Platelet-derived Growth Factor Dimers Research Articles

PDGF as an Important Initiator for Neurite Outgrowth Associated with Fibrovascular Membranes in Proliferative Diabetic Retinopathy

ABSTRACT Purpose The formation of fibrovascular membranes (FVMs) is a serious sight-threatening complication of proliferative diabetic retinopathy (PDR) that may result in retinal detachment and eventual blindness. During the formation of these membranes, neurite/process outgrowth occurs in retinal neurons and glial cells, which may both serve as a scaffold and have guiding or regulatory roles. To further understand this process, we investigated whether previously identified candidate proteins, from vitreous of PDR patients with FVMs, could induce neurite outgrowth in an experimental setting. Materials and methods Retinal explants of C57BL6/N mouse pups on postnatal day 3 (P3) were cultured in poly-L-lysine- and laminin-coated dishes. Outgrowth stimulation experiments were performed with the addition of potential inducers of neurite outgrowth. Automated analysis of neurite outgrowth was performed by measuring β–tubulin-immunopositive neurites using Image J. Expression of PDGF receptors was quantified by RT-PCR in FVMs of PDR patients. Results Platelet-derived growth factor (PDGF) induced neurite outgrowth in a concentration-dependent manner, whilst neuregulin 1 (NRG1) and connective tissue growth factor (CTGF) did not. When comparing three different PDGF dimers, treatment with PDGF-AB resulted in the highest neurite induction, followed by PDGF-AA and -BB. In addition, incubation of retinal explants with vitreous from PDR patients resulted in a significant induction of neurite outgrowth as compared to non-diabetic control vitreous from patients with macular holes, which could be prevented by addition of CP673451, a potent PDGF receptor (PDGFR) inhibitor. Abundant expression of PDGF receptors was detected in FVMs. Conclusion Our findings suggest that PDGF may be involved in the retinal neurite outgrowth, which is associated with the formation of FVMs in PDR.

Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells.

Platelet-derived growth factor (PDGF) has recently been demonstrated to control the expression of alkaline phosphatase and proteoglycan synthesis of odontoblastic cells in dental pulp tissues. Although PDGF appears to be closely related to dentinogenesis, much about the mode of action of PDGF on odontoblast differentiation remains unclear. In this study, we examined the effects of three PDGF dimers (PDGF AA, AB, and BB) on odontoblastic differentiation of dental pulp cells in long-term mineralized cultures. Dental pulp cells isolated from rat lower incisors were continuously treated with each of PDGF AA, AB, and BB in separate cultures for 20 days. The three PDGF dimers suppressed alkaline phosphatase activity, osteocalcin and calcium content, and the formation of dentin-like nodules. The expression of mRNA for dentin sialoprotein (DSP) in the cells was inhibited by PDGF AA treatment, whereas PDGF AB and BB treatment stimulated the expression of DSP, even though the dentin-like nodule formation was inhibited. Although the effects of PDGF on odontoblastic differentiation varied among the dimers, the cells expressed both PDGF alpha and beta receptors, whose quantities were similar. These results suggest that PDGF exerts diverse effects on odontoblastic differentiation depending on its dimeric form. These in vitro findings explain, at least in part, the in vivo action of PDGF in dentinogenesis during the repair process of damaged dental pulp.

Platelet-Derived Growth Factor Receptors Are Essential in the Development of Asbestos-Induced Fibrosis

