Epidermal Growth Factor Stimulates the Rapid Accumulation of Inositol

Vol. 262, No. 7, Issue of March 5 , pp. 2951-2956, 1987 Printed in U.S.A.

THEJOURNALOF BtoLOClCAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Epidermal Growth Factor Stimulatesthe Rapid Accumulation of Inositol (1,4,5)-Trisphosphate anda Rise in Cytosolic Calcium Mobilized from Intracellular Stores in A431 Cells* (Received for publication, September 17, 1986)

John R. HeplerSgll, Norimichi Nakahatae, TimothyW.Lovenberge, James DiGuiseppiII**, Brian HermanII**$$, H. Shelton Earp$$OQ, and T. Kendall Harden$OlIT From the $Program inNeurobiology, the Departments of §Pharmwology, IlAnatomy, and §§Medicine,the **Laboratoriesfor Cell Biology, and $$TheLineberger Cancer Research Center, The University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514

transmembrane glycoprotein that possesses intrinsictyroExposure of A431humanepidermoidcarcinoma cells to epidermal growth factor (EGF), bradykinin, sine-specific protein kinase activity (1-5). Activation of the and histamine resulted in a time- and concentration- EGF receptor initiates a complex series of events thatincludes dependentaccumulation of the inositolphosphates the phosphorylation of several protein substrates (6, 7) in(InsP) inositol monophosphate, inositol bisphosphate, cluding autophosphorylation of the receptor (2), a rapid inand inositol trisphosphate (InsP3). Maximal concentraternalization of the EGF.receptor complex (8, 9), and mitotions of EGF (316 ng/ml; -50 nM), bradykinin (1p ~ ) , genesis (10). Because the products of many oncogenes code and histamine (1 mM) resulted in 3-, 6-, and %fold increases, respectively, inthe amountsof inositol phos- for tyrosine kinases, including the v-erb B oncogene which phates formed over a 10-min period. The KO, values codes for a truncated version of the EGF receptor (3), many for stimulation were approximately10 nM, 3 nM, and studies have focused on understanding the relationship between tyrosine kinase activity and the controlof cell growth 10 p~ for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid ac- and proliferation. However, other biochemical events may be cumulation of the two isomers of InsP3, Ins(1,3,4)P3, important in the action of EGF. For example, activation of and Ins(1,4,5)P3 as determined by high performance EGF receptors on A431 human epidermoid carcinoma cells liquid chromatography analysis; maximal accumulahas been reported to result in an increased rate of radiolabeltion of Ins(1,4,5)P3 occurred within 15 s. EGF and ing of plasma membranephosphoinositides (11,12), including bradykinin also stimulated a rapid (maximal levels PIP and PIP, (13), the production of diacylglycerol (13), the attained within 30 s after addition of hormone) and a activation of protein kinase C (14), the uptakeof 45Ca2+(11, sustained 4- and 6-fold rise, respectively, in cytosolic 12), a rapid but transient rise in cytoplasmic Ca2+ (15), and free Ca2+levels as measured by Fura-2 fluorescence. the activation of an amelioride-sensitive Na+/H+ exchange EGF and bradykinin also produced a rapid, although (13, 16). transient, 3- and 5-fold increase, respectively, in cyHormone-stimulated rises in intracellular Ca2+concentratosolic free Ca2+after chelation of extracellular Ca2+ tion occur either by influx of extracellular Ca2+ and/or by with 3 mM EGTA. These data are consistent with the release of stored Ca2+ fromintracellular organelles. Only idea that EGFelevatesintracellularCa2+levelsin A431 cells, at least in part, as a result of the rapid limited informationis available on themolecular mechanisms formation of Ins( 1,4,5)P3 and consequential the release that serve to link the activation of hormone receptors to the influx of extracellular Ca2+.In contrast, understandingof the of Ca2+from intracellular stores. molecular events underlying hormone-mediated mobilization of Ca2+ from intracellularsources has been advanced markedly in recent years. Stimulation of a variety of hormone receptors results in the rapidhydrolysis of plasma membrane The EGF’receptor is asingle-chained,170,000-dalton PIP, to form the twosecondmessengers Ins(1,4,5)P3 and * This work wassupported by United States Public Health Service diacylglycerol (17). Ins(1,4,5)P3has beenshown inmany Grant GM29536.The costs of publication of this article were defrayed tissues to release Ca2+ from intracellular stores, most imporin part by the payment of page charges. This article must therefore tantly the endoplasmic reticulum (17). The occurrence of a be hereby marked “advertisement” in accordance with 18 U.S.C. second InsP, isomer,Ins(1,3,4)P3, has been demonstrated Section 1734 solely to indicate this fact. 11 Supported by a graduate student fellowship from Lilly Research recently (18-20), although the cellular actions, if any, of this Laboratories. compound have not been defined. 17 Established Investigator of the American Heart Association. To Several recent reports provide evidence that activation of whom correspondence and reprint requests should be addressed. EGF receptors does not stimulate a measurable accumulation The abbreviations used are: EGF, epidermal growth factor; InsP, total inositol phosphates including inositol monophosphate, inositol of inositol phosphates in BALB/c/3T3 fibroblasts (21) or a bisphosphate, and inositol trisphosphate; InsP1, inositol monophos- measurable change in theradiolabeling of polyphosphoinosiphate;InsPz, inositol bisphosphate; InsP3, inositol trisphosphate; tides in rat hepatocyte membranes (22).However, data have Ins(1,3,4)P3 and Ins(1,4,5)P3, the two isomers of InsP,; PIP, phos- been reported thatsuggest that EGF stimulates the hydrolysis phatidylinositol 4-monophosphate; PIPz, phosphatidylinositol 4,5of plasma membrane phosphoinositides to form inositol phosbisphosphate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; phates in A431 cells (11-14), although this response has not HPLC, high performance liquid chromatography; BSA, bovine serum been shown directly. To this end,we have initiated studies to albumin. as well as other Ca2+ mobilizing determinewhetherEGF

