Epidermal Growth Factor (EGF) - The Journal of Biological Chemistry

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 27, Issue of September 25, pp. 13868-13874,1988 Printed in U.S. A.

Epidermal Growth Factor(EGF) and Hormones Stimulate Phosphoinositide Hydrolysis and IncreaseEGF Receptor Protein Synthesis andmRNA Levels in Rat Liver Epithelial Cells EVIDENCE FOR PROTEIN KINASE C-DEPENDENT AND -INDEPENDENT PATHWAYS* (Received for publication, November 18, 1987)

H. Shelton Earp,n,b*C*d John R. Hepler,”.”Leslie A. Petch,’ Aileen Miller,b*fAlice R. Berry,’ JoAnna Harris,’ Victoria W. Raymond,”g Bryan K. McCune,‘ Lester W. Lee,’.’ Joe W. Grisham,a*h and T. Kendall Hardencre From the Lineberger Cancer Research Center “Cell Biology and Chemical Carcinogenesis Programs, ‘Program in Neurobiology, the Departments of *Medicine, ‘Pharmacology,and hPathology, Universityof North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514

Epidermal growth factor(EGF) stimulated the rapid sin 11. We conclude that in WB cells EGF receptor accumulation of inositol trisphosphate in WB cells, a synthesis can be regulated EGF by and otherhormones continuous line of rat hepatic epithelialcells. Since we that stimulate PtdIns(4,5)Pzhydrolysis. In thesecells, previously had shownthat EGF stimulates EGF recep- EGF receptor synthesis appears to be regulated by tor synthesis in these cells, we tested whether horseveral mechanisms: one pathway is dependent upon mones that stimulate PtdIns(4,B)Pz hydrolysis would EGF receptor activation and can operate independincrease EGF receptor protein synthesis and mRNA ently of protein kinase C activation; another pathway levels. Epinephrine, angiotensin 11, and [Ar$]vasois correlated with PtdIns(4,5)Pz hydrolysis andis depressin activate phospholipase C in WB cells as evi- pendent, at least in part, upon protein kinase C actidenced by the accumulation of the inositol phosphates, vation. inositol monophosphate, inositol bisphosphate,and inositol trisphosphate. A 3-4-h treatment with each hormone also increased therate of EGF receptor protein Stimulation of cells by mitogenic peptides involves a comsynthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [36S]methionine-labeled plex series of events that begin within the first minute of cells. Northern blot analyses of WB cell EGF receptor growth factor exposure and extend for 6-12 h (1-4). EGF’ is mRNA levels revealed that agents linked to the phos- no exception. Almost immediately upon binding to its cell phoinositidesignalingsystemincreasedreceptor surface receptor, EGF alters ion fluxes (5-8); within several mRNA content within 1-2 h. A maximal increase of hours the accumulation of specific mRNAs and thesynthetic 3-7-fold was observed after a 3-h exposure to EGF rate of specific proteins (9-13) is increased, and within 12-18 and hormones. h DNA synthesis ensues (1).In rat liver epithelial cells ( l l ) , The phorbol ester, 12-0-tetradecanoylphorbol 13- KB cells (12), and breast carcinoma cells (13), one protein acetate (TPA), which activates protein kinase C also whose synthesis appears to be regulated by EGF is the EGF stimulated EGF receptor synthesis. Pretreatment of receptor. Increased EGF receptor protein synthesis is due at WB cells for 18 h with high concentrations of TPA least in part to an enhanced accumulation of EGF receptor “down-regulated” protein kinase C and blocked TPA- mRNA (11,12). Themechanisms by which EGF-EGF recepdirected EGF receptor mRNA synthesis. In contrast, tor coupling initiates these changes are unknown. However, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Ep- the receptor, a 170,000-dalton transmembrane glycoprotein, inephrine-induced increases in EGF receptor mRNA contains an intrinsic ligand-activated, tyrosine-specific prowere reduced from 4- to 2-fold. Similarly, 18 h TPA tein kinase (14-16). Abolition of the kinase activity by sitepretreatment abolished theeffect of TPA on EGF directed mutagenesis results in a receptor that is deficient in receptor protein synthesis but did not affectEGF-de- many aspects of EGF-dependent signal transduction (17,18). pendent EGF receptor protein synthesis. The 18-h TPAThe ability of EGF to stimulate cell proliferation varies pretreatment diminished by 30-50% the induction of from cell type to cell type and often depends on the presence receptor protein synthesisby epinephrine or angioten- of additional hormones or growth factors (2, 4). In certain mouse fibroblast lines, EGF by itself is a relatively ineffective * This work was supported in partby National Institutes of Health mitogen, whereas in certain cells of epithelial origin, e.g. rat Grants DK 30002 (to H. S. E.), GM29536 (to T. K. H.), and CA hepatocytes and keratinocytes, EGF treatment is the major 29323 (to J. W. G.). The costs of publication of this article were determinant of DNA synthesis (2, 19-22). This may be due defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 to anintrinsic difference in theresponse of a specific cell type to the intracellular signal generated by the EGF receptor or U.S.C. Section 1734 solelyto indicate this fact. to a difference in the type or number of intracellular signals To whom correspondence should be addressed. ~~

