BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 183, No. 3, 1992
Pages ] 2 1 6 - ] 2 2 3
March 31, 1992
EFFECTS OF E P I D E R M A L G R O W T H FACTOR ON GLYCOLYSIS IN A431 CELLS
Josep Baulida, Rafael Onetti and Anna Bassols*
Departament de Bioqufmica i Biologia Molecular, Facultat de Veterinhria, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Spain Received
February
II,
1992
SUMMARY: A431 cells were treated with epidermal growth factor (EGF) to study the mechanism by which this factor accelerates the glycolytic flux. After EGF treatment, fructose-2,6-bisphosphate (Fru-2,6-P2) levels rose up to 2-fold. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or translation of the enzyme, neither in the amount of enzyme. PK-C does not appear to be involved in the signalling mechanism since EGF was equally potent in PK-C depleted cells than in control cells. The increase in Fru-2,6-P 2 levels was lower and more transient in cells treated with EGF in a calcium-free medium than in the presence of the cation, and it was restored by the addition of calcium to the medium. These results suggest a possible role for calcium-mediated pathways in the control of Fru-2,6-P 2 levels in A431 cells. ® 3_992Academic P..... Inc.
Accelerated glycolysis is a common feature of transformed and rapidly growing cells. Although this characteristic has been known for many years, the molecular basis of such effect is not clear. Epidermal growth factor (EGF) accelerates glucose consumption and this effect has been related to its ability to enhance glucose uptake into the cells [1]. However, it has been known for several years that the high rate of anaerobic glycolysis caused by growth promoting factors is independent of glucose uptake, as was demonstrated in experiments performed in glucose-free medium, and presents a striking parallel with the activity of 6-phosphofructo-l-kinase (PFK-1), the key enzyme on the glycolytic pathway [2,3]. The most potent activator of PFK-1 is the bisphosphorylated sugar fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P 2 concentration is controlled by the balance between the activities of 6-phosphofructo-2-kinase (PFK-2) and Fru-2,6-P 2 bisphosphatase (Fru-2,6-P2ase). Both activities are present in the same molecule of the multifunctional enzyme PFK-2/Fru-2,6-P2ase in most tissues and cells, although different isozymes have been identified in liver, heart and muscle [4]. * To whom correspondence should be addressed. Abbreviations used: Fru-2,6-P2, fructose-2,6-bisphosphate; PFK-1, 6-phosphofructo-l-kinase; PFK-2,6-phosphofructo-2-kinase; Fru-2,6-P2ase, fructose-2,6-bisphosphatase; EGF, epidermal growth factor; PK-C, protein kinase C; TPA, 12-O-tetradecanoylphorbol 13-acetate; cyclic AMP, adenosine 3',5'-phosphate. 0006-291x/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1216
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EGF acts on the target cell after binding to a specific membrane receptor. EGF binding to the receptor induces the activation of its tyrosine kinase activity [5-7]. A recently described mechanism suggests that a main substrate of the receptor kinase is phospholipase C [8,9] which will hydrolyse phosphatidyl-inositol 4,5-bisphosphate, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In turn, diacylglycerol will activate PK-C, leading to the phosphorylation and regulation of different protein substrates. Therefore, PK-C is considered to be involved in the mechanism by which EGF exerts some of its effects. In this paper we have used A431 cells to study the role of Fru-2,6-P 2 in the stimulation of glycolysis by EGF. Our data indicate that PK-C is not involved in the mechanism of action of EGF and suggest a possible role for calcium ions.