2951

2952

EGF-stimulated Accumulationof Inositol (1,4,5)-Trisphosphate

passed through the column for an additional 8 min before a new sample was injected. The eluate containing InsP3 isomers was collected at 0.3-min intervals. The retention timesof inositol phosphates and adenine nucleotides gradually changed after repeated use of the HPLC columns. Therefore, the retention timesof adenine nucleotides were checked each day prior to injection of radiolabeled inositol phosphates, and adenine nucleotides were added to each sample as internal standards. The overall elution profile of 3H-labeledinositol compounds isolated from stimulated A431 cells was very similar to that previously reported for rat parotid gland (19) and for 1321N1 human astrocytoma cells (25). The identity of the radiolabeled InsP3 peaks isolated from A431 cells was verified based on their positions relative to AMP, ADP, and ATP as well as the similarity of their retention times with the reported retention times of Ins(1,4,5)P3(18, 19,25) and of Ins(1,3,4)P3(18-20,251. It has been reported previously that hs(1,3,4)P3coelutes with ATP and that parotid glands stimulated with carbachol under the conditions described here predominantly containIns(1,3,4)P3(19). The sixth peak of radioactivity from EXPERIMENTALPROCEDURES stimulated A431 cells was found to coelute with ATP, with the Materials-Dulbecco's modified Eagle's medium, Eagle's minimal predominant peak from carbachol-stimulated parotid glands, and essential medium, and fetal calf serum were purchased from Grand with a retention time and AF/P concentration consistent with its Island Biological Co. (Grand Island, NY). EGF was prepared from identity as Ins(1,3,4)P3. Irvine et al. (19) reported that Ins(1,4,5)P3 mouse maxillary glands as described previously (23). Bradykinin was eluted with a somewhat greater retention time thanATP and purchased from Boehringer Mannheim, and orthophosphoric acid Ins(1,3,4)P3. Stimulation of erythrocyte ghosts with Ca2+also pro(HPLC grade) was purchased from Fisher. Fura-2 and Fura-2-AM duces a large amount of Ins(1,4,5)P3 (26). The seventh peak of were obtained from Molecular Probes (Junction City, OR). All other radioactivity from stimulated A431 cells was found to coelute with reagents, including histamine, EGTA, bovine serum albumin (recrys- InsPs from Ca2+-stimulated human erythrocyte ghosts and with a tallized and lyophilized), lithium chloride, ATP, ADP, AMP, formic retention time and AF/P buffer concentration consistent with its acid, and ammonium formate were purchased from Sigma. m y ~ - [ ~ H ]identity as Ins(1,4,5)P3.Consistent with this conclusion, the seventh Inositol (15 Ci/mmol) was obtained from American Radiolabeled peak also was found to coelute with authentic [3H]Ins(1,4,5)P3.An 1,4,5-trisphos- eighth peak of radioactivity was also observed. Battyet al. (27) Chemicals, Inc. (St. Louis, MO), and my~-[~H]inositol phate (1Ci/mmol) was obtained from Amersham Corp. A431 epider- recently have reported that stimulation of rat cortical slices with moid carcinoma cells were obtained from the Lineberger Cancer carbachol results in the rapid formation of the novel inositol phosResearch Center Tissue Culture Facility a t the University of North phate, inositol (1,3,4,5)-tetrakisphosphate(InSP4). Based on shared elution properties off HPLC with a peak enzymatically converted Carolina, Chapel Hill. Cell Cultures"A431 human epidermoid carcinoma cell stock cul- from exogenous [3H]Ins(1,4,5)P3,as described by Nakahataand tures were maintained a t 37 "C in an8%COz humidified atmosphere Harden (25), we have tentatively identified this eighth peak to be in Dulbecco's modified Eagle's mediumcontaining high glucose (4500 InSP4. mg/liter) supplemented with 5% fetal calf serum and antibiotics. All Intracellular Calcium Measurements by Fura-,!?-Intracellular Ca2+ stock plates of cells were confluent and 6-8 days old a t the time of levels of A431 cells were measured with the fluorescent Ca2+indicator subculture. Subculture was accomplished by aspirating the medium Fura-2 (28). A431 cells were grown on individual glass coverslips (22and detaching the cells by the addition of 3 mM EGTA in isotonic mm square, Corning) for 3-4 days to a preconfluent state in medium NaC1-citrate buffer (pH 7.8) for 30 min. Cells were seeded at a 1:5 containing 5% fetal calf serum as described above. The cells were dilution onto 150-mmplastic culture plates (Nunc),and the medium serum-deprived in the same medium for 24 h, and the serum-free was replaced every 4th day. For experiments, cells were routinely medium was replaced with Eagle's minimal essential medium-HEPES (25 mm) medium containing no serum. Prior to loading with Fura-2, grown