Secretion of Factor VIII inChinese Hamster Ovary Cells

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1989, p. 1233-1242 0270-7306/89/031233-10$02.00/0 Copyright ©D 1989, American Society for Microbiology

Vol. 9, No. 3

Effect of von Willebrand Factor Coexpression on the Synthesis and Secretion of Factor VIII in Chinese Hamster Ovary Cells RANDAL J. KAUFMAN,'* LOUISE C. WASLEY,1 MONIQUE V. DAVIES,' ROBERT J. WISE,2 DAVID I. ISRAEL,' AND ANDREW J. DORNER' Genetics Institute, 87 Cambridge Park Drive, Cambridge, Massachusetts 02140,1 and Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Boston, Massachusetts 021152 Received 12 September 1988/Accepted 16 December 1988

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.

The factor VIII complex has two distinct biologic functions: coagulant activity and a role in primary hemostasis (for reviews, see references 32 and 43). The analysis of factor VIII deficiency diseases, classic hemophilia and von Willebrand's disease, have contributed to the understanding that factor VIII is a complex of two components: the factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related antigen (von Willebrand factor [vWF]). The factor VIII molecule is an important regulatory protein in the blood coagulation cascade. After activation by thrombin, it accelerates the rate of factor X activation by factor IXa, eventually leading to the formation of the fibrin clot (18, 28, 39, 49, 50). Factor VIII is synthesized as a large precursor which is cleaved to generate an amino-terminalderived heavy chain of 200 kilodaltons (kDa) in a metal ion-stabilized complex with the carboxy-terminal-derived light chain of 80 kDa (9, 13, 41, 51). Factor VIII has binding sites for factor IXa, factor X, Ca2 , phospholipid, and vWF. Although there are do known natural cell lines that express factor VIII, most evidence suggests that the protein is synthesized in hepatocytes and possibly in endothelial cells (2, 27, 31, 33, 56, 58, 61). The vWF molecule is an adhesive glycoprotein that plays a central role in platelet aggregation and facilitates plateletvessel wall interactions in response to vascular injury (43). It also serves as a carrier for factor VIII in plasma. Most evidence to date indicates that the vWF has either a stabilizing effect on the factor VIII in plasma, or that the vWF can elicit the release of factor VIII from storage depots or stimulate the synthesis or secretion or both of factor VIII (4, 8, 36, 48, 57). vWF is synthesized in endothelial cells and megakaryocytes. In these cell types the highest-molecularweight forms of vWF are stored in specific granules, termed *

Weibel-Palade bodies and alpha granules. vWF is translated as a large precursor of approximately 300 kDa, from which a 100-kDa propeptide is cleaved. The mature vWF molecule of 226 kDa is assembled into large disulfide-bonded multimers which range from 5 x 105 to over 107 daltons. The propeptide of vWF is required to direct the intracellular multimerization of vWF (53, 59). Discrete domains of vWF bind to platelet receptor sites on glycoprotein lb and on the glycoprotein Ilb-Illa complex as well as to binding sites on collagen (for reviews, see references 15 and 42). Recently, a factor VIII-binding site has been identified within the first 272 amino acids of the mature vWF protein (12). A variety of abnormalities in vWF activity can result in von Willebrand's disease (15, 42). The isolation of the cDNA and genomic sequences for both factor VIII (16, 47, 51) and vWF (3, 30, 44, 52) have made available the primary amino acid sequence and the ability to analyze functional domains. In addition, it has made feasible the production of recombinant factor VIII and vWF preparations which are essentially free of contaminating viruses (3, 24, 52, 60). The production of factor VIII through recombinant DNA technology has been achieved by expression of the cDNA-encoding factor VIII in mammalian cells. These cells have provided for the first time the ability to study the synthesis and the intracellular and extracellular processing of factor VIII (24). We have previously demonstrated the requirement for the presence of vWF in the medium to promote the stable accumulation of secreted factor VIII (24). It appears that vWF can promote the association of the heavy and light chains of factor VIII and thereby result in a stable complex resistant to proteolysis (11, 24; A. J. Dorner, unpublished observations). Here we show that cells can be engineered to express both factor VIII and vWF to result in the stable accumulation of secreted factor VIII activity in the absence of serum.

Corresponding author. 1233

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KAUFMAN ET AL.