MATERIALS AND METHODS Cell culture. A431 human epidermoid carcinoma cells were grown at 37°C in the presence of 5% CO 2 in Dulbecco's Modified Eagle Medium containing 10% calf serum with 100 IU penicillin/ml and 100 lag/ml streptomycin. This cell line has been described to contain 2.6x106 EGF receptors/cell [10]. Treatment of the cells with EGF and other effectors. Cells were plated by triplicate in 35 or 60 mm plates and the experiments were performed after 48 hours of confluency. In these conditions, cells are quiescent and the glycolytic flux is low. Sixteen hours before EGF treatment, the medium was removed and 2 or 5 ml of a low glucose, glutamine-free medium containing 126 mM NaCI, 14 mM NaHCO3, 38 mM KC1, 0.9 mM Na2HPO4, 0.6 mM KH2PO 4, 0.6 mM MgSO4, 0.3 mM CaC12, 6 mM glucose, 20 mM Hepes pH 7.2 (buffer A) was added to the plates. This medium was renewed 90 minutes before the treatment with EGF to eliminate possible secreted molecules and restore the initial glucose concentration. EGF 100 ng/ml was added to the cells for different times. Controls received 100 laM acetic acid. The triplicate plates were treated, processed and analyzed separately. At the end of the incubation, the medium was removed and used for lactate determination. Monolayers were immediately frozen in liquid nitrogen and stored at -80°C. Down regulation of PK-C was achieved by culturing the monolayers in the presence of 500 nM TPA during the 16 hours of preincubation in buffer A. The effect of extracellular calcium was examined by incubating the cells with calcium-free buffer A after rinsing the monolayers for five times with the same buffer supplemented with 0.5 mM EGTA. Cycloheximide was prepared in ethanol and actinomycin D and the calcium ionophore A23187 in DMSO. In all the cases, control cells received an identical volume of the diluent. Measurement of metabolites and enzyme activities. Glucose 6-P was determined in acid extracts by the glucose 6-P dehydrogenase method as previously described [11]. Fru-2,6-P 2 was measured in alkaline extracts by the method described by Van Schaftingen et al. [12]. Cell extracts for PFK-2 activity were prepared in 100 mM KCI, 5 mM MgC12, 1 mM EGTA, 1 mM DTr, 50 mM Hepes pH 7.1 (buffer B). Total PFK-2 activity was measured as described by Mojena et al. [13]. Protein concentration was determined as described by Lowry et al. [14]. Determination of flux through PFK-1. The flux through PFK-1 was estimated from the rate of 3H20formation from [3-3H]glucose as described in [15]. Phosphoenzyme assay of PFK-2/Fru-2,6-P a2.~.gse. PFK-2/Fru-2,6-P2ase concentration was determined in samples precipitated with polyethylene glycol (4-15 % w/v) by measuring the amount of phosphoenzyme intermediate as described by E1-Maghrabi et al. [16]. Briefly, I00 lag of protein were incubated with 1 laM [2-32p]Fru-2,6-P2 (25 x 103 cpm/pmol) in buffer B in the presence of 25 mM potassium phosphate. Immediately, samples were spotted on P81 phosphocellulose paper (Whatman) and washed extensively in 72 mM phosphoric acid before counting. Recombinant liver PFK-2 [17] was used as standard. Materials. EGF from mouse submaxillary glands was from Boehringer Manheim (grade I) or a generous gift from Dr. J. Massagu6 (Sloan Kettering Cancer Institute, New York, USA). 1217
V o l . 183, N o . 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Reagents for cell culture were from Gibco. All other reagents were from Boehringer Mannheim or Sigma.
RESULTS Effects of E G F on Fru-2,6-P2 metabolism in A431 cells Fru-2,6-P 2 levels in A431 cells were increased about 2-fold after E G F treatment. Dose-response experiments showed a maximal-effect of E G F at 100 ng/ml (results not shown), similar to other effects of the factor. The effect was apparent at 30 min of treatment and persisted for at least 4 hours (Fig. 1). Lactate production was linear along the incubation time and was enhanced about 2.5-fold by E G F (control 329 _+ 32 vs E G F 871 -+ 74 pmols/mg x h, n=3). In order to see whether the increase in Fru-2,6-P 2 had a direct effect on the glycolytic pathway, we measured the flux through PFK-1 as the release of 3H20 from [3)H]-glucose. E G F caused a 2.7-fold increase in the flux (1.40 _+ 0.16 vs 3.86 -+ 0.18 nmols transformed glucose/min x mg). The increase in Fru-2,6-Pz cannot be attributed to an increase in the uptake of glucose by the cell, since no difference in the cellular concentration of glucose 6-P was found between control and EGF-treated cells (data not shown). Total PFK-2 activity was assayed in extracts from control and EGF-treated cells and a small but consistent increase in PFK-2 activity was found after E G F treatment. The effect in PFK-2 showed a similar time-course to that observed for Fru-2,6-P 2 levels
(Fig. 2).
~, ,o 22
105 E
go
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- l
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I
.--
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~14 Q.
30
LL)
12 I
I
0
50
[
I
I
I
1 0 0 1 5 0 2 0 0 2 5 0
~
Time (min)
~
I
I
0
50
I
I
I
1 0 0 1 5 0 2 0 0 2 5 0
Time (rain)
FiE. ]. Time course of Fru-2,6-P2 concentration in A431 cells incubated with ( n ) or without (~)
addition of 100 ng/ml EGF.
Values are means -+ S.E.M. for 7 independent experiments performed by triplicate. Fig. 2. Time course of PFK-2 activity in A431 cells incubated with (m) or without (o) addition of 100 ng/ml EGF.