to confluency on 35-mm plastic culture dishes. Analysis of Inositol Phosphutes-Confluent cultures of A431 cells the cells were maintained a t 4 "C for 5 min. Preincubation of the cells were labeled for 24-48 h with 2-3 pCi/ml [3H]inositolin inositol-free for a brief period at low temperatures was found to reduce signifiDulbecco's modified Eagle's medium containing high glucose and no cantly the background fluorescence localized to subcellular compartfetal calf serum. After labeling, the labeling medium was replaced by ments as observed by microscopic examination. The cells then were Eagle's minimal essential medium-HEPES (25 mM) containing 10 placed at room temperature in the dark and exposed to 1 p~ Fura-2mM LiCl and incubated for 10 min at 37 "C in the presence of room AM for 20 min. Following Fura-2 loading, the cells were rinsed twice air. LiCl was not included in experiments designed to investigate the with phosphate-buffered saline containing HEPES, the rinse buffer time course of agonist-stimulated accumulation of InSPa isomers as was replaced by Eagle's minimal essential medium-HEPES (pH 7.4), determined by HPLC. Agonists were prepared in a vehicle of the and the cells were maintained at approximately 37 "C using an air same medium containing 0.1% BSA final and added for the indicated curtain incubator. Single cells were monitored for their change in times, and the reactions were terminated by the addition of 1.0 ml of Fura-2 fluorescence in response to vehicle in the presence or absence cold 5% trichloroacetic acid. Cells were scraped and collected from of drug using digital fluorescence video microscopy and the results the dish and the dish rinsed once with 0.5 ml of water. The trichlo- analyzed by digital video image processing as described previously roacetic acid precipitate was removed by centrifugation (4000 X g), (29, 30). Fura-2 fluorescence displays marked Ca2+sensitivity when and thetrichloroacetic acid was extracted from each sample by three excited at 340 nm; 380-nm excitation is used to quantitate the washes with 2 mlof diethyl ether. Residual ether was removed by hydrolyzed Fura-2 pool that is notCa2+bound (28). The ratio of these aspiration and exposure to nitrogen gas. Samples used for HPLC two wavelengths then provides a measure of cytosolic free Caz+over analysis of InsP3 isomers were frozen a t -20 'C and saved for analysis a 25 nM-1 p M range and corrects for differences in accessible volume at a latertime. [3H]Inositol-labeledinositol phosphates in theaqueous or cytoplasmic pathlengths. Following each experiment, changes in sample were determined by anion exchange chromatography as de- ratioed Fura-2 fluorescence (340 nM/380 nM) were calibrated against EGTA-buffered solutions containing defined free Cas+ concentrations scribed previously (24). Determination of Imp, Isomers by HPLC-InsP3 isomers were (29). analyzed by the method of Irvine et al. (19) and asdescribed previously with minor modifications (25). Samples were injected into a Waters RESULTS HPLC system equipped with a Whatman Partisil SAX 10 (4.6 X 250 mm) anion exchange column equilibrated with Hz0 and maintained EGF (100 ng/ml; -17 nM), bradykinin (1 p M ) , and histaat a flow rate of 1.25 ml/min. ATP, ADP, and AMP (5 nmol of each) mine (1 mM; data not shown) each stimulated a rapid rise i n were used as UV markers. After the sample was injected, a linear t h e level of total inositol phosphates, i.e. InsP, InsP2 + gradient of 0-0.7 M ammonium formate (pH 3.7) buffered by H3PO1 15 s (Fig. 1).Inositol (AF/P buffer) was eluted over the first 6 min. The concentration of InsP,, that was readily measurable within AF/P buffer then was increased linearly to 1.8 M until 32 min and phosphate levels continued to rise for up to 10 min in the the elution with 1.8 M buffer maintained until 37 min. Water was presence of each hormone(data not shown). EGF, bradykinin, hormones stimulate the formation of inositol phosphates in A431 cells and to provide a detailed characterization of the inositol phosphates formed.We report that EGF, bradykinin, rapid accumulation of InsP,, and histamine stimulate the InsP2,and InsP3 i n A431 cells i n a time- and concentrationdependent manner. Furthermore, data are presented toshow that EGF and bradykinin stimulate the rapid but transient accumulation of both isomers of Inspa, InsP(1,4,5)P3 and Ins(1,3,4)P3, and that the observed increases in inositol phosphate levels i n response to EGF and bradykinin arecorrelated with a rapid but transient rise in cytoplasmic Ca2+ i n the absence of extracellular Ca2+.These data are consistent with the idea thatEGF stimulates the formation of Ins(1,4,5)P3 t o release Ca2+from intracellular stores in A431cells.