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’Fellowship supported by National Institutes of Health Training Grant AM 07129. Fellowship supported by National Institutes of Health Training Grant CA 09156. ’ Fellowship supported by National Institutes of Health Training Grant ES 07126.

* The abbreviations used are: EGF, epidermal growth factor; InsP, inositol phosphates; InsPs, inositol trisphosphates; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; TPA, 12-0-tetradecanoylphorbol 13-acetate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; kb, kilobase(s).

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generated by EGF in a particular cell type. For example, the failure of EGF to trigger the proliferative response in quiescent mouse BALB/c3T3 fibroblasts has been correlated with an inability of EGF tostimulatePtdIns(4,5)Pzhydrolysis (23). In contrast, we and others have recently demonstrated EGF-dependent generation of Ins(1,4,5)P3 and mobilization of intracellular Ca2+ in cells of epithelial origin, including A431 cells (8, 24, 25), MDA468 cells (25), and hepatocytes (26). To determine whether EGF would stimulate PtdIns(4,5)P2 hydrolysis in another epithelial cell, we analyzed hormone-stimulated accumulationof InsP in WB cells, a continuous line of rat hepatic epithelial cells that exhibit a surface complementof EGF receptors comparable to hepatocytes, approximately 200-300,000/cell (27). EGF stimulated phospholipase C activity as determined by the rapid accumulation of inositol phosphates. This observation led u s to investigate the potential role of PtdIns(4,5)Pz hydrolysis and protein kinase C activation inthe regulation of EGF receptor synthesis. The results indicate that other hormones which stimulate phospholipase C i n rat hepatic epithelial cells also stimulate EGF receptor mRNA accumulation and protein synthesis. EXPERIMENTALPROCEDURES

Cell Culture and Analysis of Inositol Phosphates, CAMP, and lz5IEGF Cell Surface Binding-WB cells were grownto confluence in 35mm plates as described previously (11, 27). Cells were labeled for 18 h with [3H]inositol (2-5 pCi/ml, 15 Ci/mmol, American Radiolabeled Chemical Co., St. Louis, MO) in inositol-free Dulbecco's minimal essential medium containing high glucose(4500 mg/liter) and 5% fetal calf serum. The labeling medium was replaced by Eagle's minimal essential medium-HEPES (25 mM) containing 10 mM LiC1, and cells were incubated for 10 min at 37 "C, prior to addition of hormone or vehicle for 0-10 min. Incubations were terminated by the addition of 1ml of cold 5% trichloroacetic acid and inositolphosphates purified by anionic exchange chromatography as described previously (8, 28). To resolve the isomers of InsP3, some samples were frozen at -20 "C and analyzed by high performance liquid chromatography at a later time using a Partisil SAX 10 anionic exchange column as described previously (8, 29). In controlexperiments the ability of EGF to stimulate InsP3 accumulation was demonstrated in medium with 100 nM free calcium; underthese conditions the capacity of EGF to stimulate inositol phosphate accumulation was comparable to that seen under the standard conditions. The effect of forskolin on WB cell adenylate cyclase was tested by studying the accumulation of [3H]cAMPfrom cells prelabeled with [3H]adenine (30). EGF surface binding was performed as described previously (27). Purified mouse EGF was iodinated using chloramine T. '"I-EGF (400,000 cpm/35-mm dish) was incubated for 2 h at 4 "C with confluent WB cells in 35-mm culture dishes. After washing in phosphatebuffered saline-albumin, bound lZ6I-EGFwas quantitated in an LKB y-counter. Binding was performed in triplicate at various times after the addition of hormones. For each sample the binding in the presence of 1 pg/ml unlabeled EGF was subtracted to correct for non-specific binding. fmmunoprecipitation-Confluent WB cells grown on 35-mm plates were exposed to the indicated agents for a total of 4 h in minimal essential medium supplemented with glutamine and containing 2% of the normal methionine concentration. For the final 2 h of the incubation 50 pCi of [35S]methionine (Du Pont-New England Nuclear) was added. After detergent lysis, centrifugation, and preclearance of supernatants with Pansorbin, immunoprecipitation was performed as described previously (11). The polyclonal anti-rat EGF receptor antibody (Ab 1382) was raised in rabbits againstpurified rat liver EGF receptor (11).Immunoprecipitates were boiled in electrophoresis sample buffer containing sodium dodecyl sulfate and subjected to 6 or7% polyacrylamide gel electrophoresis. Gels were treated for 1 h with 1 M sodium salicylate and after drying were exposed to Kodak XAR film. After fluorography the p160 to p170 region was identified. In some experiments the receptor regionwas cut from dried gels.Gel pieces digested in 1 ml of 30% H'O,, 1% NHaOH overnight, and 100pl of the sample were counted using 5 ml of Scintiverse 11. In parallel experiments, total incorporation of [36S]

