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MOLECULAR AND CELLULAR BIOLOGY. JUly 1988. p. 2753-2762 Vol. 8, No. 7 0270-7306/88/072753-10$02.00/0 Copyright © 1988. American Society for Microbiology Expression of Recombinant Platelet-Derived A- and B-Chain Homodimers in Rat-1 Cells and Human Fibroblasts Reveals Differences in Processing and Autocrine Effects MARGARET BYWATER,' FREDRIK RORSMAN.' ERIK BONGCAM-RUDLOFF, GEORGE MARK,2 ANNET HAMMACHER,3 CARL-HENRIK HELDIN , BENGT WESTERMARKl AND CHRISTER BETSHOLTZ'* Department of Pathology, Univ'ersit' Hospital,' and Ludwvig Institute for Cancer ResearcIh, Uppsala Brancsh,3 Uppsala, Sweden, and Laboratory of Humnan Carcinogenesis, National Canocer Institute, Bethesda, Maryland2 Received 22 December 1987/Accepted 8 April 1988

The autocrine effects of platelet-derived growth factor (PDGF) A- and B-chain homodimers (PDGF-AA and PDGF-BB) on rat-1 cells and human fibroblasts have been investigated by using human PDGF A- and B-chain cDNA clones expressed in a retroviral vector. Infection with replication-defective virus carrying the B-chain cDNA resulted in a phenotypical transformation resembling that induced by simian sarcoma virus. The resulting cells were focus forming in monolayer cultures, grew to high saturation densities, and formed large colonies in soft agar. The PDGF A-chain transfectants showed no transformed morphology and lacked focus-forming activity but grew to high saturation density in monolayer culture and formed small colonies in soft agar. A similar but weaker effect was obtained with an A-chain cDNA variant containing a 69-base-pair insertion in the 3' end of the protein-coding domain. A- and B-chain transfectants released PDGF receptor- competing activity into the medium, but only the medium conditioned by the B-chain transfectants possessed potent mitogenic activity on human fibroblasts. Both types of transfectants had downregulated levels of PDGF receptors; however, the B-chain transfectants were downregulated to significantly lower levels. Metabolic labeling and immunoprecipitations with PDGF antiserum showed that the PDGF B-chain protein was processed to a 24-kilodalton cell-associated and a 30-kilodalton secreted dimeric protein. The A-chain protein was rapidly secreted as a 31-kilodalton dimeric protein. The present study shows a marked difference in the autocrine effects of PDGF-AA and -BB expressed under the control of a retroviral promoter and suggests that different biological properties may be assigned to these two PDGF isoforms.

