Expression of human epidermal growth factor precursor cDNA in ...

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lomavirus-based vector in which the human kidney EGF precursor cDNA has been ... the submandibular gland and kidney and -1000 times lower in lung, spleen ...
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 126-130, January 1988 Cell Biology

Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells (bovine papilloma viral vector/preproepidermal growth factor)

BARBARA MROCZKOWSKI*t, MARTHA REICH*, JONATHAN WHITTAKERt, GRAEME I. BELLI, STANLEY COHEN*

AND

*Vanderbilt University, School of Medicine, Department of Biochemistry, Nashville, TN 37232; and tHoward Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637

Contributed by Stanley Cohen, September 8, 1987

ABSTRACT Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ,uM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is 4150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

the submandibular gland and kidney and -1000 times lower in lung, spleen, brain, and ovary. Interestingly, in some tissues such as the kidney, it is the EGF precursor rather than EGF that accumulates, suggesting that the precursor itself might have additional physiological functions. Numerous proteins share sequence homology with EGF. These include human pancreatic secretory trypsin inhibitor (13), a-transforming growth factor (a-TGF) (14, 15), the 19-kDa early protein of vaccinia virus (16-18), and two homeotic gene products, the Drosophila notch gene product (19) and the lin-12 gene product of the nematode Caenorhabditis (20). A portion of the EGF precursor also exhibits limited homology with the sequence of the low density lipoprotein receptor, suggesting perhaps that the EGF precursor and the low density lipoprotein receptor both descended from a common ancestral transmembrane protein (21, 22). To gain a better understanding of the function of the EGF precursor and the factors resulting in its tissue-specific processing to EGF, we have used a bovine papillomavirus (BPV) mouse metallothionein I promoter-based expression vector together with a human kidney cDNA encoding the EGF precursor to produce high levels ofthis protein in mouse NIH 3T3 cells. The ability of BPV vectors to propagate extrachromosomally as multicopy plasmids (23) in conjunction with their ability to efficiently express foreign proteins (24-28) has allowed us to establish stably transfected cell lines that express high levels of the hEGF precursor. The EGF precursor synthesized by these cells is predominantly a membrane-associated polypeptide, which is consistent with predictions based on its amino acid sequence as well as transient expression studies of the EGF precursor cDNA in monkey kidney cells. However, the conditioned medium from cells in which expression of the EGF precursor has been induced with butyric acid also contains a high molecular weight form of EGF as well as material that is capable of competing with iodinated EGF for binding to the EGF receptor.

Murine epidermal growth factor (EGF) is a polypeptide of 53 amino acid residues with three intrachain disulfide bonds (1). The homologous human protein (hEGF) has been isolated from urine (2, 3); it is structurally and functionally identical to urogastrone, a human antisecretory hormone (4). Although the in vitro and in vivo growth-promoting properties of exogenous EGF have been thoroughly described in many biological systems (5) and EGF has been demonstrated to be an effective inhibitor of gastric acid and pepsin secretion (6), the normal physiological function(s) of this growth factor remains to be determined. Analysis of cDNA clones derived from mRNA transcripts encoding murine EGF and hEGF has revealed that these peptides are synthesized as large precursor molecules of 1217 and 1207 residues, respectively, and that they contain a hydrophobic transmembrane domain (7-10). It is of additional interest that these precursors contain nine regions of partial sequence homology to mature EGF (7-11). Whether these EGF-related sequences are ever processed to serve a biological function is not known. Messenger RNA encoding the mouse EGF precursor has been detected in many adult tissues, including submandibular gland, kidney, mammary gland, pancreas, duodenum, pituitary, lung, spleen, brain, ovary, and uterus, implying that the EGF precursor is synthesized by each of these tissues (12). However, the levels of mRNA vary widely, being highest in

MATERIALS AND METHODS Construction of hEGF Precursor and BPV Recombinants.