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methionine into trichloroacetic acid-precipitable protein was assessed. In other experiments the relative amounts of immunoprecipitatedEGF receptor were estimated by densitometric analysis of autoradiograms using an LKB laser densitometer with software for peak integration. Analysis of EGF Receptor mRNA Content-WB cells were grown to confluence on 100-mm plates and treated for 0.5-4 h with EGF, TPA, or hormones that stimulate PtdIns(4,5)Pz hydrolysis in WB cells. Total cellular RNA was isolated by the method of Chirgwin et al. (31) using 2-4 plates/time point. Cells were lysed in guanidinium isothiocyanate, layered over a cushion of 5.7 M cesium chloride in an SW 50.1 tube, and centrifuged at 40,000 rpm at 16 "C overnight. The supernatant was carefully decanted. The clear RNA pellet was resuspended in diethylpyrocarbonate-treatedwater and was precipitated with the addition of ethanol and sodium acetate. After resuspension, the absorbance ratio was determined at 260/280 nm. Twenty pg of total RNA was subjected to 1.0% agarose gel electrophoresis in formamide and transferred to nitrocellulose (32). The RNA blots were hybridized with nick-translated cDNA insert. One of two cDNAs were used 64.1, a probe of the 5"coding sequence of the human EGF receptor in pUCl0 (16) (generously provided by Axel Ullrich, Genentech), or 10.1, a 1.8-kb cDNA in pEMBL, selected with 64.1 from a Sprague-Dawley rat cDNA library.' When stated, blots were stripped and reprobed with a class I MHC probe (pH-Plla; Ref. 33) to confirm that equal amounts of mRNA were loaded in each lane. Autoradiograms were scanned as noted above. RESULTS

The addition of purified mouse EGF t o WB cells prelabeled with [3H]inosit~l resulted in a time- and concentration-dependent hydrolysis of PtdIns(4,5)P2 as determined by the accumulation of inositolphosphates(InsP).TheEGF-induced increase in InsP levels was readily measurable within 2 min (Fig. 1) and InsP accumulation plotted as a percent of maximal accumulation indicate a K0.5 between 10 and 30 n g of EGF/ml (Fig. 1).This is similarto the half-maximal effect of EGF o n the induction of EGF receptor protein synthesis (11). Other hormones known to stimulate PtdIns(4,5)Pz hydrolysis in primary culturesof hepatocytes includingepinephrine, angiotensin11, and [Ar$]vasopressin (34-36) were tested for their capacity to stimulate the accumulation of InsP i n WB cells. As shown in Fig.2, epinephrine, angiotensin11,and [Ar$]vasopressin, in addition to EGF, each significantly increased levels of inositol monophosphates, inositol bisphosphate, and InsP3 during a 5-min stimulation. High performance liquid chromatography analysis of the InsP formed in the absence of LiCl revealed that levels of Ins(1,4,5)P3, inositol1,3,4-trisphosphate, and inositol1,3,4,5-tetrakisphosphate all were elevatedafter a 60-s incubation withEGF (data not shown) or angiotensinI1 (data not shown, and Ref. 39). Previous studies demonstrated that EGF-directed EGF receptor synthesis, while barely detectableat 2 h, was stimulated by 4-&fold after a 4-h treatment with EGF (11). Therefore, WB cells were incubated for 4 h with EGF, angiotensin 11, epinephrine, TPA, or forskolin, an activator of adenylate cyclase. Isolation of [35S]methionine-labeled receptor by immunoprecipitation demonstrated that EGF, angiotensin 11, epinephrine (Fig. 3), as well as [Ar$]vasopressin and bradykinin (data not shown) each increased EGF receptor synthesis. The protein kinase C agonist TPA, an agent that does not stimulate InsP3 generation in WB cells, also increased EGF receptor synthesis. Forskolin was ineffectiveat concentrations (30 pM) that produced a 30-fold increase in [3H] CAMP production (data not shown).Theincreased EGF receptor synthesis was not due t o generalized increases in protein synthesis. Stimulating agents did not produce major changes in total [35S]methionine incorporation into trichloJ. Harris, L. A. Petch, Z. W. Raymond, D. C. Lee, and H. S. Earp, unpublished results.