Asbestos exposure results in the formation of fibroproliferative lung abnormalities similar to those encountered in subjects with idiopathic pulmonary fibrosis. Our prior publications demonstrate that platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for lung fibroblasts and is elaborated in response to asbestos fibers. Stimulation of alveolar macrophages or lung fibroblasts with asbestos fibers in vitro induces the release of three biologically active PDGF dimers (AA, AB, BB). Likewise, we reported that exposure of rats or mice to chrysotile asbestos results in upregulation of PDGF-A and PDGF-B chain messenger RNA and peptide expression, as well as the PDGF-α receptor that binds all three PDGF ligands, at the sites of fiber deposition and subsequent lesion formation. Now, we are employing the PDGF-receptor–selective tyrosine kinase inhibitor to test whether PDGF is essential in the formation of asbestos-induced lesions. We report herein that the development of asbestos-induced scars is blocked by 64% in PDGF-receptor–selective tyrosine kinase inhibitor-treated mice. PDGF ligand or receptor knockout mice are not viable. Thus, in an effort to determine which PDGF ligand-receptor interactions are important to asbestos-induced scar formation, we exposed “Patch” mice, which exhibit a 50% reduction in PDGF-α–receptor expression, and βRH mice with similar reductions in PDGF-β–receptor expression, along with their wild-type littermates, to a regimen of chrysotile asbestos (15 mg/m3) for 5 h/d on 3 consecutive days. Fibrotic lesions developing at the alveolar duct bifurcations were analyzed 4 weeks following the onset of exposure and were found to be reduced in volume by approximately 50% in two separate experiments in the βRH mice, but not in Patch mice. Our in vivo findings could not be explained by differences in proliferative responses to PDGF isoforms using lung fibroblast isolates from βRH mice and their wild-type littermates. Thus, our findings demonstrate that PDGF does play a significant role in asbestos-induced scar formation, and that reduction in expression of PDGF-β, but not PDGF-α, receptor inhibits asbestos-induced fibrogenesis.

Increased plasma levels of platelet-derived growth factor (PDGF-BB + PDGF-AB) in patients with never-treated mild essential hypertension

Platelet-derived growth factor (PDGF) could play a role in both vascular hypertrophy and atherosclerotic disease associated with hypertension. To assess whether plasma PDGF level is increased in mild essential hypertension, we measured plasma PDGF concentration in 25 never-treated patients with uncomplicated mild essential hypertension and in 22 normotensive healthy subjects. To evaluate the contribution of platelets to plasma PDGF in the two groups, we also measured plasma β-thromboglobulin (BTG). Measurement of PDGF was carried out through an enzyme-linked immunoadsorbent assay, which detects two PDGF dimers, namely PDGF-BB and PDGF-AB. Both plasma PDGF and BTG were higher in the hypertensive than in the normotensive subjects. The ratio of PDGF to BTG was similar in the two groups. Plasma PDGF was weakly correlated with plasma BTG in the normotensive subjects, whereas this relationship was lost in the hypertensive patients. Our results suggest that the increase in plasma PDGF (PDGF-AB + PDGF-BB) in never-treated essential hypertension is mainly due to platelet activation. The increased circulating level of PDGF could play a role in the vascular structural changes associated with hypertension.

Gene expression and specific binding of platelet-derived growth factor and its effect on DNA synthesis in human decidual cells

To clarify the biological significance of platelet-derived growth factor (PDGF) in human decidual cell function, which is important for the maintenance of pregnancy, we investigated gene expression of the PDGF subunits, PDGF-A and PDGF-B, specific binding of the PDGF isoform, and the effect of PDGF dimers on DNA synthesis in human decidual cells. We detected in decidua from early pregnancy the expected DNA bands of PDGF-A and PDGF-B by reverse transcriptase-polymerase chain reaction (RT-PCR) as well as mRNAs of each PDGF subunit by Northern blot hybridization, demonstrating that both PDGF subunits exist in this tissue. Scatchard plot analysis showed that decidual cells had both PDGF- α and PDGF- β receptors. PDGF-AA, -AB and -BB stimulated [ 3H]-thymidine incorporation in cultured decidual cells in a dose-dependent manner. These results indicate the importance of PDGF in human decidua.

Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds.

