©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Autocrine Regulation of Membrane Transforming Growth Factor- Cleavage (*)

(Received for publication, October 23, 1995)

José Baselga (§) John Mendelsohn Young-Mee Kim Atanasio Pandiella (1)

From the Receptor Biology Laboratory and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 and the Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas, Universidad de Salamanca, 37007 Salamanca, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor alpha (TGF-alpha) is biosynthesized as a membrane-bound precursor protein, pro-TGF-alpha, that undergoes sequential endoproteolytic cleavages to release a soluble form of the factor. In the present study, we have analyzed the biosynthesis and regulation of TGF-alpha production in human tumor-derived cell lines that endogenously express pro-TGF-alpha and the epidermal growth factor (EGF) receptor. These cells biosynthesized membrane-anchored forms of the TGF-alpha that accumulated on the cell surface. Membrane-bound pro-TGF-alpha interacted with the EGF receptor, and complexes of receptor and pro-TGF-alpha contained tyrosine-phosphorylated receptor. Activation of the EGF receptor by soluble EGF or TGF-alpha had a dual effect on TGF-alpha production: an increase in pro-TGF-alpha mRNA levels and an increase in pro-TGF-alpha cleavage. These effects were largely prevented by preincubation with an anti-EGF receptor monoclonal antibody that blocked ligand binding. Growth factor autoinduction of cleavage could be stimulated by several second messenger pathways that are activated by the EGF receptor, including protein kinase C and intracellular calcium, and by other alternative mechanisms. EGF-stimulated cleavage of pro-TGF-alpha could be partially blocked by inhibition of these second messenger pathways. These results suggest that juxtacrine stimulation takes place in human tumor cells that coexpress both the EGF receptor and membrane-anchored TGF-alpha and that TGF-alpha is able to induce its own endoproteolytic cleavage by activating the EGF receptor.


INTRODUCTION

Transforming growth factor alpha (TGF-alpha) (^1)is a 50-amino acid single polypeptide, initially isolated from the culture medium of several oncogenically transformed cell lines(1) , that is structurally and functionally related to the epidermal growth factor (EGF). TGF-alpha binds to the 170-kDa EGF receptor, a transmembrane glycoprotein with an extracellular ligand-binding domain and an intracellular domain that contains a tyrosine-specific protein kinase(2) . Upon binding of TGF-alpha to the EGF receptor, the tyrosine kinase is activated, resulting in a cascade of biochemical and physiological responses that are involved in the mitogenic signal transduction pathway of cells(2) . TGF-alpha has gained attention because of its predominant expression in tumor-derived cell lines and in human tumors(3, 4) , suggesting that TGF-alpha contributes to neoplastic growth through autocrine and paracrine mechanisms(5) . In fact, coexpression of high levels of TGF-alpha and the EGF receptor leads to a transformed cellular phenotype (3, 6) , and increased expression of the precursor for TGF-alpha in transgenic mice causes hyperplasia of several tissues and even neoplastic transformation(7, 8, 9) . Furthermore, the expression of EGF receptors is elevated in many epithelial tumor-derived cell lines(3) , and many types of epithelial malignancies display increased EGF receptors on their cell surface membranes(5) . This overexpression correlates with a poor clinical outcome in a number of malignancies (10) .

TGF-alpha is derived from a larger 20-22-kDa transmembrane precursor (11, 12) , pro-TGF-alpha, which undergoes several posttranslational modifications that include N- and O-linked glycosylation (13, 14, 15) and palmitoylation(13) . Several molecular forms of soluble TGF-alpha have been reported, and this heterogeneity appears to be due to both the type and degree of ectoglycosylation and the preference for different sites of proteolytic cleavage of the precursor (14, 16, 17) . In transfected fibroblasts, proteolytic maturation of TGF-alpha occurs in two steps(13, 15, 18) . At the plasma membrane or in a cellular compartment very close to it, the first cleavage of pro-TGF-alpha occurs between Ala and Val (by an activity referred to as pro-TGF-alpha-ase-I). This removes the NH(2)-terminal glycosylated extension leaving a cell-associated 17-kDa pro-TGF-alpha form that contains the mature sequence of TGF-alpha within the precursor. This cleavage step occurs rapidly (t= 15 min)(18) . Release of soluble forms of TGF-alpha occurs only after a second enzymatic activity (referred to as pro-TGF-alpha-ase-II) cleaves the Ala-Val peptide bond that links TGF-alpha to the rest of the precursor molecule. This cleavage results in the generation of a 6-kDa soluble fragment, which accumulates in the culture medium and corresponds to mature TGF-alpha, and a cell-associated 15-kDa residual terminal fragment often referred to as a tail.