The discovery of a close structural homology between the press). Human osteosarcoma- (18) and melanoma-derived platelet-derived growth factor (PDGF) B-chain and the pre- (44) growth factors have been identified as PDGF-AA, as has dicted protein product of the simian sarcoma virus (SSV) the major part of the activity secreted by human glioma cells oncogene, v-sis (11, 13, 43), provided the first direct evi- (A. Hammacher, M. Nistdr, B. Westermark, and C.-H. dence that the uncontrolled expression of a growth factor Heldin, submitted for publication 2; M. Nistdr, A. Hamma- may alter cell growth patterns in a way that might contribute cher, K. Mellstrom, A. Siegbahn, L. Ronnstrand, B. to malignant transformation. Additional examples of growth Westermark, and C.-H. Heldin, Cell, in press). The v-sis factor that under transcriptional control of viral pro- product (32) and porcine PDGF (39) are made up as homo- moters provide a transforming signal to relevant cells include dimers of B-chain homologs (PDGF-BB). the colony-stimulating factors GM-CSF (27) and CSF-l Recent studies have suggested that the various isoforms of (M-CSF) (37), transforming growth factor-Cx (TGF-oL) (15. 36, PDGF may have different functional activities. Thus, PDGF- 42), (EGF) (38), and basic fibroblast AA has been shown to possess relatively low mitogenic growth factor (bFGF) (34). Two potential oncogene prod- activity and be devoid of chemotactic activity and ability to ucts, the hst (41) and int-2 (12) , have recently been induce tyrosine phosphorylation in porcine PDGF receptor shown to be homologous to bFGF and may be further preparations and cytoskeletal reorganization in cultured examples of transforming growth factor genes. human fibroblasts (Nist&r et al., in press). Binding studies Human PDGF is made up as a dimer of one B-chain have indicated the existence of at least two types of PDGF disulfide-linked to a second polypeptide, the A-chain (22). receptors, one (type A) which binds all three dimers, and The sequence of human PDGF A-chain cDNA clones another (type B) which binds only PDGF-BB and PDGF-AB showed a 50% amino acid sequence identity between the A- G. A. A. and B-chain precursors (4). Whereas the B-chain is (C.-H. Heldin, Backstrom, Ostman, Hammacher, located on human 22 (10, 40), the A-chain gene L. Ronnstrand, K. Rubin, M. Nistdr, and B. Westermark, is encoded by a distinct gene (35) on human EMBO J., in press). Thus, the functions specified above (4). which are specific for B-chain containing PDGF dimers All three possible dimeric isoforms of PDGF have been appear to be triggered through the type B receptor. identified. PDGF purified from human platelets is predomi- The different biological properties of PDGF-AA and nantly a heterodimer (PDGF-AB) (A. Hammacher, U. Hell- PDGF-BB are of interest in relation to the differential man, U. Johnsson, A. Ostman, A. Gunnarsson, B. Wester- expression of the PDGF-A and -B genes in human tumor cell mark, A. Wasteson, and C.-H. Heldin, J. Cell. Biochem., in lines (4). The autocrine action of a growth factor is depen- dent not only on its biological activity in the extracellular space, but also on its synthesis rate, secretion, and process- * Corresponding author. ing. In order to experimentally determine and compare the 2753 2754 BYWATER ET AL. MOL. CELL. BIOL. autocrine effects of PDGF-AA and -BB, we have transfected of a heat-denatured radiolabeled DNA probe per ml was rat-1 cells and human fibroblasts by using human PDGF added, and incubation was continued overnight at 42°C. cDNA clones in a retrovirus vector. Differences were re- Probes were radiolabeled according to Feinberg and Vogel- vealed between the two PDGF proteins in the ability to stein (14). The filters were washed in 0.1x SSC-0.1% SDS at induce a transformed phenotype, growth in semisolid me- 60°C for 30 min and exposed at -70°C to Kodak XAR-5 films dium, and downregulation of PDGF receptors. Analysis of for 1 to 3 days. the PDGF A- and B-chain proteins produced by the trans- PDGF receptor measurements and the PDGF radioreceptor fected cells demonstrates differences in processing and con- assay. Pure PDGF-AB was prepared from human platelets as firmed the differences in biological activity of the two described (19). PDGF-AA was purified to apparent homoge- molecules. neity from supernatants of yeast cells transfected with a PDGF A-chain construct (A. Ostman et al., manuscript in MATERIALS AND METHODS preparation). The concentration of PDGF-AA was deter- mined by amino acid composition analysis. Both factors Construction of infectious retrovirus carrying coding se- were labeled with 1251 by the chloramine T method to quence for PDGF-A and PDGF-B. The human PDGF-A specific activities of 40,000 (PDGF-AB) and 50,000 (PDGF- cDNA clones 13.1 (35) and Dl (4) corresponding to a major AA) cpm/,ug. and a minor form of the PDGF-A mRNA, differing only by Binding of [1251]PDGF-AB was performed as described the absence or presence of exon 6 of the human gene (35), (30). Nonspecific binding was determined as the amount of respectively, were inserted into the BainHI site of the bound [125I]PDGF-AB in the presence of a 100-fold molar retroviral vector pLJ (26). This vector maintains the RNA excess of unlabeled PDGF-AB. packaging site qP. The cDNAs were inserted downstream Serum-free conditioned medium was harvested from ex- from the 5' Moloney murine leukemia virus (Mo-MuLV) ponentially growing G418-selected rat-1 cell cultures carry- long terminal repeat (LTR) and upstream from the neomycin ing the various recombinant retrovirus genomes. For assay- resistance gene, with the help of BarmHI linkers. The two ing the content of PDGF receptor-competing activity in cDNA clones, 1,302 and 1,233 bases long, respectively, these media, human diploid fibroblasts were used as test contain the complete protein coding sequence as well as 387 objects (30). Briefly, confluent cultures of fibroblasts were bases of 5' untranslated sequence (both 13.1 and DI) and 278 incubated with conditioned medium for 2 h at 4°C, followed (Dl) and 254 (13.1) bases of 3' untranslated sequence. The by washings and an additional incubation in buffer contain- Dl clone encodes a 211-amino-acid PDGF-A precursor with ing 2 ng of [125I]PDGF-AB per ml for 60 min at 4°C. After a highly basic and charged C-terminus. The 13.1 clone extensive washing, cells were solubilized in a Triton X-100- predicts a 15-amino-acid-shorter precursor lacking the basic containing buffer, and released radioactivity was determined C-terminus. in a Beckmann gamma counter. A human PDGF-B cDNA (sis-5; C. Betsholtz and A. Mitogenic and growth assays. Mitogenic activity of the Johnsson, unpublished) was cleaved with BamHI to gener- serum-free conditioned medium from marker-selected rat-1 ate a 2,082-base fragment containing the entire protein cultures was measured as [3H]thymidine incorporation in coding domain as well as 254 bases of 5' and 1,105 bases of human fibroblasts (3). The PDGF-specific nature of the 3' untranslated sequence, which was inserted into the same mitogenic activities was determined by neutralization with site of pLJ. anti-PDGF immunoglobulin G (IgG) prepared from rabbit These constructs were first transfected into the amphotro- polyclonal antisera (17) by chromatography on protein A- pic retrovirus-packaging cell line +-am (9) and the virus titer Sepharose (Pharmacia). was amplified by shuttle infection into PA12 cells (25). The Growth properties of G418-selected rat-1 transfectants cells carrying the construct were selected in medium con- were tested as growth rates in Eagle minimal essential taining G418 (GIBCO) (500 pug/ml). Helper virus-free stocks medium (MEM) supplemented with 1 or 10% fetal calf serum of infectious virus were produced and titrated on rat-1 cells. (FCS; GIBCO). Stocks containing 103 to 104 FFU/ml were used for