Platelet-derived growth factor (PDGF), a major mitogen for mesenchymal cells, is a disulfide-bonded dimer of two subunit polypeptides named A and B. All of the three possible dimeric forms, i.e. AA, BB, and AB, exist in nature. The dimeric structure has been presumed to be necessary for biological activity, since reduction of the dimer results in loss of activity and simultaneous conversion to monomeric form as determined by SDS-gel electrophoresis. However, reduction of the native molecule destroys intrachain, as well as interchain, disulfide bonds, and it is possible that the former rather than the latter are critical for proper conformation of the active protein. We show here that PDGF-B polypeptides in which all 8 cysteines or the 2nd, 4th, 5th, and 8th cysteines have been mutated to serines fail to form covalent dimers and possess dramatically less mitogenic activity than native PDGF-BB. Another mutant, PDGF-B(C2,4S), in which just the 2 cysteines involved in interchain disulfides were converted to serine, ran as a monomer on SDS-polyacrylamide gels as expected. Somewhat unexpectedly, however, the mitogenic activity of the PDGF-B(C2,4S) analog was similar to the activity of wild-type PDGF-BB disulfide-bonded dimer under physiological conditions. The activity of the analog was more sensitive to the effect of low pH than was the activity of wild-type PDGF-BB. Molecular weight analysis utilizing light scattering and sedimentation equilibrium demonstrated that the PDGF-B(C2,4S) analog exists as a noncovalent dimer at pH 4-7 but dissociates to a monomer at pH 2.5. Disulfide analysis of the mutant protein demonstrated that the intrachain disulfide bonds are the same as those formed in wild-type PDGF-BB homodimers. We conclude that proper formation of intrachain disulfide bonds is critical to maintaining the correct conformation of PDGF monomers, but that appropriately folded monomers can associate into active noncovalent dimers in the absence of interchain disulfide bonds. Interchain disulfide bonds thus appear to increase the stability of the PDGF dimer rather than being crucial to its existence.

Characterization of the retention motif in the C-terminal part of the long splice form of platelet-derived growth factor A-chain.

Platelet-derived growth factor (PDGF) A-chain is found in two different splice variants; the long A-chain variant differs from the short one in that it contains a stretch of basic amino acid residues in the C-terminal part that mediates retention of the growth factor inside the producer cell and to the cell matrix. By analyzing mutants in which different amino acid residues in the retention motif had been changed to alanine residues, we found that the total positive charge of the sequence is of importance for the function of the retention motif. Moreover, we showed that retention also occurs if only one of the polypeptides in the PDGF dimer carries the retention motif. Surface iodination and competition with a peptide having the sequence of the retention motif revealed that the long A-chain variant, in contrast to the short A-chain variant, is localized on the outside of the cells and is also associated to the cell matrix. The association is likely to be mediated partially through heparan sulfate proteoglycans since treatment of matrix with heparitinase released the long A-chain variant.

Diabetes mellitus induces accelerated growth of aortic smooth muscle cells: association with overexpression of PDGF β‐receptors

The mechanism of diabetic macroangiopathy was studied from the view point of phenotypic change of aortic smooth muscle cells (SMC). The growth rates of cultured SMC of diabetic rats or rabbits were higher than those of non-diabetic animals (controls). This difference of the growth responses was observed specifically with platelet-derived growth factor (PDGF). Of the three PDGF dimers, PDGF-AB heterodimer (PDGF-AB) and PDGF-BB homodimer (PDGF-BB) stimulated growth of diabetic SMC more than that of control SMC but PDGF-AA homodimer (PDGF-AA) did not. The binding of 125I-PDGF to the diabetic SMC was greater than that to control SMC. This was due to increase in the number of cell surface receptors for PDGF. On in vitro culture, SMC from diabetic rats expressed more PDGF beta-receptor mRNA than SMC from non-diabetic rats. Moreover, in vivo, the aortic media of diabetic rabbits expressed PDGF beta-receptor mRNA, but that from non-diabetic rabbits did not. Thus diabetic SMC over-react on PDGF stimulation through over-expression of the PDGF beta-receptor gene. The significance of this fact in development of diabetic macroangiopathy is discussed.