In transformed cells such as retrovirally transformed fibroblasts (14, 16) and hepatoma cells(17) , indirect evidence indicates that the basal activity of both enzymatic activities can be relatively high. In these cells, primary activity of pro-TGF-alpha-ase-II can lead to production of an additional soluble 20-kDa form of TGF-alpha, designated meso-TGF-alpha, which retains the glycosylated NH(2) extension(14) .

Endoproteolytic cleavage by pro-TGF-alpha-ase-II is highly regulated and depends on the activity of several second messenger systems such as protein kinase C (PKC), free cytosolic calcium ([Ca]), and other still undefined pathways that can be stimulated by serum factors(18, 19) . The pro-TGF-alpha-ase-II cleaving activity is quite distinct from other protein maturation or degradation processes(20) . Biochemical evidence suggests that the enzyme belongs to the serine protease family (21) and requires ATP and membrane association for activity(22) , and topologically, its regulated activity depends on the presence of the enzyme and substrate at the plasma membrane. This endoproteolytic system is likely to be involved in the cleavage of other transmembrane proteins that undergo regulated release of their ectodomains(23) .

Since knowledge of the molecular properties and factors regulating release of pro-TGF-alpha and other membrane-anchored growth factors is of significant biological and potentially therapeutic interest, we have analyzed the expression, biosynthesis, and cleavage of pro-TGF-alpha in several different tumor cell lines that endogenously express both the EGF receptor and TGF-alpha. We show that membrane-bound pro-TGF-alpha accumulates at the plasma membrane and is able to interact with the EGF receptor. Moreover, TGF-alpha or EGF, by acting through the EGF receptor, is able to increase TGF-alpha production by a dual mechanism that involves mRNA increase and cleavage of the membrane-bound precursor into a soluble factor. Some of these effects are prevented by a monoclonal antibody (mAb) that binds to the extracellular domain of the EGF receptor and blocks ligand binding(24, 25) . This antibody induces tumor xenograft regression in nude mice, which bear neoplasias caused by injection of human tumor cells that overexpress both the EGF receptor and its ligand TGF-alpha(26, 27) .


EXPERIMENTAL PROCEDURES

Materials

Anti-EGF receptor mAbs 225 and 528 and anti-pro-TGF-alpha antibodies have been described previously(24, 25, 28) . EGF was from Collaborative Research (Waltham, MA) and TGF-alpha from Intergen Company (Purchase, NY). Anti-phosphotyrosine 4G10 antibody was from Upstate Biotechnology Inc. (Lake Placid, NY); phorbol 12-myristate 13-acetate (PMA) and EGTA were from Sigma; A23187 was from Calbiochem.

Cell Lines

Chinese hamster ovary cells (CHO) had been previously transfected with a cDNA that encodes the rat pro-TGF-alpha (CHO-TGF-alpha cells)(15) . A431 squamous carcinoma cells, MDA-468 breast adenocarcinoma cells, SKRC-29 renal carcinoma cells, ME-180 cervix carcinoma cells, C4I cervix carcinoma cells, and DU-145 prostate adenocarcinoma cells were obtained from ATCC. DiFi colorectal cells were generously provided by Dr. B. Boman (Creighton University, Omaha, NE). MCF-10A cells were obtained from the Michigan Cancer Foundation. Unless otherwise specified, cells were grown at 37 °C in monolayer culture with Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1). ME-180 and C4I cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium with 300 mg/liter glutamine. All culture medium were supplemented with 10% fetal bovine serum added to the medium in all cultures. MCF-10A cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium, 5% horse serum, 0.5 mg/ml hydrocortisone, 10 µg/ml insulin, and 100 µg/ml cholera enterotoxin.