infection Focus-forming properties were tested by seeding 500 of exponentially growing rat-1 cells and human fibroblasts infectant cells on a subconfluent layer of normal rat-1 cells, (AG1523, obtained from the Human Genetic Cell Mutant after which the cultures were incubated in 5% FCS for 2 Repository, Camden, N.J.). Cells having the viral genome weeks, with a medium change every second day. integrated were then selected with G418 (500 ,ug/ml for rat-1 Ability to grow in soft agar was tested by plating 105 cells and 250 ,ug/ml for human fibroblasts). Individual neomycin- in 1 ml of Eagle MEM supplemented with 10% FCS and resistant clones were picked in some cases, but to avoid 0.3% agarose on top of a 2-ml layer of the same medium with effects of clonal variations, most of the experiments were 1.5% agarose. Dishes were then monitored for 2 weeks, and done on mass-selected cultures. colonies were scored in a microscope. Standard methods were used for construction and propa- Metabolic labeling, immunoprecipitations, and polyacryl- gation of the PDGF gene containing expression vectors (28). amide gel electrophoresis. Roller bottles (850 cm2) with RNA preparation and Northern (RNA) blot analysis. Total exponentially growing cells were incubated in 10 ml of cellular RNA was prepared form trypsinized and pelleted cysteine-free Eagle MEM containing 1 mCi of [35Slcysteine cells by the LiCl-urea method (1). Electrophoresis in dena- (600 Ci/mmol, 1 Ci = 37 GBq; Amersham) for the indicated turing formaldehyde-agarose gels and blotting to nitrocellu- periods of time, followed by chases of various lengths in 10 lose membranes (Schleicher & Schull, type BA85) were ml of cysteine-containing medium. Pulse and chase media, done as described (28). Filters were prehybridized in 50% as well as a cell lysate prepared by freeze-thawing in a 0.01 formamide-100 mM Tris hydrochloride (pH 7.4-S5x SSC M phosphate buffer containing 0.5 M NaCI and 1% Triton (1x SSC = 150 mM NaCl, 15 mM sodium citrate)-l x X-100, were then sequentially immunoprecipitated with a Denhardt solution (28)-0.1% sodium dodecyl sulfate (SDS)- control rabbit serum and PDGF antiserum (Hammacher et 250 ,ug of sonicated and denatured salmon sperm DNA per al., in press) as described (2). Immunoprecipitates were then ml overnight at 42°C. In the hybridization mixture, 106 cpm analyzed on 15% SDS-polyacrylamide gels (6) under reduc- VOL. 8, 1988 RECOMBINANT PDGF HOMODIMER EXPRESSION DIFFERENCES 2755 ing or nonreducing conditions. Dried gels were exposed to Kodak XAR-5 films for 3 to 14 days. *0)U) az -, A RESULTS Construction of expression vectors for PDGF A- and B- chains. We used cDNA clones corresponding to the PDGF A- (4) and B-chains (C. Betsholtz and A. Johnsson, unpub- lished) isolated from a human glioblastoma cDNA library to construct expression vectors for the two PDGF chains. The 28S outline of the vectors is shown in Fig. 1. A 2.0-kilobase (kb) BamHI fragment of the B-chain cDNA containing the com- 18S- plete protein coding domain as well as 254 bases of 5' and 1,105 bases of 3' untranslated sequence was inserted in both the sense and antisense orientation in the unique BamHI site of the retrovirus vector pLJ (26). This vector expresses the selectable transposon TnS neomycin phosphotransferase B gene (neo) through the use of an internal simian virus 40 (SV40) promoter and the inserted sequence under the con- trol of the viral LTR. Two forms of A-chain cDNAs, 1,296 0 and 1,227 bp in length and differing by the presence (PDGF- 28S- AL) or absence (PDGF-As) of a 69-bp insertion, were ligated into the same restriction site of pLJ. The presence of the 18S- 69-bp insertion predicts an A-chain precursor with a 15- amino-acid C-terminal extension which is highly charged and basic (4). This difference is likely to be due to differential splicing, as the 69-bp insertion forms a separate exon in the FIG. 2. Northern blot analysis of 10 pug of total RNA prepared A-chain gene (35). Both A-chain cDNAs have full protein from G418-selected cells infected by the various recombinant retro- coding capacity and include 387 bases of 5' and 278 (PDGF- viruses. Human cDNA clones corresponding to A- (A) and B-chains AL) or 254 bases of 3' untranslated sequence. (B) of PDGF were used as probes. Films were exposed for 3 days. (PDGF-A,) rat-1, Uninfected parental line; rat-lB, -AL, and -As, infected with The resulting constructs were transfected into +i-am retro- the B-chain virus and the long and short A-chain viruses, respec- virus-packaging cells (9), followed by shuttle infection into tively. rat-lAst is a clonal selectant of the rat-lAs culture. PA12 cells (29) to increase virus titers. Stocks of replication- defective virus produced were used to infect rat-1 cells and human fibroblasts. no rearrangements of the viral genome appear to have Different cellular effects of transfected PDGF A- and B- occurred during integration into the cellular genome. No chain cDNAs. Exponentially growing rat-1 cell cultures were expression of the endogeneous rat A- and B-chain genes was infected with recombinant virus carrying PDGF B-chain, found in the parental rat-1 cells or in any of the transfectahts. PDGF-B-antisense, PDGF-AL, PDGF-As, or the neomycin The B-chain construct was much more potent than the resistance gene only. Of these viruses, only the B-chain A-chain constructs in inducing a change in cellular pheno- construct was found to induce foci of phenotypically trans- type (Fig. 3). The morphology of PDGF-B/rat-1 resembled formed cells (not shown). G418 selection verified that ap- closely that of SSV-transformed or PDGF-stimulated rat-1 proximately the same number of cells were infected with cell cultures (not shown). Incubation of PDGF-B/rat-1 cells each type of virus (not shown). with suramin (100 ,ug/ml) resulted in reversion to a normal To determine whether the other constructs had effects morphology (Fig. 3). Analogous to studies performed on which were not revealed as focus formation in a monolayer SSV-transformed cells (5), this implies that the transformed of normal cells, we studied the cellular morphology of morphology of the fl-chain transfectants is the result of the G418-mass-selected cultures. Northern blot analysis of total extracellular autocrine action of a PDGF-like growth factor. cellular RNA revealed expression of mRNA hybridizing to a The growth behavior of the various transfectants is pre- radiolabeled A-chain probe in the PDGF-A/rat-1 cells and sented in Fig. 4 and 5. Both PDGF-A/rat-1 and PDGF-B/ mRNA hybridizing to a B-chain probe in the PDGF-B/rat-1 rat-1 cells grew to higher saturation densities than cells cells (Fig. 2). The transcript sizes correspond to the size of infected with the virus carrying the G418 resistance gene the retroviral genome (4.6 kb) plus inserted cDNA (5.9 and only (Fig. 4A). This difference persisted in low serum 6.6 kb for the A- and B-chain viruses, respectively). Thus, concentrations (1%) (Fig. 4B). The decreased growth rate of the PDGF-AL/rat-1 cells in this condition may relate to the lower production of PDGF A-chain protein (see below). I PDGF-B Finally, both PDGF-As/rat-1 and PDGF-B/rat-1 cells showed an increased ability to grow in soft agar compared PDGF-As. I with uninfected and control virus-infected cells (Fig. 5). However, the PDGF-13/rat-1 cells formed significantly larger colonies than PDGF-As/rat-1 cells (Fig. 5). BamHI Downregulation of PDGF receptors and production of Neo I pBR ori LTR PDGF-like activities in PDGF-A/rat-1 and PDGF-B/rat-1 cells. pLJ Table 1 shows the specific binding of [1251]PDGF-AB to FIG. 1. Outline of the reltroviral constructs used in the present confluent cultures infected by the various recombinant vi- study. These were constructLed as described in the text. ruses and mass selected with G418. Only the cells infected 2756 BYWATER ET AL. MOL. CELL. BIOL.