Synthetic Xho I oligonucleotide linkers were ligated to a 6.4-kilobase-pair (kbp) Sma I fragment of XhEGF116 (10) containing 62 bp of the 5' untranslated region, 3621 bp of coding sequence, and 791 bp of 3' untranslated region as well as 2.0 kb of the right arm of AgtlO. After Xho I digestion and removal of excess linkers, the 6.4-kb fragment containing the entire hEGF precursor cDNA coding region was ligated into the Xho I site of pBPV-MTH-Xho (kindly provided by D. Hamer, National Istitutes of Health, Bethesda, MD). This

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: EGF, epidermal growth factor; hEGF, human EGF; NP-40, Nonidet P-40; BPV, bovine papillomavirus. tTo whom reprint requests should be addressed. 126

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Proc. Natl. Acad. Sci. USA 85 (1988)

vector is similar to that described by Pavlakis and Hamer (29). It contains the following structural elements: the 5.5-kb subgenomic fragment of BPV, the mouse metallothionein gene (30) modified by replacement of the Bgl II site upstream of the initiator ATG by an Xho I site, and the origin of replication and ampicillin-resistance gene of pML2 (31). The physical map of this construct, pBPV-MTH-hEGF, is shown in Fig. lA.

Cell Transfection and Selection of Transformants. Mouse NIH 3T3 cells (2-4 X 105 cells) were cotransfected with a calcium phosphate precipitate of S pug of pBPV-MTH-hEGF and the plasmid pSV2Neo (32) in a 10:1 ratio followed by glycerol treatment as described by Gorman (33). After 24 hr in culture, cells were split 1:5 and cultured thereafter in the presence of selective medium [Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum/gentamycin (50 ;kg/ ml)] containing 600 jug of G418 per ml. Two weeks later, G418-resistant clones were isolated and grown in 12-well plates and tested for the expression of EGF precursor mRNA and protein by RNARNA hybridization and immunoblot

analysis, respectively. RNA Isolation and Blot Hybridization Analysis. Cytoplasmic RNA was isolated from the various cell lines by detergent lysis and phenol/chloroform/isoamyl alcohol extractions. Briefly, 100-mm dishes were washed and scraped in calciumfree magnesium-free phosphate-buffered saline (CMF-PBS), cells were pelleted at 1500 rpm for 10 min and resuspended in 0.02 M Tris, pH 7.5/0.15 M KCI/0.005 M MgCl2/0.5% Nonidet P-40 (NP-40)/0.005 M 1,4-dithiothreitol/1000 units of RNasin per ml (Promega Biotec). Lysates were spun in an Eppendorf Microfuge (13,000 x g) for 10 min and the RNA was purified from the supernatants with phenol/chloroform/ isoamyl alcohol (1:1:50). Slot blots (Schleicher & Schuell) were prepared on nitrocellulose membranes. RNA samples A

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FIG. 1. Expression of hEGF precursor in transfected NIH 3T3 cells. (A) Structure of the expression construct pBPV-MTH-hEGF. This construct consists of DNA segments from various sources: a 5.5-kb subgenomic fragment of BPV (hatched area), the mouse metallothionein I (MT.1) gene (solid line), the prokaryotic pML DNA sequences (open area), and a 6.4-kb Sal I fragment encoding the hEGF precursor to which Xho I linkers had been added (solid area). (B) Quantitation of EGF precursor mRNA levels in transfected NIH 3T3 cells. Total RNA (10 ,ug) was immobilized on a nitrocellulase membrane using a slot blot manifold and then hybridized with a 32P-labeled RNA transcript complementary to EGF precursor mRNA (see Materials and Methods). hEGF 1-36 designates individual G418-resistant clones from which RNA was isolated.