MATERIALS AND METHODS Derivation of the factor VIII and vWF expression vector. The factor VIII expression vector pRXPyVIII-1 has been described previously (24). The factor VIII transcription unit within this vector comprises the polyomavirus origin of replication and transcriptional enhancer, the adenovirus major late promoter (including the adenovirus 5' splice site from the first leader of adenovirus late mRNAs), a 3' splice site, the factor VIII-coding region, a dihydrofolate reductase (DHFR)-coding region within the 3' end of the transcript, and the simian virus 40 (SV40) early polyadenylation signal. The vWF expression plasmid pMT2-ADA-vWF has been described previously (3). This plasmid contains two transcription units encoding human vWF and human adenosine deaminase (ADA). vWF is transcribed from the adenovirus major late promoter and SV40 transcriptional enhancer to yield an mRNA which contains the majority of the adenovirus tripartite leader sequence which is present on adenovirus late mRNAs, the same 5' and 3' splice site present within the factor VIII transcription unit, the entire vWF cDNA, a DHFR sequence within the 3' end of the transcript, and the SV40 early polyadenylation signal. ADA is transcribed from the SV40 early promoter, and the mRNA production utilizes the SV40 late polyadenylation signal. Cell culture, DNA transfection, and cell line selection. The derivation of the IOAl Chinese hamster ovary (CHO) cell line which expresses factor VIII by cotransfection with DHFR and coamplification by selection for resistance to 1 mM methotrexate (MTX) has been described previously (24). Plasmid pMT2ADA-VWF was introduced into factor VIII-producing 1OAl cells by protoplast fusion as described previously (19). At 48 h after fusion, the cells were subcultured into alpha medium lacking nucleosides and containing 10% dialyzed fetal bovine serum, 20 F.M MTX, and AAUselective medium (1.1 mM adenosine, 10 FM alanosine, and 10 ,ug of uridine per ml) containing 0.1 p.M 2'-deoxycoformycin (dCF) (20). After 14 days, a pool of transformants (lOA13a) was propagated in stepwise increasing concentrations of dCF (0.5, 1.0, and 2.0 ,uM). The pool resistant to 2.0 puM dCF and 1 mM MTX was subcloned by dilution plating. One representative clone, designated 10A1C6, produced over 1 U/106 cells per day of factor VIII in either serum-free medium or 10% fetal bovine serum-containing medium and produced 4 pLg/106 cells per day of vWF. A CHO cell line expressing only vWF was previously derived and produces vWF at 0.5 ,ug/ml per day (3). Assay for factor VIII and vWF. Conditioned media were analyzed for factor VIII activity by the Kabi Coatest method. Similar values were determined by the one-stage activated partial thromboplastin time assay (Clotek activity) (24). Quantitation of factor VIII antigen was performed by an enzyme-linked immunosorbent assay with a monoclonal antibody (Hybritech, Inc., La Jolla, Calif.) that recognizes the light chain of factor VIII. vWF expression was monitored by an enzyme-linked immunosorbent assay using affinity-purified rabbit antihuman vWF-specific antiserum (Calbiochem-Behring Diagnostics, La Jolla, Calif.) and purified vWF antigen from normal human plasma pools to serve as standards and controls and immunoglobulin isolated from goat anti-rabbit serum conjugated with alkaline phosphatase (CalbiochemBehring). vWF and factor VIII synthesis. The synthesis and secretion of vWF and factor VIII were monitored as described previously (6, 24) by labeling cells in methionine-free medium

MOL. CELL. BIOL.