Metabolic Labeling

Cells were grown in monolayer culture either in 60-mm (CHO-TGF-alpha) or 100-mm dishes (all other cell lines). When cells became near confluent, the medium was replaced with cysteine and serum-free modified Eagle's medium. After two 20-min incubations and decantations, cells were labeled for 30 min in medium with [S]cysteine (200 µCi/ml; ICN). The radioactive medium was then replaced with modified Eagle's medium, and the incubations were continued for the indicated times.

After metabolic labeling and culture, cells were rinsed with ice-cold phosphate-buffered saline (PBS) and lysed with immunoprecipitation buffer (Hepes, containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM sodium orthovanadate, and 1% Triton X-100). Lysates were incubated for 10 min on ice, and cell debris was pelleted at 10,000 times g for 10 min at 4 °C. Supernatants were transferred to fresh tubes and incubated for 2 h with rabbit anti-pro-TGF-alpha antibodies raised against a mixture of two synthetic peptides corresponding to residues 138-151 and 145-159 in the cytoplasmatic domain of the rat pro-TGF-alpha sequence (28) at a 1:100 dilution. Immunocomplexes were harvested with protein A-Sepharose and washed 4 times with immunoprecipitation buffer. The beads were heated in 30 µl of electrophoresis sample buffer, and proteins were analyzed in 13% SDS-PAGE. Gels were fixed and fluorographed using Enlightning (DuPont NEN).

Northern Analysis

Total cellular RNA was extracted from A431 cells using guanidine thiocyanate as previously described(29) . Total cellular RNA (10 µg/lane) prepared as above was electrophoresed on a 1% formaldehyde-agarose gel and transferred onto nitrocellulose (Schleicher and Schuell) membranes. The membranes were hybridized to DNA probes that were radiolabeled by random priming. Hybridizations and autoradiography were performed as previously described(29) . At least 1 times 10^8 [P]dCTP total counts were added for each hybridization. The radiolabeled probe was isolated from a plasmid containing a 1.35-kilobase pair EcoRI cut human TGF-alpha (11) .

Western Immunoblotting

Three confluent dishes (100 mm) of A431 cells were washed twice with PBS containing Ca and Mg and then lysed with 1 ml of immunoprecipitation buffer. Lysates were cleared by a 15-min centrifugation (10^4 times g, 4 °C), followed by immunoprecipitation with either the anti-pro-TGF-alpha antibody or anti-EGF receptor mAb 528 for 2 h at 4 °C. Immunocomplexes were harvested by the addition of 50 µl of a 1:1 slurry of protein A-Sepharose in PBS, washed four times with the immunoprecipitation buffer, and boiled into 30 µl of electrophoresis sample buffer. The samples were subjected to SDS-PAGE followed by overnight transfer to polyvinylidine difluoride membranes. Membranes were blocked with a solution containing 150 mM NaCl, 1% bovine serum albumin, 0.1% Tween 20, and 20 mM Tris, pH 7.4, for 1 h, then incubated with the anti-phosphotyrosine antibody (1:5000) for an additional hour, washed four times for 5 min each in the same solution, and incubated for 45-60 min with a 1:10,000 dilution of a horseradish peroxidase-conjugated secondary antibody. The membrane was vigorously washed four times for 5 min each in the same solution, followed by a 1-min incubation with a luminol-based solution and chemiluminescent detection.