EGF caused a significant increase of PFK-2 activity (p
Vol. 183, No. 3, 1992
Pages ] 2 1 6 - ] 2 2 3
March 31, 1992
EFFECTS OF E P I D E R M A L G R O W T H FACTOR ON GLYCOLYSIS IN A431 CELLS
Josep Baulida, Rafael Onetti and Anna Bassols*
Departament de Bioqufmica i Biologia Molecular, Facultat de Veterinhria, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Spain Received
February
II,
1992
SUMMARY: A431 cells were treated with epidermal growth factor (EGF) to study the mechanism by which this factor accelerates the glycolytic flux. After EGF treatment, fructose-2,6-bisphosphate (Fru-2,6-P2) levels rose up to 2-fold. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or translation of the enzyme, neither in the amount of enzyme. PK-C does not appear to be involved in the signalling mechanism since EGF was equally potent in PK-C depleted cells than in control cells. The increase in Fru-2,6-P 2 levels was lower and more transient in cells treated with EGF in a calcium-free medium than in the presence of the cation, and it was restored by the addition of calcium to the medium. These results suggest a possible role for calcium-mediated pathways in the control of Fru-2,6-P 2 levels in A431 cells. ® 3_992Academic P..... Inc.
Accelerated glycolysis is a common feature of transformed and rapidly growing cells. Although this characteristic has been known for many years, the molecular basis of such effect is not clear. Epidermal growth factor (EGF) accelerates glucose consumption and this effect has been related to its ability to enhance glucose uptake into the cells [1]. However, it has been known for several years that the high rate of anaerobic glycolysis caused by growth promoting factors is independent of glucose uptake, as was demonstrated in experiments performed in glucose-free medium, and presents a striking parallel with the activity of 6-phosphofructo-l-kinase (PFK-1), the key enzyme on the glycolytic pathway [2,3]. The most potent activator of PFK-1 is the bisphosphorylated sugar fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P 2 concentration is controlled by the balance between the activities of 6-phosphofructo-2-kinase (PFK-2) and Fru-2,6-P 2 bisphosphatase (Fru-2,6-P2ase). Both activities are present in the same molecule of the multifunctional enzyme PFK-2/Fru-2,6-P2ase in most tissues and cells, although different isozymes have been identified in liver, heart and muscle [4]. * To whom correspondence should be addressed. Abbreviations used: Fru-2,6-P2, fructose-2,6-bisphosphate; PFK-1, 6-phosphofructo-l-kinase; PFK-2,6-phosphofructo-2-kinase; Fru-2,6-P2ase, fructose-2,6-bisphosphatase; EGF, epidermal growth factor; PK-C, protein kinase C; TPA, 12-O-tetradecanoylphorbol 13-acetate; cyclic AMP, adenosine 3',5'-phosphate. 0006-291x/92 $1.50 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1216
Vol. 183, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EGF acts on the target cell after binding to a specific membrane receptor. EGF binding to the receptor induces the activation of its tyrosine kinase activity [5-7]. A recently described mechanism suggests that a main substrate of the receptor kinase is phospholipase C [8,9] which will hydrolyse phosphatidyl-inositol 4,5-bisphosphate, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In turn, diacylglycerol will activate PK-C, leading to the phosphorylation and regulation of different protein substrates. Therefore, PK-C is considered to be involved in the mechanism by which EGF exerts some of its effects. In this paper we have used A431 cells to study the role of Fru-2,6-P 2 in the stimulation of glycolysis by EGF. Our data indicate that PK-C is not involved in the mechanism of action of EGF and suggest a possible role for calcium ions.