+

EGF-stimulated Accumulation

of Inositol (1,4,5)-Trisphosphate

2953

elution profile for Ins(1,3,4)P3 andIns(1,4,5)P3 isolated from EGF- and bradykinin-stimulated A431 cells is presented in Fig. 3. Details of the characterization of these two peaks as Ins(1,3,4)P3 and Ins(1,4,5)P3are described above (see “Experimental Procedures”). Exposure of A431 cells to EGF (300 ng/ml) orbradykinin (1 p ~ for ) 15 s resulted inmarked increases in the amounts of both Ins(1,4,5)P3 and Ins(1,3,4)P~. The time coursefor hormone-stimulated accumulation of these two isomers is shown in Fig. 4. Maximal levels were attained for both Ins(1,3,4)P3 and Ins(1,4,5)P3 within 15 s. Whereas Ins( 1,4,5)P3 levels returned to control within5 min of exposure to EGF, Ins(1,3,4)P3 levels remainedelevated over this period (Fig. 4, A and B ) . Treatment of A431 cells with bradykinin(1 p ~ also ) resulted in a time-dependent TIME ( s e d FIG. 1. Time course of accumulation of inositol phosphates increase in the amount of each isomer (Fig. 4, C and D).As (Imp)in the presence of EGF and bradykinin ( B K ) .A431 cells with the response to EGF, bradykinin-stimulated levels of were grown to confluency and incubated for 2 days with 2 pCi/ml Ins(1,4,5)P3were maximal within 15s and returned to control [3H]inositol in inositol-free medium in the absence of serum. Cells within 5 min. A peak of radioactivity tentatively identified as were incubated with EGF (O),bradykinin (W), or vehicle ( 0 , O ) for the indicated times in the presence of 10 mM LiCl and were assayed Ins(1,3,4,5)P4also was observed to increase in A431 cells in shown). for the accumulation of inositol phosphates as described under “Ex- response to EGF and bradykinin (data not perimental Procedures.” Each point represents the mean of three Since EGF and braydkinin each stimulated the rapid fordishes & S.E., and the data arerepresentative of three experiments. mation of Ins(1,4,5)P3,which has been shown to be a potent releasing agent of Ca2+ from intracellular stores, it was important to determine whether thesetwo hormones were also capable of raising cytosolic Ca2+levels by mobilizing Caz+ in the absenceof extracellular Ca2+.A431 cells were loaded with the fluorescent Ca2+ indicator, Fura-2, and then exposed to EGF or bradykinin in presence the or absence of 3 mM EGTA. As illustrated in Fig. 5A, exposure of A431 cells to EGF (300 ng/ml) in the presence of 1.8 mM extracellular Ca2+resulted levels. Maximal in a rapid 4- to 5-fold risein intracellular Ca2+ Ca2+levels were attained within 30 s after addition of EGF, and levels remained elevated for up to 4 min (Fig. 5 A ) . In the presence of 3 mM EGTA in the extracellular medium, EGF also elicited a rapid 3- to 4-fold rise in intracellularCa2+levels with a peak time of 30 s following the addition of hormone. However, the maximal rise in cytosolic Ca2+levels in response to EGF in the presence of 3 mM EGTA was somewhat lower -log [AGONIST] FIG. 2. Concentration-dependent formation of inositol than that observed in the presence of extracellular Ca2+. phosphates (ZnsP) by EGF, bradykinin ( B K ) ,and histamine Furthermore, EGF-stimulatedelevation of Ca2+levels in the (HA) in A431 cells. A431 cells weregrown to confluency and presence of 3 mM EGTA returned to baseline within 4 min, incubated for 2 days with 2 pCi/ml [3H]inositol in inositol-free in contrast to the elevated levels of Ca2+ maintained in the medium in the absence of serum. Cells weretreated with the indicated presence of extracellular Ca2+over the sameperiod (Fig. 5A). or histamine (A) various concentrations of EGF (O),bradykinin (a), for 10 min in the presence of 10 mM LiCl and were assayed for the These data suggest that the EGF-stimulatedrise in cytosolic accumulation of inositol phosphates as described under “Experimen- Ca2+ in A431 cells is composed of both an intracellular and tal Procedures.” The control level of radioactivity accumulated in the an extracellular component. Similar results were obtained presence of vehicle (0.1% BSA) over the same period was 993 & 22 when A431 cells were incubated with bradykinin (Fig. 5B). cpm; this value wassubtracted from each value presented. Eachpoint Exposure of A431cells with bradykinin (1 PM) in thepresence represents the mean of three determinations, and the data are repof 1.8 mM extracellular Ca2+ resulted in a rapid 6-fold rise in resentative of three experiments. cytosolic Ca2+ levels. Bradykinin-stimulated levels peaked and histamine each increased inositol phosphate accumula- within 30 s and slowly decreased over the next 3.5 min (Fig. tion in a concentration-dependent manner with approximate 5B). In thepresence of 3 mM extracellular EGTA, bradykinin values of 10 nM, 3 nM, and 10 pM, respectively (Fig. 2). also was capable of rapidly raising intracellularCa2+levels by Exposure of A431 cells to maximal concentrations of EGF 5-fold within 30 s after the addition of hormone. However, than that observed inthe (316 ng/ml, -50 nM), bradykinin (1 p ~ ) and , histamine (1 the elevationwasusuallyless presence of Caz+, and hormone-elevated Ca2+levels returned mM) for 10minresultedin a 3-, 6-, and %fold increase, respectively, in the levels of inositol phosphates (Fig. 2). As to baseline within 90 s after the addition of hormone (Fig. shown in Table I, separation of the hormone-stimulated ino- 5B). Chelation of extracellular Ca2+ with3 mM EGTA did not sitol phosphatesby anion exchange chromatography revealed block EGF-, bradykinin-, or histamine-stimulatedaccumulathat EGF, bradykinin, and histamine eachsignificantly ( p c tion of inositol phosphates (data not shown). 0.05) increased the levels of InsP1, Inspa, and InsP3 above DISCUSSION control (vehicle) during a 5-min stimulation. Since EGF was capable of stimulating the formation of The present work demonstrates that in A431 epidermoid InsPB (Table I) A431 in cells, it was important to identify the carcinoma cells, EGF and other Ca2+ mobilizing hormones of stimulate the rapid formationof Ins(1,4,5)P3 anda concomiInsP3 products formed. To this end, the HPLC method Irvine et al. (19) was used to separate InsP3 isomers. The tant rise in cytosolic Ca2+, apparently mobilized from intraI 3.0