containing 0.5 mCi/ml of [35S]methionine (7,800 Ci/mmol; Dupont, NEN Research Products, Boston, Mass.) and aprotinin (0.5%; Sigma Chemical Co., St. Louis, Mo.) and by immunoprecipitation of cell extracts and conditioned medium. Immunoprecipitates were analyzed by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels (29). Immunoprecipitations were performed with a monoclonal antibody specific for the heavy chain of factor VIII (kindly provided by W. B. Foster, Genetics Institute, Cambridge, Mass.), a rabbit polyclonal antiserum specific to human vWF (Calbiochem-Behring), or anti-BiP (GRP78) monoclonal antibody culture supernatant (kindly provided by D. Bole, Yale University, New Haven, Conn.) (1). After electrophoresis, gels were fixed, treated with En3Hance (Dupont, NEN), dried, and exposed to film (XAR; Eastman Kodak Co., Rochester, N.Y.) at -70°C. Band intensities on autoradiograms were quantitated by using an ultrascan laser densitometer (model 2202; LKB Instruments, Inc., Gaithersburg, Md.). Southern and Northern (RNA) blot hybridization. Highmolecular-weight DNA was isolated, and 10 ,ug was digested to completion and separated by electrophoresis on 1.1% agarose gels for Southern blot transfer (21) and hybridization to either DHFR, factor VIII, or VWF radiolabeled probes. Total RNA was prepared by guanidine thiocyanate extraction, electrophoresed on formaldehyde-1% agarose gels, transferred to nitrocellulose, and hybridized with doublestranded nick-translated DNA fragments by standard techniques (7). Transcription analysis. Nuclei were prepared from subconfluent monolayers of cells as described previously, except that CaCl2 was omitted from the reticulocyte swelling buffer (23). Transcription reactions were performed as described previously (23) with minor modifications. Nitrocellulose filters were prepared by denaturing purified DNA restriction fragments in 0.2 M NaOH-1 M ammonium acetate for 10 min on ice and then filtering them onto nitrocellulose with a Slot Blot apparatus (Schleicher & Schuell, Inc., Keene, N.H.). Filters were then washed first with 4x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and then with 5 x Denhardt solution and baked under vacuum at 80°C for 2 h. Restriction fragments were as follows: for adenosine deaminase, a 1.53-kilobase (kb) EcoRI fragment from pSV2ADA (35); for DHFR, a 1.2-kb PstI fragment from pAdD26SVOD (22); for GRP78, a 1.6-kb PstI-EcoRI fragment from p3C5 (46); for vWF, a 5.2-kb XmnI-EcoRI fragment from pMT2VWF (3); for 5' factor VIII, a 4-kb SalI-EcoRI fragment from pSP64VIII (47); for 3' factor VIII, a 3.3-kb EcoRV-SalI fragment from pSP64VIII (47); for chicken beta actin, a 1.6-kb PstI fragment from pAl (5). Restriction fragments were purified after agarose gel electrophoresis by using a glass powder purification procedure. RNA was incubated in 9% formaldehyde and 75% formamide for 3 min at 65°C immediately before hybridization. Hybridization was done in 5 x SSC at 37°C for 60 h. Washes were done in 2x SSC and 0.2x SSC at 65°C. Immunofluorescent localization of vWF. Cells growing on glass cover slips were analyzed by immunofluorescence essentially as described previously (40). The cells were washed three times with phosphate-buffered saline and fixed by treatment with 1% formaldehyde in phosphate-buffered saline for 20 min at room temperature. Cells were then permeabilized by acetone treatment. The primary antibody was rabbit anti-vWF (Calbiochem-Behring) which had been purified over a protein A-Sepharose column and used at 6 to

VOL. 9, 1989

EFFECT OF vWF COEXPRESSION ON FACTOR VIII

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TABLE 1. Coexpression of vWF and factor VIII in CHO cell lines' Selection lin _____________ CellCell line Slcon dCF MTX

(RM) 1OAl

1OA13a

(RM)

Factor VIII

VWF antigen [pg/cell]) (jig/ml (pgm p/el)

0.38

1,000 0.1 0.5 2.0

20 20 1,000

(pU/cell) U/el No

Serum

serum

0.07 (0.1) 0.8 (1.1) 7.4 (24)

0.93

E 0.63 1.4

0.89 1.5

a The derivation of cell lines lOAl and lOA13a is described in Materials and Methods. At each step in the growth selection in MTX and dCF, cells were subcultured for factor VIII and vWF assay. Cells were rinsed with serum-free medium 24 h later, and then either serum-free medium or 10%o fetal bovine serum-containing medium was applied at approximately 5 x 10' cells per ml. Conditioned medium was taken for factor VIII and vWF assay 24 h later, and the cells were taken for quantitation by a Coulter counter (Coulter Electronics, Inc., Hialeah, Fla.). Factor VIII activity was determined by the Kabi Coatest method, and vWF levels were determined by an enzyme-linked immunosorbent assay as described in Materials and Methods. Quantitation of factor VIII antigen by an enzyme-linked immunosorbent assay method gave results comparable with those obtained by the activity assay.