MATERIALS AND METHODS Cell culture. A431 human epidermoid carcinoma cells were grown at 37°C in the presence of 5% CO 2 in Dulbecco's Modified Eagle Medium containing 10% calf serum with 100 IU penicillin/ml and 100 lag/ml streptomycin. This cell line has been described to contain 2.6x106 EGF receptors/cell [10]. Treatment of the cells with EGF and other effectors. Cells were plated by triplicate in 35 or 60 mm plates and the experiments were performed after 48 hours of confluency. In these conditions, cells are quiescent and the glycolytic flux is low. Sixteen hours before EGF treatment, the medium was removed and 2 or 5 ml of a low glucose, glutamine-free medium containing 126 mM NaCI, 14 mM NaHCO3, 38 mM KC1, 0.9 mM Na2HPO4, 0.6 mM KH2PO 4, 0.6 mM MgSO4, 0.3 mM CaC12, 6 mM glucose, 20 mM Hepes pH 7.2 (buffer A) was added to the plates. This medium was renewed 90 minutes before the treatment with EGF to eliminate possible secreted molecules and restore the initial glucose concentration. EGF 100 ng/ml was added to the cells for different times. Controls received 100 laM acetic acid. The triplicate plates were treated, processed and analyzed separately. At the end of the incubation, the medium was removed and used for lactate determination. Monolayers were immediately frozen in liquid nitrogen and stored at -80°C. Down regulation of PK-C was achieved by culturing the monolayers in the presence of 500 nM TPA during the 16 hours of preincubation in buffer A. The effect of extracellular calcium was examined by incubating the cells with calcium-free buffer A after rinsing the monolayers for five times with the same buffer supplemented with 0.5 mM EGTA. Cycloheximide was prepared in ethanol and actinomycin D and the calcium ionophore A23187 in DMSO. In all the cases, control cells received an identical volume of the diluent. Measurement of metabolites and enzyme activities. Glucose 6-P was determined in acid extracts by the glucose 6-P dehydrogenase method as previously described [11]. Fru-2,6-P 2 was measured in alkaline extracts by the method described by Van Schaftingen et al. [12]. Cell extracts for PFK-2 activity were prepared in 100 mM KCI, 5 mM MgC12, 1 mM EGTA, 1 mM DTr, 50 mM Hepes pH 7.1 (buffer B). Total PFK-2 activity was measured as described by Mojena et al. [13]. Protein concentration was determined as described by Lowry et al. [14]. Determination of flux through PFK-1. The flux through PFK-1 was estimated from the rate of 3H20formation from [3-3H]glucose as described in [15]. Phosphoenzyme assay of PFK-2/Fru-2,6-P a2.~.gse. PFK-2/Fru-2,6-P2ase concentration was determined in samples precipitated with polyethylene glycol (4-15 % w/v) by measuring the amount of phosphoenzyme intermediate as described by E1-Maghrabi et al. [16]. Briefly, I00 lag of protein were incubated with 1 laM [2-32p]Fru-2,6-P2 (25 x 103 cpm/pmol) in buffer B in the presence of 25 mM potassium phosphate. Immediately, samples were spotted on P81 phosphocellulose paper (Whatman) and washed extensively in 72 mM phosphoric acid before counting. Recombinant liver PFK-2 [17] was used as standard. Materials. EGF from mouse submaxillary glands was from Boehringer Manheim (grade I) or a generous gift from Dr. J. Massagu6 (Sloan Kettering Cancer Institute, New York, USA). 1217
V o l . 183, N o . 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Reagents for cell culture were from Gibco. All other reagents were from Boehringer Mannheim or Sigma.
RESULTS Effects of E G F on Fru-2,6-P2 metabolism in A431 cells Fru-2,6-P 2 levels in A431 cells were increased about 2-fold after E G F treatment. Dose-response experiments showed a maximal-effect of E G F at 100 ng/ml (results not shown), similar to other effects of the factor. The effect was apparent at 30 min of treatment and persisted for at least 4 hours (Fig. 1). Lactate production was linear along the incubation time and was enhanced about 2.5-fold by E G F (control 329 _+ 32 vs E G F 871 -+ 74 pmols/mg x h, n=3). In order to see whether the increase in Fru-2,6-P 2 had a direct effect on the glycolytic pathway, we measured the flux through PFK-1 as the release of 3H20 from [3)H]-glucose. E G F caused a 2.7-fold increase in the flux (1.40 _+ 0.16 vs 3.86 -+ 0.18 nmols transformed glucose/min x mg). The increase in Fru-2,6-Pz cannot be attributed to an increase in the uptake of glucose by the cell, since no difference in the cellular concentration of glucose 6-P was found between control and EGF-treated cells (data not shown). Total PFK-2 activity was assayed in extracts from control and EGF-treated cells and a small but consistent increase in PFK-2 activity was found after E G F treatment. The effect in PFK-2 showed a similar time-course to that observed for Fru-2,6-P 2 levels
(Fig. 2).
~, ,o 22
105 E
go
•
___------
~
~ 20
E
- l
::I. 18
I
.--
60
tO
"~ 16 ~:
45
_
±
~
i ._._____~" _
~14 Q.
30
LL)
12 I
I
0
50
[
I
I
I
1 0 0 1 5 0 2 0 0 2 5 0
~
Time (min)
~
I
I
0
50
I
I
I
1 0 0 1 5 0 2 0 0 2 5 0
Time (rain)
FiE. ]. Time course of Fru-2,6-P2 concentration in A431 cells incubated with ( n ) or without (~)
addition of 100 ng/ml EGF.
Values are means -+ S.E.M. for 7 independent experiments performed by triplicate. Fig. 2. Time course of PFK-2 activity in A431 cells incubated with (m) or without (o) addition of 100 ng/ml EGF.
EGF caused a significant increase of PFK-2 activity (p