RESULTS

Molecular Forms of TGF-alpha in Human Tumor Cell Lines

The presence of high M(r) soluble TGF-alpha forms in the culture medium from oncogenically transformed cells (14, 16) led us to address whether human tumor-derived cell lines have distinctive biosynthetic and/or enzymatic cleaving activity. Monolayers of different cell lines were metabolically labeled with [S]cysteine, lysed at different chase times, and immunoprecipitated with an antiserum raised against the last 14 amino acids of the cytosolic domain of pro-TGF-alpha. In the human epidermoid carcinoma cell line A431 (Fig. 1A), three major pro-TGF-alpha forms were detected at the beginning of the chase: an 18-kDa form together with two lower mobility forms of 20 and 22 kDa. When compared to CHO-TGF-alpha biosynthetic products, these forms comigrated with the nascent (18 kDa) and heterogeneously glycosylated (20-22 kDa) pro-TGF-alpha, respectively. Within the first 30 min of chase, the glycosylated 20-22-kDa forms rapidly disappeared to become a 17-kDa membrane-bound form, which remained cell-associated for up to 4 h of chase. Analysis of pro-TGF-alpha in the other tumor cell lines showed that they followed a similar pattern of pro-TGF-alpha biosynthesis (Fig. 1B and data not shown) and also predominantly accumulated the 17-kDa form. A 15-kDa band, which corresponds to the COOH-terminal fragment (tail), was seen in the CHO-TGF-alpha cells after 2 h of chase. In some experiments, this 15-kDa band could also be detected in the tumor cell lines 1 h or more after the chase, but this always represented a small fraction of the total. That these bands were all true pro-TGF-alpha biosynthetic products was verified by performing the immunoprecipitations in the presence of an excess (1 µM) of competing peptide against which the anti-pro-TGF-alpha antibody was raised (Fig. 1C).


Figure 1: Molecular forms of TGF-alpha in tumor cell lines expressing EGF receptors. A, CHO-TGF-alpha and A431 cells were metabolically labeled with [S]cysteine for 30 min and chased for the depicted time points. pro-TGF-alpha was immunoprecipitated from cell lysates with rabbit anti-pro-TGF-alpha antibodies (directed at a pro-TGF-alpha COOH-terminal sequence). Immunocomplexes were processed by SDS-PAGE and analyzed by autoradiography. The molecular weights of the different growth factor species are shown at the left. B, a panel of malignant cell lines known to express the EGF receptor was analyzed for pro-TGF-alpha expression 4 h after metabolic labeling as above. Cell lines include: A431 squamous carcinoma; DiFi colon carcinoma; SKRC-29 renal cell carcinoma; ME-140 and C4I cervix carcinoma; DU-145 prostate carcinoma; MCF-10A non-malignant breast cells; MDA-468 breast carcinoma. C, immunoprecipitations were performed in the presence of an excess (1 mM) of competing peptide against which the anti-pro-TGF-alpha antibody was raised. The competing peptide prevented immunoprecipitation of pro-TGF-alpha by the antibody.



Interaction of Pro-TGF-alpha with the EGF Receptor in A431 Cells

In cocultures of cells artificially engineered to overexpress either the EGF receptor or mutant pro-TGF-alpha resistant to endoproteolytic cleavage, these two molecules have been found to be biologically interactive, resulting in activation of receptor tyrosine kinase(30, 31, 32) . The situation with cells that endogenously express both proteins may be significantly different. First, these cells typically have a lower complement of pro-TGF-alpha than transfected cell lines; second, they produce pro-TGF-alpha that can be readily cleaved; and third, the receptor and the ligand may be present in the same cell. To analyze whether membrane-anchored pro-TGF-alpha could interact with the EGF receptor, lysates from confluent cultures of A431 cells were immunoprecipitated with either anti-pro-TGF-alpha or anti-EGF receptor antibodies, and Western blots of these immunoprecipitates were probed with an anti-phosphotyrosine antibody. As shown in Fig. 2A (left lane), a tyrosine-phosphorylated 170-kDa band was immunoprecipitated using the anti-pro-TGF-alpha antibody. This band comigrated with the EGF receptor immunoprecipitated from the same cells (Fig. 2B, left lane). Treatment of these cells with exogenous EGF induced further phosphorylation of the 170-kDa EGF receptor band (Fig. 2B, right lane) but, as anticipated, did not appreciably affect the phosphotyrosine content of EGF receptor coimmunoprecipitated with the anti-pro-TGF-alpha antibodies (Fig. 2A, right lane). These results suggest that cell membrane-bound pro-TGF-alpha partially activates EGF receptors on the A431 cells. Receptors that are not activated by endogenous pro-TGF-alpha can be activated by addition of exogenous ligand.