FIG. 3. Micrographs of G418-selected rat-i cell cultures of cells infected by the various recombinant retroviruses; vector control virus (A); B-chain virus (B); short-A chain virus (C); long A-chain virus (D); B-chain antisense virus (E); B-chain virus-infected cells treated with suramin (200 pug/ml) for 48 h (F). with the A- or B-chain viruses showed a significant reduction transfectants appears to be due to downregulation of the of ['25I]PDGF-AB binding. PDGF-B/rat-1 showed more than type A receptors only. 90% reduction in ['25I]PDGF-AB binding compared with That rat-1 cells carry fewer type A PDGF receptors than cells infected with the vector control and PDGF-B-antisense human fibroblasts (Heldin et al., in press) was confirmed by virus. The PDGF-A/rat-1 cells showed PDGF receptor [1251]PDGF-AA binding (not shown). However, their biolog- downregulation, but to a lesser extent; PDGF-AL/rat-1 ical significance was demonstrated by exogenous additions showed 10% and PDGF-As/rat-1 showed 25% reduction in of PDGF-AA. Exposed rat-1 cells grew to higher saturation the binding to [I251]PDGF-AB (Table 1). In analogy with densities and showed increased survival under serum star- experiments performed on SSV-transformed cells (5, 16, 25), vation (not shown). This is in agreement with the in vitro the binding of [1251I]PDGF-AB was restored to normal levels behavior of the PDGF-A/rat-1 transfectants presented following a 48-h incubation with suramin (Table 1). In order above. to get an idea of the relative numbers of type A and type B Conditioned medium harvested from PDGF-A/rat-1 and PDGF receptors on rat-1 cells and the transfectant cells, the PDGF-B/rat-1 competed with [1251I]PDGF-AB for binding to binding of [1251]PDGF-AB (which binds to both types of human fibroblasts, indicating that PDGF-like proteins are receptors) was competed with by PDGF-AB as well as released by the cells. As shown in Table 2, PDGF-B/rat-1 PDGF-AA (which only competes for binding to the type A released the equivalent of 20 ng of PDGF-AB per ml. receptor). Whereas [125I]PDGF-AB was most efficiently PDGF-As/rat-1 produced similar levels of PDGF receptor- competed with by PDGF-AB, with a 75% reduction in competing activity, whereas PDGF-AL/rat-I released the binding at 50 ng/ml, the same concentration of PDGF-AA equivalent of 5 ng of PDGF-AB per ml (Table 2). These resulted in a much smaller reduction (15%) (Table 1). The values are probably underestimations of the actual concen- corresponding figures for human foreskin fibroblasts were trations of PDGF-BB and PDGF-AA released into the me- 75% reduction by PDGF-AB at 50 ng/ml and 50% reduction dium, since both types of homodimers compete less effi- by the same concentration of PDGF-AA (Heldin et al., in ciently with [125I]PDGF-AB than does PDGF-AB itself press). [1251]PDGF-AB binding to the PDGF-A/rat-1 trans- (Heldin et al., in press). fectants was efficiently competed for by PDGF-AB but not The PDGF-B/rat-1 medium was strongly mitogenic for to any significant degree by PDGF-AA. Thus, our data human fibroblasts (Table 2). The PDGF-A/rat-1 medium suggest that the major fraction of the PDGF receptors on was, however, in comparison with receptor-competing ac- rat-1 cells are of type B. These were effectively downregu- tivities, relatively weakly mitogenic. Both the high mitogenic lated in the PDGF-B/rat-1 transfectants. The relatively small activity of the PDGF-B/rat-1 medium and the lower activities decrease in ['251]PDGF-AB binding to the PDGF-A/rat-1 of the PDGF-A/rat-1 media were blocked by the addition of VOL. 8, 1988 RECOMBINANT PDGF HOMODIMER EXPRESSION DIFFERENCES 2757