Platelet-derived growth factor AB heterodimer interchain interactions influence secretion as well as receptor binding and activation.

Platelet-derived growth factor (PDGF) is a disulfide-linked dimer comprised of two related polypeptide chains. To investigate the effects of an inactivating lesion introduced into one chain of the nascent PDGF dimer, approaches were developed to optimize synthesis, assembly, secretion, and purification of heterodimers between normal PDGF A and wild-type or mutant PDGF B. PDGF AB heterodimers were released into culture fluids less efficiently than PDGF AA, but to a greater degree than the cell-associated PDGF BB. These results suggest that interactions between two chains influence PDGF secretion. Analysis of heterodimers between PDGF A and disabled PDGF B mutants on cells that express either alpha or beta PDGFRs demonstrated that the impaired biologic activity of the mutant PDGF B chain was ameliorated with respect to binding and triggering of alpha PDGFRs. In cells that expressed both receptor types, heterodimers of mutant PDGF B and wild-type PDGF A gained substantially in their ability to recruit and trigger alpha, but not beta, PDGFRs. Partial rescue of impaired PDGF B mutant chain function by dimer formation with a wild-type PDGF A chain implies that interchain interactions markedly affect PDGFR binding and activation.

Differential expression of PDGF A- and B-chain genes and production of AA and AB dimers by activated human blood monocytes

Platelet-derived growth factor (PDGF) is composed of two chains (A and B) bound by disulfide bridges. Blood monocytes (BM) express the c- sis proto-oncogene, the gene coding for PDGF B-chain, and release PDGF. To evaluate PDGF release and A- and B-chain gene expression by BM, normal BM were cultured with LPS and specific transcripts for PDGF A- and B-chain genes were detected by Northern analysis and PDGF dimers by specific Elisas. Normal BM did not spontaneously express either A- or B-chain gene while, when activated. PDGF B-chain expression was precocious (maximum at 1 h) and decreased over 24 h; PDGF A-chain transcripts were present after 4 h and progressively increased over 24 h. Furthermore, activated BM released more AB dimers than AA dimers and almost no BB dimers. This observation confirms the concept of PDGF A- and B-chain separate gene regulation, and defines the specific molecular pattern of PDGF released by activated BM.

Platelet-derived growth factor AA homodimer is the predominant isoform in human platelets and acute human wound fluid.

Platelet-derived growth factor (PDGF) is a dimeric protein composed of A- and B-chains (AA, AB, and BB). PDGF purified from human platelets has been shown to be composed primarily of the AB heterodimer. Immunoblots of total platelet extracts, cell-bound PDGF from the platelet extracts, and acute human wound fluid with PDGF A- and B-chain-specific antisera all demonstrate that the PDGF A-chain is the predominant peptide. Chemotactic and immunochemical assays of chromatographic fractions during PDGF isolation support these observations and demonstrate that PDGF AA can be separated from PDGF AB and BB by ion exchange chromatography. These studies indicate that the AA isoform constitutes the major PDGF dimer contained in human platelets and is the major form present at sites of injury during the acute phase of the wound repair response.

Proteins derived from platelet α granules modulate the uptake of oxidized low density lipoprotein by macrophages