Figure 2: ProTGF-alpha binds to and activates the EGF receptor in A431 cells. Confluent cultures of A431 cells were incubated in the absence or presence of EGF (10 nM) for 15 min and cell lysates immunoprecipitated with anti-pro-TGF-alpha antibodies (A) or with anti-EGF receptor mAb 528 (B). Western blots were probed with anti-phosphotyrosine antibody (Anti-p tyr). IP, immunoprecipitate; EGF-R, EGF receptor; Ab, antibody.



Autocrine Regulation of TGF-alpha Production

In several cell lines that biosynthesize both the EGF receptor and pro-TGF-alpha, the mRNA coding for these proteins is usually up-regulated by increasing the level of activity of the receptor(33, 34, 35) . We asked whether there was a dual regulation of pro-TGF-alpha production, i.e. if certain treatments (stimulatory or inhibitory) could affect both synthesis on the one hand and cleavage to produce soluble factor on the other. The effects of receptor activation on pro-TGF-alpha regulation were studied by adding either EGF or TGF-alpha. As an inhibitor of receptor activity, anti-receptor mAb 225 was used. This antibody binds with high affinity to the extracellular domain of the EGF receptor, blocks binding of ligand(s) to the receptor, and decreases ligand-induced receptor phosphorylation(24, 25) .

First, the effects of EGF receptor stimulation on pro-TGF-alpha mRNA accumulation were analyzed. Addition of saturating concentrations of TGF-alpha (30 nM) increased the production of a 4-kilobase pair mRNA that hybridized with a specific human pro-TGF-alpha probe in A431 cells (Fig. 3). Having established that EGF receptor stimulation resulted in increased pro-TGF-alpha mRNA, we next analyzed the effects of EGF receptor inhibition on pro-TGF-alpha levels. Addition of mAb 225 (10-100 nM) decreased the basal pro-TGF-alpha mRNA level in A431 cells (Fig. 3). Thus, the prevention of ligand-induced activation of the receptor by a receptor-blocking antibody resulted in down-regulation of TGF-alpha mRNA expression.


Figure 3: Pro-TGF-alpha mRNA expression is induced by soluble TGF-alpha and reduced by an EGF receptor blocking antibody. Northern analysis of pro-TGF-alpha expression in total RNA isolated from A431 cells after treatment with soluble TGF-alpha or anti-EGF receptor antibody (mAb) 225 for 4 h is shown.



Studies with metabolically radiolabeled A431 cells showed that addition of exogenous TGF-alpha induced cleavage of membrane pro-TGF-alpha (Fig. 4). As expected from a precursor-product relationship, either a decrease of the 17-kDa pro-TGF-alpha form or an increase in the 15-kDa tail form is indicative of pro-TGF-alpha cleavage, and the kinetics can be followed by analyzing the labeled 17-kDa/15-kDa ratio. Treatment with EGF or TGF-alpha for as short as 15 min induced the appearance of the 15-kDa cytosolic tail of pro-TGF-alpha with a concomitant decrease in the intensity of the 17-kDa precursor form (Fig. 4A). This cleavage did not increase by further prolonging incubation times with either growth factor for up to 1 h. The persistence of a significant proportion of the 17-kDa form indicates that cleavage was not complete. Incubation with TGF-alpha also induced cleavage of membrane pro-TGF-alpha in other tumor-derived cell lines (Fig. 4B).


Figure 4: Soluble TGF-alpha induces pro-TGF-alpha cleavage in a panel of EGF receptor-expressing cell lines. A, A431 cells were metabolically labeled with [S]cysteine for 30 min and chased for the depicted time points either with no additions or treatment with TGF-alpha (10 nM) or anti-EGF receptor mAb 225 (100 nM). Pro-TGF-alpha was immunoprecipitated from cell lysates with rabbit anti-pro-TGF-alpha antibodies directed at a pro-TGF-alpha COOH-terminal sequence. Immunocomplexes were processed by SDS-PAGE and analyzed by autoradiography. B, a panel of malignant cell lines known to express the EGF receptor was analyzed as above. See Fig. 1for a description of cell lines. Cells were metabolically labeled with [S]cysteine for 30 min and chased for 45 min.