10 p,g/ml. Secondary antibody was fluorescein-conjugated immunoglobulin G fraction of goat anti-rabbit immunoglobulin (Organon Teknika, Malvem, Pa.) used at a concentration of 0.24 mg/ml. Both antibodies were diluted, and incubations were performed in phosphate-buffered saline containing 1 mg of bovine serum albumin per ml. Immunofluorescence photography was done with a Leitz VarioOrthomat system. Nondenaturing agarose gel electrophoresis for analysis of vWF multimers. Electrophoresis of VWF immunoprecipitates on SDS-1% agarose (FMC BioProducts, Seakem, Me.) gels was performed as described previously (59). RESULTS Coexpression of vWF and factor VIII eliminated the requirement for exogenously added vWF. Our initial experiments demonstrated that vWF secreted from COS-1 monkey cells transfected with a human vWF expression plasmid can serve to promote accumulation of factor VIII activity from CHO cells (data not shown). Thus, we next investigated whether coexpression of vWF with factor VIII in the same cell may also yield stable factor VIII accumulation and alleviate the requirement for exogenously added vWF. Cells which coexpress both factor VIII and vWF were obtained by introduction of a vWF expression vector into factor VIIIproducing lOAl CHO cells. pMT2ADA-vWF was introduced into the factor VIII-expressing 1OAl cells by protoplast fusion and selection for ADA expression by growth in cytotoxic concentrations of adenosine and low levels of dCF, an inhibitor of ADA (20). A pool of transformants were subsequently selected for amplification of the transfected ADA gene by propagation in increasing concentrations of dCF in the presence of cytotoxic concentrations of adenosine (20). At each step of the amplification process, the production of vWF and of factor VIII was measured after 24 h in the presence of 10% fetal calf serum or in serum-free medium. The results demonstrated that vWF expression increased over 200-fold with increasing selection for ADA (Table 1). As the vWF expression level increased, there was a corresponding increase in the level of factor VIII activity accumulated in the conditioned medium when cells were cultured in the absence of serum. At the high level of vWF

I-

Time (hrs) FIG. 1. Accumulation of factor VIII in the presence and absence of vWF coexpression. The accumulation of factor VIII in the 1OAl and 10A1C6 cells over 3 days was determined by rinsing cells and then adding medium with or without the addition of 10%o fetal calf serum. Samples of the conditioned medium were taken at 24, 48, and 72 h for factor VIII activity determination by the Kabi Coatest method. The CHO cell line 1OAl expressed only factor VIII. 10A1C6 produced factor VIII and vWF and was selected in 1 mM MTX and 2.0 F.M dCF. At the 72-h time point, the cellular productivities of the 10A1C6 and 1OAl cells were 5.9 and 0.3 p.U per cell, respectively, in the presence of serum and 3.2 and 0.06 ,uU per cell, respectively, in the absence of serum. At 72 h there were 30 and 22 ,ug of vWF per ml present in the conditioned medium of the 10A1C6 cells in the presence and absence of serum, respectively. The lower productivities of factor VIII and vWF may have resulted from the physiologic state of the cells growing in serum-free medium.

expression, similar levels of factor VIII activity were obtained in the presence or absence of serum. In contrast to factor VIII, vWF expression was not dramatically influenced by the presence of fetal bovine serum at any expression level (data not shown). The accumulation of factor VIII activity in the medium of the original lOAl cell line and in the vWF coexpressing 1OA1C6 cell line was compared in the presence and absence of 10%o fetal calf serum (Fig. 1). Conditioned medium from 1OAl cells exhibited low-level factor VIII activity which reached a peak by 24 h and then subsequently declined in either the presence or absence of serum. In contrast, conditioned medium from 10A1C6 cells exhibited a high accumulation of factor VIII activity for up to 72 h and was independent of the presence of fetal bovine serum. High productivity per cell in the coexpressing cell line was observed in the presence or absence of serum (see the legend to Fig. 1). The twofold-reduced rate of factor VIII accumulation observed in the absence of serum may have resulted from the physiology of the cells propagated in the serum-free medium. The cellular productivities of both factor VIII and vWF were reduced to the same degree in serum-free medium (see the legend to Fig. 1). These experiments show that the ability of the 10A1C6 cells to supplement the medium with

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MOL. CELL. BIOL.

KAUFMAN ET AL.

-ADA

A.

B. PROTEIN

RNA

__-DHFR

-GRP 78

-Actin

(c

(41,

, I

C)

.1-1 f