EGF-stimulated Accumulation of Inositol (1,4,5)-Trisphosphate

2954

TABLEI Hormone-stimulated accumulationof ImpLImpa and Imp3in A431 cells A431 cells were labeled overnight with 3 pCi/ml of [3H]inositolin inositol-free medium in the absence of fetal calf serum. Cells weretreated with vehicle (0.1% BSA), EGF, bradykinin, or histamine at the indicated concentrations for 5 min. Individual inositol phosphateswere separated and collected by anion exchange chromatography as described under "Experimental Procedures." The data are the mean f S.E. of four determinations and are representative of three experiments. Initial ( t = 0) levels of radioactivity, in cpm,for InsP,, InsPz,and InsP3were 1487 f 187, 771f 5, and 1894 f 90, respectively, and were subtracted from the values presented. InsPl

InsP,

Addition

cpm k S.E.

InsPI Control

cpm f S.E.

%

Control

cpm k S.E.

Control %

%

1731Vehicle 453 100 f 280 f 33 100 100117 f 76 (500 EGF ng/ml) 3239187" f 402 1277 468282" f 227 f 120 400" Histamine (1mM) 3229 1373 185" f 239 708303"f 66 f4 605" Bradykinin (1p ~ ) 4634 1341 267" f 432 765296" f 240 653" f 45 The increase in inositol phosphate formation dueto hormone was significantly greaterthan control withp < 0.05. Statistical analysis of the data was performed using a two-tailed Student's t test. EGF (300ng/ml)

Bradykinin (IJJM)

c

2400

s

.L

0

100-

-400

-"""."".