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were resuspended in 7.5x SSC (lx SSC = 0.15 M NaCl/ 0.015 M sodium citrate) containing 4.3 M formaldehyde and incubated at 80'C for 10 min prior to spotting onto nitrocellulose. Blots were baked at 80'C for 30 min under vacuum and prehybridized for 3 hr at 550C in the following solution: 50% (vol/vol) formamide/0.75 M NaCl/0.15 M Tris'HCl, pH 8.0/0.01 M EDTA/0.2 M sodium phosphate/ix Denhardt's solution (0.02% bovine serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone)/10%o dextran suflate/0.1% NaDodS04. Hybridizations were carried out for 16 hr at 550C in fresh buffer containing [32P]CTP-labeled prepro-EGF RNA. The hybridization probe was a 1015-bp EcoRI/BamHI fragment from X-hEGF116 [corresponding to nucleotides 547-1562 in the sequence presented by Bell et al. (10)] subcloned into the vector pGEM4 (Promega Biotec). High specific activity 32P-labeled probes of both sense and anti-sense strands were prepared by using SP6 and 17 RNA polymerases according to the manufacturer's protocol. The hybridization and washing conditions were those suggested by Promega Biotec and Deleon et al. (34). Immunoblotting. Cell cultures were lysed in detergent containing buffer (0.02 M Tris, pH 7.5/0.15 M KCl/0.005 M/MgCl2/0.5% NP-40) and clarified by centrifugation at 13,000 x g for 10 min at 4°C. Aliquots (25 ,ul) from single 100-mm dishes of cells suspended in 500 ,ul of buffer were mixed with NaDodSO4 sample buffer, heated for 2 min at 100°C, and subjected to NaDodSO4/polyacrylamide gel electrophoresis (35). Electrophoretic transfer to nitrocellulose paper was carried out with a buffer consisting of 0.025 M Tris, pH 8.3/0.192 M glycine/20%o (vol/vol) methanol in a Bio-Rad Trans-Blot apparatus (36). The nitrocellulose paper was incubated in a Tris buffer containing 3% bovine serum albumin at 37°C to block nonspecific binding followed by incubation in a buffer composed of 0.05 M Tris, pH 7.4/0.005 M EDTA/0.150 M NaCl/0.25% gelatin/0.5% Triton X100/0.1% NaDodSO4 containing a 1:600 dilution of antihEGF rabbit antiserum (37) for 1 hr at room temperature. The nitrocellulose membrane was then washed with this same buffer minus the antiserum and was further incubated with 125I-labeled protein A (106 cpm/ml) for 1 hr. The blot was extensively washed, air-dried, and exposed to Kodak XOmat film at -70°C. Cell Fractionation. Single 100-mm dishes were washed three times with 10.0 ml of CMF-PBS, scraped, and pelleted at 1500 rpm for 10 min. The pellets were resuspended in 0.5 ml of 0.02 M Hepes, pH 7.4/0.05 M MgCl2/0.004 M iodoacetic acid and homogenized by gently passing the suspension through a 22-gauge hypodermic needle 10 times. The cell homogenate was spun for 10 min in an Eppendorf Microfuge (13,000 x g). The supernatant was removed, and the pellet was washed once with 0.5 ml of buffer prior to final resuspension in 0.5 ml of the Hepes buffer. Aliquots (20 Jl) of the supernatant fraction (soluble) and the pellet fraction (containing membranes) were assayed for protein content by the Bradford procedure (38) using y-globulin as a standard and Bio-Rad reagents according to the manufacturer's directions. Preparation of Conditioned Medium for Immunoblot Analysis. Single 100-mm dishes were extensively washed (six times) with CMF-PBS, followed by a 30-min wash at 37°C in serum-free DMEM. To the washed dishes was added 5.0 ml of fresh serum-free DMEM. Cells were induced by the addition of 5 mM butyric acid. At 12 and 24 hr postinduction, the conditioned medium was collected, dialyzed extensively against 0.02 M Hepes (pH 7.4) at 4°C, lyophilized in a Savant Speed-Vac, and reconstituted with lx Laemmli NaDodSO4 sample buffer to 1/10th the original volume. Aliquots (35 .l) from a total of 500 ILI were denatured and reduced and then electrophoresed in either a 7% or a 12% NaDodSO4/polyac-

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rylamide gel prior to immunoblot analysis, unless stated otherwise.