Activated platelets secrete from their α granules a protein-like factor which stimulates the uptake of oxidized low-density lipoprotein (Ox-LDL) by macrophages. The aim of the present study was to evaluate the effect of three purified proteins obtained from platelet α granules: platelet-derived growth factor (PDGF), platelet factor-4 (PF-4), and β-thromboglobulin (B-TG), on the uptake of Ox-LDL by macrophages. Cellular degradation of Ox-LDL by the J-774 A.1 macrophage-like cell line, that was preincubated for 18 h at 37°C, with increasing concentrations of partially purified PDGF, (designated PDGF-CMS-III) was increased by up to 36% in comparison to control cells preincubated without PDGF. This effect was due to PDGF-mediated increase in the number of macrophage receptors for Ox-LDL. The enhanced uptake of Ox-LDL by PDGF resulted in an increase in cellular cholesterol content. Preincubation of macrophages with two types of recombinant PDGF dimers (10 ng/ml), revealed that PDGF-BB stimulated Ox-LDL cellular degradation by 64%, whereas PDGF-AB demonstrated only 34% stimulation, in comparison to control cells that were not treated with PDGF. The stimulatory effect of PDGF-CMS-III and PDGF-AB were reduced by 20% and 28%, respectively, when incubated in the presence of H-7, a specific protein kinase C inhibitor. When macrophages were preincubated with B-TG, cellular uptake of Ox-LDL was reduced by up to 30% at 100 ng B-TG/ml. This effect, however, was obtained only when B-TG was present in the incubation medium. Cellular degradation of Ox-LDL was not affected by preincubation of the cells with PF-4. Pretreatment of PCM with anti-PDGF or anti-B-TG antibodies abolished the effects of PCM on Ox-LDL degradation by macrophages. PDGF, thus, may represent the protein-like factor present in PCM which stimulates Ox-LDL degradation by macrophages, whereas B-TG may have a role in the recognition of PCM particles by the macrophage scavenger receptor. Modulation of macrophage cholesterol content by proteins secreted from activated platelets may have an important role in foam cell formation and atherosclerosis.

Developmental distribution of platelet-derived growth factor in the mouse central nervous system

Immunoblotting and immunohistochemical techniques have been used to characterize the developmental changes in the distribution and relative quantity of platelet-derived growth factor (PDGF), an important mitogen and growth regulator for glial (and possibly neuronal) cells. PDGF exists as a dimer of two chains, A and B, and antibodies which are relatively specific for one chain or the other can be used to localize PDGF isoforms during development. We have also studied the distribution of PDGF receptor β subunit (PDGF-Rβ)-like immunoreactivity using an antibody probe. All 3 isoforms of PDGF are found in neural structures during development, beginning at about the midpoint of embryogenesis. Immunoblotting studies confirm the presence of PDGF isoforms in brain during embryonic and postnatal development, with the distribution and relative abundance of each isoform appearing to be independently regulated. Similarly, immunoblotting studies have verified the relative abundance and specificity of PDGF receptor β subunit. The immunohistochemical findings confirm and extend these biochemical observations. Each PDGF chain (A and B) has a discrete localization during nervous system development, and the immunohistochemical distribution of PDGF-Rβ is distinct from each of the PDGF isoforms. PDGF A-chain (localized with an antibody to PDGF(AA) dimers) appears to be found in growth cones of developing neurons in mid-embryonic brain development. By 11.5 days post-conception (embryonic day 11.5, E11.5) to E12, PDGF isoforms are found in apparent neurons in the basal plate (future ventral horn) of spinal cord. PDGF-Rβ-like immunoreactivity is localized to the boundary cap region of the developing spinal cord at the same age. Similarly, at E13.5, all 3 PDGF isoforms are found, to varying extents, within cells of the dorsal root ganglia and trigeminal ganglia. At the same developmental stage, PDGF receptor protein is most prevalent in the nerves accompanying these structures. By E15, both PDGF isoform and PDGF receptor β subunit immunoreactivity have declined to near-background levels in the sensory ganglia, while in the spinal cord and developing forebrain, levels of all PDGF-related proteins remain high.

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. I. Evidence for reversible binding and inactivation of the platelet-derived growth factor by heparin.