Second Messengers Regulate the Cleavage of Membrane Pro-TGF-alpha in A431 Cells

In A431 cells, stimulation of the EGF receptor leads to tyrosine phosphorylation and activation of phospholipase C (36) . This in turn induces the hydrolysis of membrane polyphosphoinositides, generating increases in both cytosolic calcium and PKC activity. This fact, together with the general importance of these pathways in the control of membrane protein ectodomain cleavage(37, 38) , led us to investigate their participation in pro-TGF-alpha release in these cells. Artificial increases in cytosolic calcium induced by treatment with the calcium ionophore A23187 augmented the conversion of 17-kDa pro-TGF-alpha to the 15-kDa tail form (Fig. 5A). The [Ca](i) rise generated by Ca ionophores is due to dual components: redistribution from intracellular stores and increased influx from the extracellular medium. To define the importance of each of these components in the [Ca](i)-induced pro-TGF-alpha cleavage, we depleted extracellular Ca from A431 cell cultures by addition of the selective Ca chelator EGTA to the culture medium 5 min prior to the addition of ionophore. This treatment, which does not affect Ca redistribution from intracellular stores(39) , completely prevented ionophore-induced pro-TGF-alpha cleavage (Fig. 5A). Therefore, agents that induce cleavage of membrane pro-TGF-alpha by raising intracellular Ca appear to require influx of the cation from the extracellular medium to the cell cytosol.


Figure 5: Effects of A23187 and PMA on pro-TGF-alpha cleavage. A, 17 kDa (uncleaved) to 15 kDa (cleaved) pro-TGF-alpha ratio in A431 cells. Cells were labeled with [S]cysteine for 30 min and chased for 45 min in complete medium containing EGF (10 nM), PMA (1 µM), or A23187 (1 µM). Where indicated, EGTA (10 mM) was added to the cultures 5 min before the addition of these agents. In lanes 7 and 8, cells had been depleted of protein kinase C by a 24-h preincubation with 1 µM PMA (PKC, -). B, control A431 cells (PKC, +) and A431 cells that had been depleted of protein kinase C by 24-h preincubation with 1 µM PMA (PKC, -) were labeled as above and chased for 30 min in the presence of EGF ± EGTA as described under A.



The participation of PKC in pro-TGF-alpha release from A431 cells was next investigated. Treatment for 45 min with the tumor-promoting phorbol ester PMA, which is known to directly activate PKC isozymes (40) , provoked 17 to 15 kDa conversion (Fig. 5A). This was prevented by prior prolonged incubation of A431 cell cultures with PMA for 24 h, a treatment that causes down-regulation of PKC activity (not shown). These PKC and Ca pathways are independent of each other, since, on the one hand, down-regulation of PKC did not prevent Ca ionophore-induced cleavage (Fig. 5A) and, on the other hand, treatment with EGTA did not prevent PMA-induced cleavage of pro-TGF-alpha (not shown). When the activation of cleavage by the two second messenger systems was compared, raising intracellular Ca was more efficient than increasing PKC activity (Fig. 5A, first 4 bars), and either pharmacological treatment was found to be more efficient than EGF/TGF-alpha in provoking processing of the 17-kDa form.

Cleavage of membrane-anchored pro-TGF-alpha by activation of the EGF receptor with EGF or TGF-alpha was only partially inhibited by pharmacological inhibition of these second messenger systems (Fig. 5A, last 4 bars, and Fig. 5B, lanes 3 and 4). Although the combined pharmacological inhibition of both second messenger systems markedly reduced growth factor-induced cleavage (Fig. 5B, lane 5), there was still a residual cleavage activity that neither the Ca nor the PKC systems could account for (Fig. 5B, compare the 17 kDa (uncleaved) to 15 kDa (cleaved) pro-TGF-alpha ratio in lanes 1 and 5).

Treatment with the anti-EGF receptor blocking mAb did not affect the general machinery responsible for pro-TGF-alpha biosynthesis or cleavage since (i) incubation with the mAb prior to and during the chase did not affect the normal pattern of pro-TGF-alpha biosynthesis (Fig. 4A) and (ii) cleavage induced by pharmacological activation of second messenger systems with PMA or A23187 was insensitive to mAb 225 (Fig. 6).