A *Rat-1-B

4

0- co03

A I-x B 0 0 1 C.) 12 6A 0 *--''*Rat-l-BRat-l-AS

5 10 15 5 10 Days in culture FIG. 4. Growth curves of G418-selected rat-1 cells infected by the various recombinant retroviruses. Growth was measured as accumulated number of cells in a 10-cm2 petri dish in 10% (A) and 1% (B) FCS. rat-i-V, Vector control virus infected; rat-i-B, -AL, and -As, infected by B-chain virus and long and short A-chain viruses, respectively.

A B

S A

It? - * v

C 4. .4 'o40 A , No.~~ a X. ~ ~ AL 0 S*w* a twZ Z~~~ av 4 * *' a.009 I &

FIG. 5. Growth in soft agar of rat-1 cells and G418-selected cells infected by recombinant retroviruses. (A) Uninfected rat-1 cells; (B) vector control-infected cells; (C) cells infected by the short A-chain virus; (D) cells infected by the B-chain virus. 2758 BYWATER ET AL. MOL. CELL. BIOL.

TABLE 1. [1251]PDGF binding to G418-selected rat-1 cells infected by retroviruses for A recombinant carrying genes PDGF` 1 2 3 4 5 6 7 8 B1 2 3 4 5 6 7 8 kDa kDa Addition to medium or Relative binding (% of control) treatment Vc AL As B Bas -r None 100 91 75 9 105 .0 -*.: 40 40b I . V.... PDQF-AB (50 ng/ml) 24 27 32 n.d.! n.d. .0 101*.." PDGF-AB (250 nglml) 12 10 12 10 11 PDGF-AA (50 ng/ml) 85 90 77 n.d. n.d. 31- _ 31- # 3- 24- aU After 48 h of incubation 96 103 95 91 105 22- with suramin (100 g/ml) PDGF-AB (250 ng/ml) and 8 8 9 10 9 48-h incubation with suramin "Cells were plated in Falcon 12-well plates and allowed to grow to FIG. 6. Metabolic labeling of G418-selected cultures of rat-1 confluence. Cultures were then washed, and the binding of [1251]PDGF-AB or cells infected by the various recombinant retroviruses. Cells were [125I]PDGF-AAs was determined as described in the text. Vc, Vector control- labeled uniformly for 2 h, and conditioned medium and cell lysates infected cells; AL, PDGF-AL infectants; AS, PDGF-As infectants; B, PDGF- were subjected to sequential immunoprecipitation by normal rabbit B infectants; Bas, PDGF-B-antisense infectants. Values are expressed as percentage of [125IIPDGF-AB bound to the vector control transfectants and serum and PDGF antiserum. Electrophoresis was run on unreduced represent means of triplicate determinations. samples. (A) Immunoprecipitates from the conditioned medium; (B) b n.d., Not determined. immunoprecipitates from the cell lysates. Lanes 1, 3, 5, and 7 are precipitates by the control serum; lanes 2, 4, 6, and 8 are precipitates by the PDGF antiserum. Lanes 1 and 2, Cells infected by the short A-chain virus; lanes 3 and 4, vector control infectants; lanes 5 and 6, 40 ,ug of anti-PDGF IgG per ml, indicating that they were due B-chain 7 to PDGF-like entities (Table 2). infectants; lanes and 8, B-chain antisense infectants. Synthesis and secretion of PDGF A- and B-chain proteins in rat-i transfectants. In order to demonstrate biosynthesis of PDGF-like proteins, mass-selected cultures infected by the B-chain protein was cell associated and in the 30-kDa form various recombinant viruses were labeled with [35S]cysteine, (Fig. 7A). After a 3-h chase, a 30-kDa B-chain protein had and conditioned medium as well as cell lysates were sequen- appeared in the medium and the cell-associated form at- tially immunoprecipitated with a control rabbit serum and tained the 24-kDa configuration (Fig. 7A). All these forms PDGF antiserum. The result of a 2-h labeling is shown in Fig. appear to be dimers, since on reduction they exhibited 6. Both PDGF-B/rat-1 arnd PDGF-As/rat-1 released proteins approximately half the unreduced molecular weight (Fig. in the 30-kilodalton (kDa) range which were specifically 7B). With shorter chase periods, it was evident that the recognized by PDGF antibodies. The cell lysates, however, secreted B-chain protein could be seen after a 15-min chase revealed a different pattern. The cell-associated A-chain (Fig. 8) and the cell-associated 24-kDa form was first seen protein was similar in size and amount to the released after a lag of 30 min (Fig. 8). A-chain protein. The cell-associated B-chain protein ap- It has recently been reported that PDGF-AL and PDGF- peared to be more abundant than the one released. A major As expression in monkey COS cells under SV40 promoter 30-kDa protein was identified as well as a less abundant control resulted in secretion of a PDGF-like protein only doublet of about 24 kDa. when encoded by the longer cDNA (8). Using LTR con- Pulse-chase experiments with PDGF-B/rat-1 cells are shown in Fig. 7. Following a 15-min labeling period, all of the A B 1 2 3 4 5 6 7 8 1 23 45 6 78 kDa :j TABLE 2. Determination of PDGF-like activities in conditioned r kDa medium from G418-selected cultures of rat-1 cells infected by I.. -#. kD recombinant viruses carrying PDGF genes" Relative activity Activity tested (PDGF-AB equivalents) and treatment 30_ .:i, Vs AL As B Bas 24 ..S 227-A -, PDGF receptor competition IW.. None <2 5 20 20 <2 15- Preincubation with anti-PDGF IgG <2 <2 <2 <2 <2 12- Mitogenic activity None 180 400 1,400 5,300 250 10- Preincubation with anti-PDGF 290 320 420 200 370 PDGF-AB (10 ng/ml) added 6,600 6,700 6,500 6,700 6,700 FIG. 7. Metabolic pulse-chase labelings of PDGF-B/rat-1 cul- tures followed by sequential immunoprecipitations with normal "The PDGF radio receptor assay was performed as described (see the text) rabbit serum and PDGF antiserum. (A) Nonreduced samples. (B) with undiluted conditioned medium. Values were compared with a PDGF-AB Reduced samples. Lanes 1, 3, 5, and 7 show precipitates by the standard and converted to PDGF-AB equivalents. Preincubation with anti- PDGF IgG (40 ,ug/ml) was for 6 h at room temperature. Mitogenicity on human control serum. Lanes 2, 4, 6, and 8 show immunoprecipitates by the fibroblasts was tested with conditioned medium diluted 1/4. Unconditioned PDGF antiserum. Lanes 1 to 4, 15-min pulse, no chase. Lanes 5 to medium tested in parallel gave 200 cpm per plate and, with PDGF-AB (10 ng/ 8, 15-min pulse, 180-min chase. Lanes 1, 2, 5, and 6 represent the ml) added, 6,500 cpm per plate. Values are means of triplicate determinations. cell lysates, and lanes 3, 4, 7, and 8 represent the conditioned See Table 1, footnote a, for abbreviations. medium. VOL. 8, 1988 RECOMBINANT PDGF HOMODIMER EXPRESSION DIFFERENCES 2759

