammalian cells and tissues by a specific, active and sodium-dependent mechanism characteristic of a system mediated by a specific protein transporter [13,18,21] and modulation of intracellular inositol concentrations via inositol transport has been proposed previously [25,26]. For example, tissues affected by the complications of uncontrolled acute and chronic diabetes have been shown to be inositol-depleted [26]: these tissues also have a reduced rate of inositol transport [25,26]. Furthermore, reactivation of inositol transport in experimental model systems of diabetes has been claimed to restore intracellular inositol concentrations to normal [26] and in some circumstances to reverse the clinical course of diabetic complications [27]. This study has investigated the role of inositol transport as a regulator of intracellular inositol concentra-
tion. The data presented here suggest that inositol uptake in HL60 cells is mediated by a specific and active process with K m and Vmax values similar to those reported in other systems. DMSO-induced growth arrest and cellular differentiation of HL60 was accompanied by an activation of inositol transport via an increase in Vmax, leading to a large rise (13-fold) in the intracellular inositol concentration. Interestingly, however, the changes in intracdlular inositol concentration and Vmax were not parallel over the 7 day treatment period. Inositol levels rose between days 1 and 5 of DMSO treatment, the period leading up to the cells growth arrest. The increase in limax preceded the rise in inositol concentration. Similarly, the decline in Vm~x from day 3 occurred 1 day prior to the decline in inositol concentration which continued gradually up to day 7. Thus, the apparent increase in the rate of inositol transport and the increased intracellular inositol concentration occur in association with growth arrest: they precede the acquisition of the differentiated phenotype, which is accompanied by a slight fall in intracellular inositol levels and a substantially reduced Vma~ for inositol transport. Cellular differentiation appears to be ~sociated with the activation of a number of cell membrane enzymes and transporters, in particular large increases in the flux of Na + ions have been noted [14,15,28]. Moreover, it has recently been suggested that Na+/K+-ATPase is a target enzyme for a number of the putative second messenger systems which are thought to play a role in cell regulation [14]. Inositol transport is sodium-dependent [16] and it may be that these apparently separate phenomena are mechanistically linked. Alternatively, their simultaneous occurrence may simply serve to highlight the importance of regulation of membrane function in the transduction of intracellular messages into altered cell function, growth or differentiation status. The exact role of regulated inositol transport in the control of intraceUular inositol during growth arrest and the induction of differentiation is unknown. However, the observation that inositol transport rates decrease as HL60 cells become terminally differentiated, has an interesting parallel in the slow rates of inositol transport in mature blood neutrophils. It is possible that the reduced rate of inositol transport seen in neutrophils and to a lesser extent in DMSO-induced HL60 cells, is the consequence of either direct inhibition of the transporters' intrinsic activity or reduced expression of the transporter protein which occurs as the precursor cells become committed to their fully differentiated forms. Thus, inositol transport, although important in sustaining the inositol supply in growing and differentiating cells, is less important in mature cells, possibly because mature, non-dividing cells have a reduced inositol requirement. The decreased inositol transport capacity of periph-
164 eral blood neutrophils and DMSO-induced HL60 cells may be caused either by interconversion of transporters between active and inactiw=-states or by alteration of the rate of synthesis of the transporters and their expression at the plasma membrane. Both types of process are involved in the superficially similar regulation of intracenular glucose levels by the glucose transporter [29]. The regulation of inositol transport in HL60 cells reported here, may provide an appropriate experimental system in which to answer these questions.
Acknowledgements This study was supported by grants from the Leukaemia Research Fund, the British Diabetic Association and the Medical Research Council. P.J.F. is the recipient of a SERC-CASE studentship and J.M.L. is a Royal Society 1983 University Research Fellow. References 1 Miehell, R,H,, Drummond, A. and Downes, C.P., eds. (1989) Inositol Lipids in Cell Signalling. Academic Press, London. 2 Berridge, M.J. (1984) Biochem. J. 220, 345-360. 3 Berridge, MJ. and h'vine, R.F. (1984) Nature 312, 315-321. 4 Nishizuka, Y.N. (1988) Nature 334, 661-665. 5 Lord, J.M., Bunce, C.M. and Brown, G. (1988) Br. J. Cancer 58, 549-555. 6 Kraft, A.S. and Anderson, W.B. (1983) Nature 303, 621-623. 7 Huberman, E. and Callaham, M.J. (1979) Proc. Natl. Acad. Sci. USA 76, 1293-1297. 8 Collins, S J , Ruscetti, F.W., Gallagher, R.E. and Galio. R.C. (1978) Pro¢, Natl. Acad. Sci, USA 75, 2458-2562. 9 Hemmi, H. and Breitman, T. (1987) Blood 69, 501-507.
10 Tanaka, H., Abe, E., Miyaura, C., Kuribayashi, T., Konno, K., Nishii, Y. and Suda, T. (1982) Biochem. J. 204, 713-719. 11 Collins, S.J. (1987) Blood 70, 1233-1244. 12 Michell, R.H., King, C.E., Piper, C.E., Stephens, L.R., Bunce, C.M., Guy, G.R. and Brown, G. (1989) in Cell Physiology of Blood. Vol. 43 Society of General Physiologists series (Gunn, R.D. and Parker, J.C., eds.), pp. 345-355. 13 Yorek, M.A., Dunlop, J.A. and Ginsberg, B. (1986) Arch. Biochem. Biophys. 246, 801-807. 14 Oishi, K., Zhengs, B. and Kuo, J.F. (1990) J. Biol. Chem. 265, 70-75. 15 Rozengurt, E. (1986) Science 234, 161-166. 16 Greene, D.A., Lattimer, S.A. and Sima, A.A.F. (1987) N. EngI.J. Med. 316, 599-606. 17 Toksoz, D., Bunce, C.M., Stone, P.C.W., Michell, R.H. and Brown, G. (1982) Leuk. Res. 6, 491-498. 18 Simmons, D., Bonford, J. and Ng, L.L. (1990) Clin. Sci. 78, 335-341. 19 French, PJ., Bunce, C.M., Brown, G., Creba, J.A. and Michell, R.H. (1988) Biochem. Soc. Trans. 16, 985-986. 20 Berridge, M.J., Dawson, R.M.C, Downes, C.P., Heslop, J.P. and Irvine, R.F. (1983) Biochem. J. 212, 473-482. 21 Greene, D.A. and Lattimer, S.A. (1982) J. Clin. Invest. 70, 10091018. 22 Joost, H.G., Weber, T.M. and Cushman, S.W. (1988) Biochem. J. 249, 155-161. 23 Cotlier, E. (1970) Invest. Opthalmol. 9, 681-691. 24 Prpic, V., Blackmore, P.F. and Exton, J.H. (1982) J. Biol. Ch~m. 257, 11315-11322. 25 Gillon, K.R.W. and Hawthorne, J.N. (1983) Biochem. J. 210, 775-787. 26 Greene, D.A. and Lattimer, S.A. (1984) Diabetes 33, 712-716. 27 Mayer, J.H. and Thomlinson, D.R. (1983) Diabetologia 25, 433438. 28 Vallance, S., Downes, C.P., Cragoe, E.J. and Whetton, A.D. (1990) Biochem. J. 26, 359-364. 29 Joost, H.G. and Weber, T.M. (1989) Diabetologia 32, 831-838.