RESULTS Expression of hEGF Precursor mRNA in Transfected NIH 3T3 Cells. The expression vector pBPV-MTH-hEGF shown in Fig. lA places the entire coding region for the kidney hEGF precursor as well as 62 bp of the 5' untranslated and 791 bp of the 3' untranslated flanking sequences under the control of the inducible mouse metallothionein I promoter. Mouse NIH 3T3 cells were cotransfected with a calcium phosphate precipitate ofthis construct and the plasmid pSV2Neo, which provides a dominant selectable neomycin-resistance marker. Thirty-six individual G418-resistant cell lines were established from the initial cotransfection, and each was tested for the expression of prepro-EGF mRNA by RNA-RNA hybridization and for the synthesis of prepro-EGF protein by immunoblot analysis. To quantitate the relative steady-state levels of preproEGF mRNA in parental (nontransfected) and transfected G418-resistant cell lines, cytoplasmic RNA was isolated by detergent lysis, followed by phenol/chloroform/isoamyl alcohol extractions, and immobilized onto nitrocellulose. Hybridizations were carried out with a 1015-bp anti-sense prepro-EGF RNA probe produced by SP6 RNA polymerase. Results of such an experiment are shown in Fig. 1B. Of the initial 36 G418-resistant cell lines established, 27 expressed various levels of prepro-EGF mRNA constitutively. In contrast, RNA isolated from nontransfected NIH 3T3 cells did not hybridize to the human prepro-EGF RNA probe. Three cell lines expressing high levels of EGF precursor mRNA, hEGF-11, -12, and -19, were selected for further characterization. These cell lines were cultured in the presence or absence of 5 mM butyric acid for 12 hr prior to isolation of RNA. Hybridization of the hEGF precursor RNA probe to RNA isolated from these cells indicated that there was at least a 10-fold increase in this mRNA after induction (Fig. 2). Growth of these cells in the presence of 100 ,uM ZnCl2 produced only a modest =2-fold increase in EGF precursor mRNA levels (data not shown). Immunoblot Analysis of prepro-EGF Content in Transfected Cell Lines. Polyclonal antibodies directed toward epitopes specific for the mature hEGF peptide were used to identify and quantitate relative levels of the hEGF precursor in hEGF-11, -12, and -19 transfected cell lines. The various sera used in these experiments showed no cross-reactivity with

Proc. NatL Acad. Sci. USA 85

(1988)

murine EGF (data not shown). The results from an immunoblot analysis of prepro-EGF content in either ZnCl2- or butyric acid-induced transfected cells is shown in Fig. 3. Lanes D-F represent equivalent aliquots of NP-40 lysates from hEGF-12 cells induced for 12 hr with either 100 ,uM ZnCl2 (lane D), 5 mM butyric acid (lane E), or in the absence of inducer (lane F). In lysates prepared from either butyric acid- or ZnCl2-induced cells, a protein of 150-180 kDa is readily identified by anti-hEGF polyclonal anti-serum. Lane G represents 1 ug of recombinant hEGF, which serves as a positive control. The levels of both prepro-EGF protein and mRNA were found to be consistently much higher in cells exposed to butyric acid as opposed to ZnCl2 (50-100 ,uM). Lanes A-C are experimentally identical to lanes D-F except that the cells used as a control (designated hIR 3.5) represent a mouse NIH 3T3 cell line transfected with a BPV construct containing the human kidney insulin receptor cDNA inserted under the control of the mouse metallothionein promoter (26). One can readily observe the absence of immunoreactive prepro-EGF protein species in this analogously transfected cell line. These results provide strong evidence that the 150to 180-kDa protein reacting with the EGF antiserum is the hEGF precursor. Interestingly, the estimated molecular mass of the EGF precursor synthesized by these cells is 20-46 kDa greater than expected from the amino acid sequence (134 kDa; the value includes the putative signal peptide as well). We feel this difference might be attributed to posttranslational glycosylation, since it has been noted that the hEGF precursor sequence contains nine potential acceptor sites for N-linked oligosaccharides (10). hEGF Precursor is Membrane Associated. The subcellular localization of the hEGF precursor was determined by cell fractionation. NIH 3T3 cells transfected with either hEGF precursor (Fig. 4, lanes A-H) or human insulin receptor (lanes I-L) were incubated in the presence or absence of 5 mM butyric acid for 12 hr. Cell homogenates were partitioned into soluble (S) and membrane-enriched fractions (M) and 50 ,ug of total protein from each fraction was resolved on an 8% NaDodSO4/polyacrylamide gel followed by electrophoretic transfer onto nitrocellulose. Immunoblot analysis clearly Cell

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