MEDIUM amino acid sequence, it is likely to be sensitive to proteolytic chase (m 0 15 30 60 90 processing. This may have occurred in the released form n p n p n P n p n p since it is similar in size to the PDGF-As protein. Both the kDa kDar conditioned medium and the cell lysate contained, in addi- tion to the 31-kDa protein, larger polypeptides of molecular @ --am-masses"_ 36 and 44 kDa specifically recognized by the PDGF- antibodies (Fig. 9A). Reduction demonstrated besides the 17-kDa mature A-chain protein, a 22-kDa form (Fig. 9B and 30- 30- ax D) which was the expected size of a precursor molecule (4). 24- The two higher-molecular-mass forms (36 and 44 kDa) seen in the medium may represent a homodimer of the precursor and a heterodimer between one precursor and one processed

UN:-IE',,'h~ molecule (Fig. 9A). 12- Infection of human diploid fibroblasts with PDGF A- and I2tBzPPr 'd' ma B-chain recombinant viruses. The human fibroblast line FIG. 8. Metabolic pulse-chase labeling of PDGF-B/rat-1 cultures AG1523, which has been extensively characterized with followed by sequential immunoprecipitations with normal rabbit regard to its response to PDGF (3) and SSV (24) and its serum (n) and PDGF antiserum (p). Th 15 min. The time was as indicated above the lane pulse was chase expression of type A and B PDGF receptors (Heldin et al., in markers. press), was used as a target cell for the amphotropic recom- binant PDGF viruses. The response was very similar to that seen in rat-1 fibroblasts. The B-chain virus had a remarkable structs in rat-1 cells, we obtained a different result. As effect on the cellular morphology, closely resembling SSV shown in Fig. 9A, PDGF-As/ralt-1 cells synthesized and transformation (24), whereas infection with the PDGF-A secreted severalfold more immunoreactive PDGF-like pro- viruses resulted in a much weaker effect, but still signifi- tein than PDGF-AL/rat-1 in spite of similar mRNA levels cantly different compared with uninfected controls (Fig. 10). (Fig. 2). Both products appeared ais 31-kDa dimeric proteins Neither A- nor B-chain transfectants grew beyond the pre- in the medium. The slightly highe r molecular weight of the dicted number of population doublings and senesced nor- intracellular PDGF-AL than of tIhe intracellular PDGF-As mally (Fig. 10E and F), indicating that expression of PDGF protein (Fig. 9C) may be accouinted for by the predicted A- or B-chains has no immortalizing effect on human fibro- longer C-terminal. Due to the very basic nature (4) of the blasts.