Figure 6: EGF receptor blocking antibody mAb 225 prevents TGF-alpha mediated pro-TGF-alpha cleavage but does not prevent A23187- and PMA-mediated pro-TGF-alpha cleavage. A431 cells were metabolically labeled with [S]cysteine for 30 min and chased for 45 min. During the last 30 min of the chase TGF-alpha (10 nM), A23187 (1 µM), or PMA (1 µM) was added as indicated. Paired cultures were incubated with saturating amounts of mAb 225 (100 nM), as indicated during the 45 min of the chase period.




DISCUSSION

In experiments with cells expressing genetically engineered mutant forms of pro-TGF-alpha resistant to cleavage by pro-TGF-alpha-ase-II, the membrane-bound 17-kDa form has been shown to be biologically active by a proposed juxtacrine mechanism(30, 31, 32) . Although the mechanism of activation of the EGF receptor by soluble ligand has been well characterized(41, 42) , little is known about the expression and activity of pro-TGF-alpha in human cancer cells with an active EGF receptor/TGF-alpha autocrine pathway. For this reason, we investigated the molecular forms of pro-TGF-alpha in a series of tumor-derived cell lines with putative active EGF receptor autocrine loops and high levels of receptor expression.

In transfected fibroblasts, generation of soluble TGF-alpha from the 17-kDa membrane-anchored form depends upon the activity of the transmembrane endoprotease pro-TGF-alpha-ase-II, which cleaves the Ala-Val peptide bond, thus eliminating the role of membrane-bound pro-TGF-alpha as a juxtacrine molecule(18) . In retrovirally transformed embryo fibroblasts (14, 16) and hepatocellular carcinoma cells(17) , the culture medium accumulates mature 6-kDa TGF-alpha as well as heterogeneous 20-kDa soluble meso-TGF-alpha. Although pulse-chase analyses of pro-TGF-alpha in these cell lines have not been reported, the high amount of large meso-TGF-alpha reflects a high pro-TGF-alpha-ase-II activity, which releases some of the pro-TGF-alpha molecules from the cell membrane before cleavage by pro-TGF-alpha-ase-I can occur. In the tumor-derived cells that we have analyzed, the biosynthesis of pro-TGF-alpha initially followed a pattern analogous to that described for transfected cellular models(13, 15, 18) . However, these cell lines accumulated the 17-kDa membrane-anchored precursor form, suggesting that the activity of pro-TGF-alpha-ase-II was low. In addition, the rapid disappearance of the NH(2)-terminal glycosylated extension to produce the 17-kDa molecular form indicates a considerable pro-TGF-alpha-ase-I activity.

The data presented here suggest that membrane-anchored pro-TGF-alpha represents a significant proportion of biosynthesized pro-TGF-alpha in the tumor cells that we have studied and, in this conformation, is associated with tyrosine-phosphorylated EGF receptor. It is possible, therefore, that membrane-anchored forms could carry out juxtacrine stimulation that would continually enhance growth of receptor-containing cells, and cleavage would terminate this function and facilitate the clearance of the factor. This could be an efficient form of receptor activation, since down-regulation of ligand and receptor, and their subsequent catabolism, would be precluded. The response to the addition of saturating concentrations of exogenous EGF (Fig. 4A) demonstrates that most EGF receptors remain inactivated when these tumor cells are only exposed to endogenous sources of ligand. Furthermore, soluble forms of TGF-alpha were reported to be more active than membrane-bound TGF-alpha(30) . Nevertheless, in these rapidly growing A431 cell cultures it is the membrane-bound form that predominates, and interestingly, exogenous soluble ligand in saturating amounts actually inhibits proliferation(24, 43, 44) .