-

nm

500

n

g

seconds

17

FIG. 4. Time course of EGF and bradykinin receptor-stimulated accumulation of Ins(1,3,4)Ps and Ins(1,4,5)Psin A431 Fraction No. h with 3 pCi/ml[3H]inositolin FIG. 3. Elution profile of InsPsisomers isolated from A431 cells. Cellswerelabeledfor48 cells. HPLC elutionof [3H]inositol phosphatesand characterization inositol-free medium in the absence of serum. The cells were incuof the radioactivepeaksas Jns(1,4,5)P3and Ins(1,3,4)P3were as bated with 300 ng/ml EGF (A and B) or 1pM bradykinin (C and D) described under "Experimental Procedures." Data are presented as for 5-320 s in the absence of LiCl and were assayed forInsP3isomers fractions collectedat 0.3-min intervals. A431 cells were labeled for2 as described under "Experimental Procedures." The time-dependent days with3 pCi/ml [3H]inositol in inositol-free medium thein absence accumulations are presentedfor Ins(1,4,5)P3 inthe presence of EGF of serum. Top panel, initial (2' = 0) amounts of 3H radioactivity in (0)or vehicle (0)(A), Ins(1,3,4)P3in the presence of EGF (A)or vehicle (A) (B), Ins(1,4,5)P3in the presence of bradykinin (0)or the cells were measured (0),or cells were treated with 300 ng/ml vehicle (0)(C), and Ins(1,3,4)P3in the presence of bradykinin (A)or EGF for 15 s in the absence of LiCl (0).Bottom panel, initial (t = 0) amounts of 3H radioactivity in the cells were measured (O), or cells vehicle (A) (D). Exposure of A431cells to vehicle (0.1% BSA) for were treated with 1 p~ bradykinin (BK) for 15 s in the absence of times less than 320 s had no effect on the levels of InsP3 isomers LiCl (0).The data are single determinations and are representative (data not shown). Ins(1,4,5)P3and Ins(1,3,4)P3were quantitated by pooling the fractions corresponding to each InsP3 isomer. The data of seven experiments. are expressed as the percent of total radioactivity (cpm) associated with the Ins(1,3,4)P3and Ins(1,4,5)P3 peaks, respectively, at t = 0 cellular stores. This conclusion is based on the observations (i.e. % basal). Initial values (t = 0; 100%)in cpm were 1488 f 395 that EGF stimulates the accumulationof total inositol phos- (A), 80 f 7 (B),1585 f 515 (C), and 232 f 25 (D). The data are the phates ina time- and concentration-dependent manner, stim-mean f S.E. of triplicate determinations and are representative of four experiments. ulates the rapid accumulation of Ins(1,4,5)P3 and Ins( 1,3,4)P3, and stimulatesa rapid, albeit transient, rise incytosolic Caz' labeling with 32P.In contrast in the absence of extracellular Ca2+. Bradykinin produced a as determined by nonequilibrium no apparent effect on inositol to the current work, EGF had qualitatively similarresponse. Previous findings withA431 cells indirectly suggested that phosphate levels. However, the earliest time point measured response to EGF EGF activates phospholipase C. Sawyer and Cohen (11)re- was 2.5 min, and perhaps, based on the ported that EGF stimulated the incorporation of 3zPand [3H] observed in the current study, a measurable elevation could inositol into phosphatidylinositol and produceda 10-fold in- have been observed at earlier times of hormone challenge. crease in the 32Plabeling of phosphatidic acid. consistent Alternatively, a different clone of A431 cells may express a different response to EGF. withthese findings, Smith et al. (12) reportedthatEGF Several reports have demonstrated that EGF increases Ca2+ stimulated a 3-to 4-fold increase in[3H]inositol-labeled phosphatidylinositol that was accompanied by a 60-70% increase fluxes and cytoplasmic Ca2+levels in A431 cells. Sawyer and in the levels of '*C-labeled diacylglycerol. Macara (13) re- Cohen (11)observed, and Macara (13) recently confirmed, ported recently that EGF did increase the turnover of the that EGF stimulates the rapid uptake and accumulation of polyphosphoinositides, PIP and PIPz, and phosphatidic acid 45Ca2'. These reports,however, did not further test the capacI