A 1 2 3 4 5 6 7 8 9 1011 12 kDa B '~~~~~~TlwI Lkna 1 2 3 4 5 6 7 8 9101112

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FIG. 9. Metabolic labeling of PDGF-As/rat-1 and PDGF-AL/rat-1 cultures. Cells were uniformly labeled for 2 h. and the conditioned media and cell lysates were sequentially immunoprecipitated by normal rabbit serum and PDGF antiserum. Two independent labelings of each cell type are shown. PDGF-Ast is a clonal selectant chosen for its high PDGF-A mRNA expression. Northern blots of the same cell cultures are shown in Fig. 3. (A) Immunoprecipitates from the conditioned media, unreduced samples. (B) Conditioned media, reduced samples. (C) Cell lysates, unreduced. (D) Cell lysates, reduced. Lanes 1, 3, 5. 7. 9. and 11 show' immunoprecipitates by the normal rabbit serum. Lanes 2, 4, 6, 8, 10, and 12 show immunoprecipitates by the PDGF antiserum. Lanes 1 to 4; mass-selected cultures infected by the short A-chain virus (two experiments). Lanes 5 to 8, clonal selectant PDGF-Ast (two experiments). Lanes 9 to 12. Mass-selected cultures infected by the long A-chain virus (two experiments). 2760 BYWATER ET AL. MOL. CELL. BIOL.

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FIG. 10. Morphology of human fibroblasts, line AG1523, infected by PDGF A- and B-chain recombinant retroviruses. (A) Uninfected cells; (B) B-chain infectants; (C) PDGF A-chain (short) infectants; (D) PDGF A-chain (long) infectants; (E) senescent A-chain (short) infectants; (F) senescent B-chain infectants.