We have investigated the effects of interactions between growth factors and heparin-like glycosaminoglycans on untransformed human arterial smooth muscle cells (hASMC) in vitro. The results indicate that heparin in the presence of serum mitogens prevents the cells from entering the S phase of the cell cycle by binding and inactivating reversibly some serum mitogen(s). Our results suggest that platelet-derived growth factor (PDGF) is one of them and that it is the most potent stimulator of hASMC growth in vitro. Thymidine incorporation as well as increase in DNA content was inhibited not only by the presence of heparin in serum-containing medium but also when serum was chromatographed on Heparin-Sepharose at physiologic salt concentrations before exposure to the cells. The mitogenic activity of the unretained serum fraction was restored by the addition of PDGF AA, AB, or BB dimers or of a fraction (RF I) that dissociated from Heparin-Sepharose at 0.2 to 0.6 M NaCl. Radiolabeled recombinant PDGF (c-sis) dissociated from Heparin-Sepharose within a concentration range of NaCl similar to that of RF I. Neither the unretained material nor the RF I or PDGF dimers were effective alone. The effect of RF I was significantly decreased by the addition of an anti-PDGF IgG that is known to neutralize the PDGF mitogenic activity partially. Addition of heparin abolished DNA-synthesis when the PDGF dimers or RF I were combined with the unretained fraction. A second fraction (RF II) bound strongly to Heparin-Sepharose and eluted between 1.1 and 1.6 M NaCl. The RF II also induced DNA synthesis but was neither as efficient as RF I nor depending on other serum fractions for growth promotion and it was not inhibited by anti-PDGF IgG. A similar strong affinity for Heparin-Sepharose was found for labeled basic fibroblast growth factor and we cannot exclude the possibility that RF II represent fibroblast growth factor. Under these culture conditions, inhibition of hASMC proliferation was directly correlated with the expression of smooth muscle specific alpha actin isoforms in stress fibers and the suppression of a proliferating cell-specific nuclear antigen. Conversely, stimulation of hASMC proliferation was associated with the opposite phenomenon. We conclude that heparin-like glycosaminoglycans influence growth and phenotype of hASMCs in vitro by binding and inactivating PDGF. Inasmuch as heparin-like substances constitute a significant proportion of the proteoglycan-associated glycosaminoglycans of the arterial wall, such mechanisms might be important for the development of atherosclerotic lesions.

The alternative-splice isoforms of the PDGF A-chain differ in their ability to associate with the extracellular matrix and to bind heparin in vitro.

Platelet-derived growth factor (PDGF) consists of disulfide-linked homo- or heterodimers of A and B chains. mRNA encoding the A chain (PDGF-A) occurs in two versions that differ by the presence or absence of a single short exon. These alternatively-spliced mRNAs encode polypeptides that differ in length by fifteen amino acids. The longer isoform (PDGF-AL) possesses a highly basic carboxy-terminal extension that is responsible for retaining PDGF-AL homodomers at the cell surface after secretion, while homodimers of the shorter isoform (PDGF-AS) are released into the extracellular medium. We have investigated the mechanism by which PDGF-AL remains in association with the cells that produce it. We expressed epitope-tagged versions of PDGF-AL and PDGF-AS in Cos cells and compared their intra- and extracellular distributions by immunofluorescence microscopy. PDGF-AL, but not PDGF-AS, was detected on and around cells in a diffuse pattern suggesting associated with the extracellular matrix (ECM). Metabolically radiolabelled PDGF-AL, but not PDGF-AS, could be eluted from ECM preparations by washing in high salt. Moreover, PDGF-AL bound reversibly to heparin-Sepharose in vitro at physiological salt concentrations, eluting at a salt concentration around 0.5 M. PDGF-AS did not bind to heparin under the same conditions. Thus, PDGF dimers that contain PDGF-AL may remain immobilized near the cells that secrete them by virtue of binding to heparin-like constituents of the ECM.

Regulation of insulin-like growth factor I messenger RNA levels in vascular smooth muscle cells.