5

9

13

21

EGF-stimulated Accumulation

of Inositol (ll4,5)-Trisphosphate

2955

intracellular calcium concentrations. Rigorous comparison of the relative capacity of EGF to increase inositol phosphate accumulation and cytosolic Ca2+ levels in A431 clones ob‘140 * O m tained fromdifferent sources should resolve this dichotomy. The present report that EGF stimulates the rapid formation of Ins(1,4,5)P3 in A431 cells demonstrates that EGF acts differently in these cells than as reported in several other tissues. Binding of EGF to BALB/c/3T3 fibroblastsresulted in mitogenesis without a measurable increase in the levels of inositol phosphates (21). Similarly, EGF stimulated an increase in tyrosine kinase activity in rat liver plasma membranes without a measurable change in the radiolabeling of membrane polyphosphoinositides (22). The reasons for the differences in the action of EGF on inositolphospholipid metabolism in A431 cells as compared to rathepatocytes and BALB/c/3T3fibroblastsare unclear. However, A431 cells express a n unusually high number of EGF receptors, 10- to seconds FIG. 5. Effect of extracellular Caz+on changes in cytosolic 100-fold greater than other tissues, and it is possible that a free Ca2+levels in response to EGF and bradykinin in A431 relationship existsbetween receptor number and thecapacity cells. Cells were grown on glass coverslips (22-mm square, Corning) of the activated receptor to functionally interact with phosand loadedwith Fura-2 as described under “Experimental Procepholipase C. Alternatively, these observed differences in the dures.” Individual cells were monitored for changesin Fura-2 fluores- action of EGF at differenttissues is consistent with the cence by digital fluorescence video microscopy and analyzed by digital possible existence of two subtypes of the EGFreceptor. Irrevideoimageprocessing as describedunder“ExperimentalProcedures.”A, A431 cells weretreated with 300 ng/ml EGFin the presence spective of the reason for these differences, the fact that EGF of 1.8 mM extracellular Ca2+ (0; n = 4), or extracellular Ca2+ was is capable of stimulating the accumulation of inositol phoschelated by the addition of 3 mM EGTA (pH 7.4, 37 “C) ( 0 n = 3); phates in A431 cells raises the possibility that EGFmay also B , A431 cells were treated with 1 p~ bradykinin in the presence of interact with phospholipase C to stimulate the hydrolysis of extracellular Ca2+(0; n = 2), or extracellular Ca2+was chelated by PIP, in other tissues. In fact, we have obtained preliminary the addition of EGTA ( 0 n = 2). The mean (S3.E.) resting level of data demonstrating the abilityof EGF to stimulate the rapid cytosolic Ca2+was calculated to be 48 k 3 nM (n = 11). formation of inositol phosphates in a rat liver epithelial cell line.’ ity of EGF to stimulate a unidirectional 45Ca2+efflux from The mechanism(s) whereby the activation of hormone re45Ca2+-preloadedcells or investigate thepossibility that EGF ceptors results in the conversion of PIP, to InsP3 has not may mobilize cellular Ca2+from intracellular as well as extra- been defined. Bradykinin and histaminehave been shown to cellular sources. It was recently demonstrated (15) that EGF stimulate the accumulation of InsP3 in several tissues, and stimulates a rapid 2- to 4-fold rise in cytoplasmic free Ca2+ recent work using washed membranes from 1321N1 human concentration in A431 cells as measured by Quin-2 fluoresastrocytoma cells has shown that carbachol (31), bradykinin, cence. This EGF-mediated rise in cytoplasmic Ca2+was not and histamine3 regulateinositol phosphate formation ina observed in the absenceof extracellular Ca2+ andwas inhib- guanine nucleotide-dependent manner. Studies with a variety ited by the Ca2+channel blockers, MnZ+ and La3+.In contrast, of other tissues and hormonereceptors also strongly suggest we observed an EGF-stimulated3-fold increase incytoplasmic the involvement of a guanine nucleotide regulatory protein in free Ca2+levels in the absenceof extracellular Ca’+. Although hormone-stimulated activation of phospholipase C (32-35). the work by Moolenaar and co-workers (15) did not include Consistent with thisidea is the recentobservation of guanine a n analysis of the effects of EGF on either inositollipid nucleotide-stimulated formation of inositol phosphatesin metabolism or inositol phosphate formation, the authors poswashed membrane preparations fromA431 cells.4 The mechtulated that perhaps EGF hydrolyzed phosphoinositides to anism(s) whereby EGF and other peptide growth factor reform diacylglycerol and an inositol phosphate that was incap- ceptors that aretyrosine-specific protein kinases, suchas the able of stimulatingthe release of Ca2+from intracellular platelet-derived growth factor receptor, regulate the breakstores. In thecells used in the current study, EGF stimulated down of PIP, are undefined; to date, a guanine nucleotide the rapid formation of Ins(1,4,5)P3,a potent releasing agent regulatory protein has notbeen implicated. A431 cells should of Ca2+ fromintracellular stores (17). provide a useful model system for comparison of the mechaThe reasons for the different results concerning effects the nisms whereby EGF and other non-tyrosine kinase receptors of EGF on inositol phosphate formation and Ca2+ mobiliza- regulate phosphoinositide metabolism and Ca2+ mobilization. tion in A431 cells are not clear. However, one possible reason Acknowledgments-We are indebtedto Elaine Lloyd for her excelmay be the existence of multiple clones of A431 cells that respond differently to EGF and other manipulations. Macaralent work in preparation of the manuscript. J. R. H. would also like to thank J. M. English for thoughtfuldiscussion,patience,and (13) reported thatit was not possible to measure intracellular constant support and encouragement. Ca2+ concentrations because the A431 clones used did not REFERENCES possess sufficient esterase activity to hydrolyze Ca2+ indicators such as Quin-2 and Fura-2. This and a previous report 1. Carpenter, G., and Cohen, S. (1978) Nature 276,409-410 (15) demonstrated that theavailable A431 clones do possess 2. Cohen, S., Ushiro, H., Stoscheck, C., and Chinkers, M. (1982)J. Bid. Chem. 257,1523-1531 sufficient esterase activity to sequester Fura-2 and Quin-2, respectively. The lack of effect of EGF to stimulatea rise in J. R. Hepler, T. K. Harden, and H. S. Earp, unpublished obsercytosolic Ca2+ in the absence of extracellular Ca2+ in A431 vations. cells (15) when measured by Quin-2 remains puzzling. HowJ. R.Hepler, N. Nakahata,and T. K. Harden,unpublished ever, it ispossible that the intracellular concentration of Quin- observation. 2wassufficientlyelevated to buffer transient changes in J. R. Hepler and T. K. Harden, unpublished observation.