for Regulation of EGF Receptor Synthesis

Multiple Pathways

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TREATMENT

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EGF TPAAng-Epi Forskolin -.

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FIG. 1. EGF-dependent accumulation of InsP in WB cells. Confluent cells were prelabeled with [3H]inositolfor 18 h. EGF was added at the indicated concentrations for 10 min (right panel)or at 300ng/mlfor the indicated times (left panel). At the end of the incubations, 5% trichloroacetic acidwasadded and total InsP assessed by anion exchange chromatography as described. Blank ( t = 0) levels of radioactivity, in counts/min, were 431 k 20 (left panel) and 585 f 21 (rightpanel) and were subtracted from the values presented. WB Cells: Separation of IP Sugars After Stimulation for

5 Minutes

; x N

I!

5

% h $ f

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35S-METHIONINE (2h)

*p95%, we have shown that WB cells treated with 600 nM TPA for 18 h no longer respond to TPA with increased mRNA levels or receptor protein synthesis. Moreover, added TPA will no longer block phospholipase C activation by EGF and other agonists, e.g. epinephrine and angiotensin I1 (Fig. 7). Therefore, it is reasonable to assume that EGF can increase receptor synthesis by a protein kinase C-independent pathway in WB cells. On the other hand, there is a protein kinase C-dependent mechanismin that TPA itself increases receptor proteinand mRNA accumulation, an effect that is blocked completely by 18 h TPA pretreatment. Bjorge and Kudlow (42) have also demonstrated that TPA increases EGF receptor protein synthesis in a human breast carcinoma cell line, MDA468 cells. However, in contrast to our finding in WB cells, prolonged treatment of MDA468 with high concentrations of TPA abolished both TPA- and EGF-dependent EGFreceptor synthesis. The question remains as to whether there is a third mechanism that is dependent upon the activation of phospholipase C but independent of protein kinase C. The existence of a third pathway is suggested by the fact that epinephrine and A. Miller, L. A. Petch, and H. S. Earp, unpublished data.

13873

angiotensin I1 still produce some increase in receptor synthesis in protein kinase C-depleted cells. Possible intracellular messages for putative a third pathway could include Ins(1,4,5)P3,one of several metabolites of Ins(1,4,5)P3, or the intracellularCa2+ signal. More direct evidence for a third pathway will be needed to confirm its existence and to understand the mechanism by which agents such as [Ar$]vasopressin and epinephrine increase receptor synthesis. A hormone-stimulated mechanism not involving phospholipase C activation cannot be ruled out. The EGF receptor is coupled by some mechanism to the enzymatic pathway which cleaves PtdIns(4,5)P2 to form diacylglycerol and InsPs in several cells of epithelial origin (8, 24-26). Others have failed to observe PtdIns(4,5)P2 hydrolysis in response to EGF in Swiss (43) and BALB/c 3T3 mouse fibroblasts (23). Our results4 using cells of mesenchymal origin, including Swiss and BALB/c 3T3 cells as well as 1321N1 human astrocytoma cells, have also failed to detect EGFdirected PtdIns(4,5)Pz hydrolysis. While far from complete, these data may suggest a lineage-specific (epithelial versus mesenchymal) or at least cell type-specific coupling of the EGF receptor to activation of phospholipase C. This finding may explain why EGF acts as an effective mitogen by itself in some cells but may only act in concert with additional mitogens in other cells. In fact, recent findings by Olashaw and Pledger (44) indicate that under certain circumstances (a 4-h incubation with cholera toxin and isobutylmethylxanthine) BALB/c 3T3 fibroblasts can be converted to cells in which EGF activatesphospholipase C. Under these conditions EGF is an effective mitogen without the prior addition of platelet-derived growth factor (44). Acknowledgments-We are indebted to Jane Radford for skillful preparation of this manuscript. We would also like to thank Axel Ullrich of Genentech who generously supplied cDNA 64.1. REFERENCES

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