DISCUSSION Betsholtz, C.-H. Heldin, B. Westermark, K. C. Robbins, and S. A. Aaronson, submitted for publication). Using human PDGF A- and B-chain cDNAs under the It is not likely that the difference in transforming ability transcriptional control of Mo-MuLV LTRs, we demon- result of differences in strated in both rat-1 cells and human diploid fibroblasts that between the two PDGF genes is the only cells expressing the B-chain protein were transformed the level of expression. Both mRNA and protein levels in the to a phenotype resembling that of SSV-transformed cells. An two types of infectants were comparable, and the differences increase in the cell saturation density was observed in in levels of cell-associated A- and B-chain proteins appeared cultures of cells infected by recombinant virus carrying to reflect a variation in processing and subcellular localiza- PDGF-A. In individual clones of PDGF-A/rat-1 cells, we tion rather than different rates of synthesis of the two noticed a correlation between A-chain mRNA expression, proteins. It is more likely that the different transforming PDGF receptor downregulation, and tendency to grow to potentials are due to different biological properties of PDGF- higher saturation densities (data not shown). This indicates AA and PDGF-BB. A recent comparison between PDGF- that overexpression of PDGF A-chains has an effect on the AA purified from human glioma cell-conditioned medium growth behavior of cells in monolayer culture. However, and PDGF-AB (human PDGF) revealed several differences; both mass-selected PDGF-A/rat-1 and individual cell clones, compared with PDGF-AB, PDGF-AA was less mitogenic some of which showed very high expression of A-chain and lacked chemotactic activity on human fibroblasts as well mRNA, differed markedly from cells infected with the B- as ability to induce autophosphorylation of the PDGF recep- chain recombinant virus. In NIH 3T3 cells, overexpression tor and reorganization of actin filaments (Nister et al., in of PDGF-AL by help of a mouse metallothionein promoter press). In contrast, PDGF-BB released by SSV-transformed induces small foci of transformed cells; however, also in cells (23) or produced as a recombinant molecule in CHO these experiments, the A-chain cDNA was markedly less cells or yeast (A. Ostman et al., unpublished) appears to be transforming than the B-chain cDNA (M. P. Beckmann, C. comparable with PDGF in inducing PDGF receptor phos- VOL. 8, 1988 RECOMBINANT PDGF HOMODIMER EXPRESSION DIFFERENCES 2761 phorylation, growth, and reorganization of actin filaments. used; clearly, however, the conclusion made by them, that Analogously, conditioned medium from PDGF-B/rat-1 was only the long PDGF A-chain variant is secreted, cannot be more mitogenic than medium from PDGF-A/rat-1 in spite of generalized. comparable protein levels, as estimated by radioreceptor The present study demonstrates that the endogenous assay and metabolic labeling. production of PDGF-AA affects the growth properties of Recent binding studies have demonstrated the presence of certain cell types but does not result in a transformed two types of PDGF receptors on human fibroblasts; type A, phenotype similar to that induced by SSV and PDGF-BB which binds all three PDGF isoforms, and type B, which expression. This is interesting in relation to the findings that binds PDGF-AB and -BB (Heldin et al., in press). The PDGF A- and B-chain gene transcription and the synthesis primary structure of a murine PDGF receptor has been of PDGF-like molecules are common properties of many determined by cDNA cloning (45). Expression of a corre- types of human tumor cell lines and frequently occur inde- sponding human cDNA in CHO cells resulted in increased pendently (reviewed in references 19 and 20). However, any specific binding of PDGF-BB and PDGF-AB but not PDGF- role of either of the two homodimeric PDGF isoforms for in AA (L. Claesson-Welsh, A. Eriksson, A. Mordn, L. Seve- vivo tumorigenicity remains to be established. rinsson, B. Ek, C. Betsholtz, and C.-H. Heldin, submitted for publication), demonstrating that this clone encodes the ACKNOWLEDGMENTS type B PDGF receptor. The nature of the type A receptor These studies were supported by grants from the Swedish Cancer remains to be elucidated. Our binding data indicate the Society. presence of a large number of type B PDGF receptors and a We thank R. C. Mulligan for making the pLJ retroviral expression relatively small but significant number of type A PDGF vector available and A. Ostman for supplying the recombinant receptors on rat-1 cells. Thus, a third possible explanation PDGF A-chain homodimers. for the different autocrine effects of PDGF-AA and PDGF- BB in these cells may relate to the relative number of the two LITERATURE CITED receptor types. However, we do not regard this as likely, 1. Auffrey, C., and F. Rougeon. 1980. Purification of mouse immu- noglobulin heavy messenger RNA from total myeloma tumor since a similar difference was noted in human fibroblasts RNA. Eur. J. Biochem. 107:303-314. which possess a relatively high number of type A PDGF 2. Betsholtz, C., C.-H. Heldin, M. Nister, B. Ek, A. Wasteson, and receptors (Heldin et al., in press). B. Westermark. 1983. Synthesis of a PDGF-like growth factor in Studies on the processing of the v-sis product have re- human glioma and sarcoma cells suggests the expression of the vealed that the end product after processing, p245s, remains cellular homologue to the transforming protein of simian sar- associated with cellular membranes (32, 33). The structural coma virus. Biochem. Biophys. Res. Commun. 117:176-182. basis for the membrane association is not known. We and 3. Betsholtz, C., and B. Westermark. 1984. Growth factor-induced others have found that SSV-transformed cells actively re- proliferation of human fibroblasts in serum-free culture depends lease a potent PDGF-like growth factor (23, 31). In the on cellular density and extracellular Ca2+ concentration. J. Cell. we show that the B-chain is Physiol. 118:203-310. present communication product 4. Betsholtz, C., A. Johnsson, C.-H. Heldin, B. Westermark, P. processed both to a cell-associated 24-kDa dimeric protein Lind, M. S. Urdea, R. Eddy, T. B. Shows, K. Philpott, A. L. and to a secreted 30-kDa dimeric protein. The exact rela- Mellor, T. J. Knott, and J. Scott. 1986. cDNA sequence and tionship between p24s5s and p3osis is not known. Several chromosomal localization of human platelet-derived growth explanations for the different subcellular localizations of the factor A-chain and its expression in tumour cell lines. Nature two B-chain proteins may be envisioned, e.g., the existence (London) 320:695-699. of receptors in the secretary apparatus carrying only the 5. Betsholtz, C., A. Johnsson, C.-H. Heldin, and B. Westermark. 30-kDa protein to the cell surface or the exposure of hydro- 1986. Efficient reversion of SSV transformation and inhibition of phobic domains in the 24-kDa form which might anchor this growth factor-induced mitogenesis by suramin. Proc. Natl. product in the membrane. The presence of PDGF antagonist Acad. Sci. USA 83:6440-6444. 6. Blobel, G., and B. J. Dobberstein. 1975. Transfer of proteins activity in the conditioned medium of the B-chain transfec- across membranes. J. Cell. Biol. 67:835-851. tants implies that the 30-kDa B-chain homodimer can inter- 7. Collins, T., J. S. Pober, M. A. Gimbrone, Jr., A. Hammacher, C. act with PDGF receptors. Whether the 24-kDa form has Betsholtz, B. Westermark, and C.-H. Heldin. 1987. Cultured PDGF receptor antagonist activity remains to be estab- human endothelial cells express platelet-derived growth factor lished. It has been suggested that cell-associated forms of the A chain. Am. J. Pathol. 127:7-12. v-sis product are mitogenic (33). 8. Collins, T., D. T. Bonthron, and S. H. Orkin. 1987. Alternative Collins and co-workers recently reported that the expres- RNA splicing affects function of encoded platelet-derived sion in monkey COS cells of the long and short versions of growth factor A-chain. Nature (London) 328:621-624. A-chain cDNA resulted in the secretion of a PDGF-like 9. Cone, R. D., and R. C. Mulligan. 1984. High-efficiency gene transfer into mammalian cells: generation of helper-free recom- growth factor only when the longer cDNA was used (8). This binant retrovirus with broad mammalian host range. Proc. Natl. finding was surprising considering that the short PDGF-A Acad. Sci. USA 81:6349-6353. transcript was the only detectable form in normal endothelial 10. Dalla-Favera, R., R. C. Gallo, A. Giallongo, and C. M. Croce. cells, which are known to release a growth factor with 1982. Chromosomal localization of the human homolog (c-sis) of properties similar to those of an A-chain homodimer (7, 8). the simian sarcoma virus onc gene. Science 218:686-688. In the present investigation, cells infected with virus carry- 11. Devare, S. G., E. P. Reddy, J. D. Law, K. C. Robbins, and S. A. ing the short form of the A-chain cDNA synthesized and Aaronson. 1983. Nucleotide sequence of the simian sarcoma released 10-fold more A-chain protein than cells infected virus genome: demonstration that its acquired cellular se- quences encode the transforming gene product p28Six. Proc. with virus carrying the longer A-chain cDNA in spite of Natl. Acad. Sci. USA 80:731-735. similar mRNA levels, indicating a lower translation rate or 12. Dickson, C., and G. Peters. 1987. Potential oncogene product increased turnover of the protein in the latter instance. The related to growth factors. Nature (London) 326:833. reason for the discrepancies between our data and those 13. Doolittle, R. F., M. W. Hunkapiller, L. E. Hood, S. G. Devare, presented by Collins et al. (8) is not known, but may be K. C. Robbins, S. A. Aaronson, and H. N. Antoniades. 1983. related to the fact that different expression systems were Simian sarcoma virus onc gene, v-sis, is derived from the gene 2762 BYWATER ET AL. MOL . CELL . B IOL .

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