2956

EGF-stimulated Accumulation. of Inositol (1,4,5)-Trisphosphute

3. Downard, J., Yarden, S. Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A., Schlessinger, J., and Waterfield, M. D. (1984)Nature 307,521-527 4. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W., Lee, J., Yarden, Y., Libberman, T. A., Schlessinger, J., Downward, J., Mayes, E. L. V., Whittle, N., Waterfield, M. D., and Seeburg, P. H. (1984)Nature 309,418-425 5. Hunter, T. (1984)Nature 311,414-416 6. Hunter, T., and Cooper, J. A. (1981)Cell 24, 741-752 7. Sawyer, S. T., and Cohen, S. (1985)J. Bwl. Chem. 260,82338236 8. Carpenter, G., and Cohen, S. (1976)J. Cell Biol. 71,159-171 9. Haigler, H., Ash, J. F., Singer, S. J., and Cohen, S. (1978)Proc. Natl. Acad. Sci. U.S. A. 74, 3918-3921 10. Carpenter, G., and Cohen, S. (1979)Annu. Rev.Biochem. 48, 193-216 11. Sawyer, S. T., and Cohen, S. (1981)Biochemistry 20,6280-6286 12. Smith, K. B., Losonczy, I., Sahai, A., Pannerselram, M., Fehnel, P., and Salomon, D.S. (1983)J. Cell. Physiol. 17,91-100 13. Macara, I. G . (1986)J.Bwl. Chem. 261,9321-9327 14. Sahai, A., Smith, K. B., Pannerselvam, M., and Salomon, D. S. (1982)Biochem. Biophys. Res. Commun. 109, 1206-1214 15. Moolenaar, W. H., Aerts, R. J., Tertoolen, L. G. J., and deLatt, S.W. (1986)J. Biol. Chem. 261, 279-284 16. Moolenaar, W. H. (1986)Annu. Reo. Physiol. 48,363-376 17. Berridge, M. J., and Irvine, R.F. (1984)Nature 312,315-321 18. Irvine, R. F., Letcher, A. J., Lander, D. J., and Downes, C. P. (1984)Biochem. J. 223,237-243 19. Irvine, R. F., Anggard, E. E., Letcher, A. J., and Downes, C. P. (1985)Biochem. J. 229,505-511 20. Burgess, G. M.,McKinney, J. S., Imine, R. F., and Putney, J.

W., Jr. (1985)Biochem. J. 232,237-243 21. Besterman, J. M., Watson, S. P., and Cuatrecasas, P. (1986)J . Bwl. Chem. 261,723-727 22. Taylor, D., Uhing, R. J., Blackmore, P. F., Pripii., V., and Exton, J. H. (1985)J. Bwl. Chem. 260,2011-2014 23. Savage, C. R.,Jr., and Cohen, S. (1972)J. BwL Chem. 247, 7609-7611 24. Nakahata, N., Martin, M. W., Hughes, A. R., Hepler, J. R.,and Harden, T. K. (1986)Mol. P h u r m o l . 29,188-195 25. Nakahata, N., and Harden, T. K. (1987)Bwchem. J. 241,337344 26. Downes, C. P., and Michell, R.H. (1981)Biochem. J. 198,133140 27. Batty, I. R.,Nahorski, S. R., and Irvine, R.F. (1985)Biochem. J. 232,211-215 28. Grynkiewicz,G., Poenie, M., and Tsien, R. Y. (1985)J. Biol. Chem. 260,3440-3450 29. DiGuiseppi, J., Inman, R., Ishihara, A., Jacobson, K., and Herman, B. (1985)Biotechnigues 3,394-403 30. Williams, D.A., Fogarty, K. E., Tsien, R. Y., and Fay, F. S. (1985)Nature 318,558-561 31. Hepler, J. R.,and Harden, T. K. (1986)Biochem. J. 239, 141146 32. Litosch, I., Wallis, C., and Fain, J. N. (1985)J. Bwl. Chem. 260, 5464-5471 33. Uhing, R.J., Prpic, V., Jiang, H., and Exton, J. H. (1986)J. Biol. Chem. 261,2140-2146 34. Straub, R.E.,and Gershengorn, M. C. (1986)J. BwL Chem. 261, 2712-2717 35. Martin, T.F. J., Bajjalieh, S. M., Lucas, D. O., and Kowalchyk, J. A. (1986)J. Bbl. Chem. 261, 10041-10049