We have previously demonstrated specific insulin-like growth factor I (IGF I) messenger RNA (mRNA) transcripts in cultured rat aortic smooth muscle cells (RASM). To define the role of IGF I in the autocrine growth program of vascular smooth muscle cells, we quantitated IGF I mRNA levels in proliferating and quiescent (serum-deprived for 48 hours) RASM. IGF I mRNA levels were markedly decreased in quiescent cells, and this effect was reversible on reexposure to serum. Since platelet-derived growth factor (PDGF) acts synergistically with IGF I to stimulate vascular smooth muscle cell growth, we exposed quiescent RASM to PDGF AB or BB and quantitated IGF I transcript levels. Both PDGF dimers caused a marked, rapid increase in IGF I message levels. To determine whether induction of IGF I mRNA levels correlated with secretion of IGF I, we measured immunoreactive IGF I in RASM conditioned medium after separation of IGF I binding proteins by gel filtration chromatography. PDGF caused a significant increase in IGF I release at 24 hours. These findings indicate that IGF I mRNA levels in vitro are regulated by serum and by growth factors such as PDGF. Serum deprivation reversibly decreases IGF I transcript levels, and exposure of quiescent cells to PDGF increases IGF I mRNA levels and IGF I release. Regulation of IGF I expression by competence growth factors such as PDGF may play an important role in the control of vascular smooth muscle cell growth.

Purification of PDGF-AB and PDGF-BB from human platelet extracts and identification of all three PDGF dimers in human platelets

We have developed a panel of monoclonal antibodies to platelet-derived growth factor (PDGF) which have variable specificities for the three dimeric forms of the molecule (AA, AB, and BB). We have used these antibodies to detect and immunoaffinity purify the individual dimers from human platelet rich plasma. Extracts of outdated platelet preparations were initially chromatographed over CM-Sepharose and then passed over the Sepharose-coupled monoclonal antibodies in series in selectively isolate the three dimeric forms of PDGF. The PDGF eluted from the affinity columns was subsequently further purified by reversed-phase HPLC. From 300 units of outdated platelet preparations, we purified 58 micrograms of PDGF-BB and 140 micrograms of PDGF-AB. Using the monoclonal antibodies to develop PDGF dimer-specific ELISAs, it was observed that all three PDGF dimer forms are present in fresh human platelet extracts and that the ratios of the three dimer forms vary depending upon the extraction conditions used. The identification of all three PDGF dimer forms in human platelets point to the need to view PDGF isolated from human platelets by conventional techniques as a mixture of all three forms and not solely as PDGF-AB.

Selective expression of mRNA encoding platelet-derived growth factor B chain following transfection of foreign genes into cell lines derived from baby hamster kidney.

The genes for platelet-derived growth factor (PDGF) A and PDGF B chains are expressed in a variety of biological situations. Active PDGF consists of two distinct but homologous polypeptide chains, PDGF A and PDGF B, which are found as heterodimers or homodimers. We report a novel situation in which there is selective expression of mRNA encoding PDGF B in cell lines derived from baby hamster kidney (BHK) following transfection with various gene/cDNA constructs and following growth selection with methotrexate. The process of transfection itself, and not expression of the proteins encoded by the transfected genes/cDNAs (hormones, enzymes, and structural proteins), induces expression of PDGF B. No PDGF B mRNA is detectable in control cell lines. Low levels of mRNA encoding PDGF A are constitutively present and are not changed by transfection and or growth selection. PDGF-like activity is present in the medium whenever PDGF B mRNA is detected. The composition of the secreted PDGF dimer cannot be established from our data, but quantitative analysis of mRNA suggests that the PDGF is a B-B dimer. However, the data show that transcription of the PDGF A and PDGF B genes in BHK cells is regulated independently, similar to that reported for some human tumor cells. Furthermore, the selective expression of PDGF B in response to the introduction of foreign genes and to growth selection may be an important aspect of the reaction of these cells to nonoptimal growth conditions, allowing survival and growth of the cells that express PDGF B.

Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.

The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately 5.7 kb) and a minor (approximately 4.8 kb) transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an approximately equal to 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF (i.e., PDGF dimers composed of two B chains or an A and a B chain). Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class.

Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species.

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.