Cleavage of membrane pro-TGF-alpha is highly regulated(37) . Mechanisms that can trigger the release of soluble TGF-alpha include rises in the intracellular free Ca concentration(19) , activation of PKC(18) , and other still undefined pathways switched on by serum factors(19) . Although these mechanisms are different, the cleavage event activated by all of them is probably the same, since identical sets of protease inhibitors block cleavage activated by the different pathways (21) and genetic mutants of pro-TGF-alpha in CHO fibroblasts are resistant to cleavage activated by several alternative mechanisms(23) . The Ca-dependent and PKC mechanisms of cleavage appear to be quite universal, since we have also found them operative in human tumor cell lines. In A431 cells, pharmacological increases in cytosolic Ca or PKC activity increased the conversion of 17-kDa pro-TGF-alpha to the 15-kDa terminal fragment tail. Since a certain degree of cross-talk between these two second messenger systems exists, it was possible that their effect was mediated by a shared pathway. This has been ruled out by using treatments that neutralize one pathway but not the other. Thus, elevated Ca activates pro-TGF-alpha cleavage in cells that have been desensitized to PMA action. On the other hand, PMA is able to induce cleavage in the presence of the Ca chelator EGTA in the culture medium, a treatment that completely prevents calcium ionophore-induced cleavage(19) . An interesting finding that comes out of our data is the relative effectiveness of these second messenger systems in inducing pro-TGF-alpha cleavage. In A431 cells, Ca is more efficient in provoking cleavage than PMA, while the opposite is the rule for cells of fibroblastic origin(18, 19) . This suggests potential mechanisms for specificity, since in some tissues a cell could be highly sensitive to the stimulation of one pathway and induce release of soluble TGF-alpha, while other cell types could be largely refractory to the activation of this pathway.

What are the mechanisms by which EGF receptor activation triggers pro-TGF-alpha cleavage? EGF receptor activation is known to induce the hydrolysis of membrane polyphosphoinositides, inducing increases in [Ca](i) and PKC activity(39, 45) . Pharmacological manipulations of these second messenger systems support the conclusion that both may participate to a certain extent. Yet, the residual activity of pro-TGF-alpha cleavage after neutralization of the [Ca](i) and PKC pathways suggests that additional unidentified mechanisms are triggered by EGF receptor activation.

We find that TGF-alpha production is autoregulated by a dual mechanism in human tumor cell lines. On the one hand, activation of the EGF receptor increased the level of pro-TGF-alpha mRNA, as has been reported in other cells(33, 34, 35) , and, on the other hand, receptor activation rapidly induced cleavage of the membrane-anchored factor. The latter is of special interest for at least two reasons. (i) It demonstrates for the first time that release of membrane-anchored pro-TGF-alpha can be triggered by a specific first messenger, in addition to the well reported effect of pharmacological manipulation of second messenger pathways; and (ii) the cleavage can be regulated by the growth factor itself. These observations point to achievement of a strong up-regulatory effect, using several different mechanisms to facilitate or perpetuate autocrine stimulation of growth. The existence of such an autocrine loop is further supported by data obtained in treatments aimed at blocking the EGF receptor activity. We observed that anti-EGF receptor mAb 225 can decrease resting pro-TGF-alpha mRNA levels in A431 cells and prevents the cleavage of membrane-bound pro-TGF-alpha induced by EGF/TGF-alpha.

The anchoring of TGF-alpha to the plasma membrane may have not only important biological consequences upon autocrine/juxtacrine pathways in malignant cells but therapeutic implications as well. For example, our laboratory has found that the Fab` monovalent fragment of anti-EGF receptor mAb 225 is effective in blocking receptor activation by exogenous TGF-alpha/EGF but is far less effective than the bivalent complete mAb in blocking activation by endogenous (autocrine/juxtacrine) TGF-alpha(46) . We postulate that the close proximity of the ``autojuxtacrine'' TGF-alpha ligand to receptors on the surface of the same cell may give the ligand preferential access to receptors, which is only overcome by the capacity of the bivalent, but not monovalent, antibody to down-regulate and remove the receptors.


FOOTNOTES

*
This work was supported by an American Society of Clinical Oncology career development award (to J. B.), Grant PB94-0075b from the Spanish Dirección General de Investigaciones Científicas y Technológicas DGICYT (to A. P.), and National Institutes of Health Grants CA 42060 and CA37641 (to J. M.).

§
To whom correspondence should be addressed: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-2162; Fax: 212-759-1494.

(^1)
The abbreviations used are: TGF-alpha, transforming growth factor-alpha; EGF, epidermal growth factor; CHO, Chinese hamster ovary; PKC, protein kinase C; [Ca], free cytosolic calcium; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Joaquin Arribas and Joan Massagué for helpful consultations. We thank Elena Diaz for her help in the coimmunoprecipitation experiments.


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