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 Growth Factor A- and B-Chain Homodimers in Rat-1 Cells and Human Fibroblasts Reveals Differences in Protein 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 genes 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), epidermal growth factor (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) proteins, 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 gene is (C.-H. Heldin, Backstrom, Ostman, Hammacher, located on human chromosome 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 chromosome 7 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.