PDGF stimulates transient phosphorylation of 180,000 dalton protein

Cell-free extracts of platelet-derived growth factor (PDGF) treated, density-arrested, quiescent BALB/c-3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P-40 solubilized cell preparations that had been incubated with [gamma-32P]ATP. When quiescent BALB/c-3T3 cell cultures were incubated at 37 degrees C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3-min exposure of the intact cells to PDGF, which was maximal after 10-15 minutes and had diminished by 30-60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4 degrees C; however, in contrast to PDGF exposure at 37 degrees C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4 degrees C. When cells exposed to PDGF at 4 degrees C were transferred to 37 degrees C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37 degrees C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions. The amount of the stimulation of PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF-stimulated phosphorylation activity in relation to the cell cycle and growth regulation.     

Modulation of PDGF mediated osteoblast chemotaxis by leukemia inhibitory factor LIF

Cell migration is a key event in tissue repair and remodeling. PDGF, a growth factor for multiple target cells, has been shown to be a potent chemoattractant for a variety of mesenchymal cells. However, it is likely that PDGF-mediated cell migration will be influenced by other cytokines that can be produced during physiological and pathological conditions. Leukemia inhibitory factor (LIF), a cytokine that is produced by a variety of cells including osteoblasts, may promote bone formation, but the mechanism is not known. Since osteoblasts are responsible for laying down new matrix during skeletal remodeling, in this report we have examined whether PDGF or LIF influences the migration of osteoblasts. Among several cytokines and growth factors tested, only PDGF was able to elicit a major chemotactic (directed migration) and a minor chemokinetic (random-migration) response in osteoblasts. LIF alone was not active in either chemotaxis or chemokinesis but when included with PDGF it caused a reduction in chemokinesis. Further, pretreatment of osteoblasts with LIF caused an increase in PDGF-driven chemotaxis. Finally, osteoblasts exposed briefly to LIF synthesized a higher level of non-collagenous proteins upon further treatment with PDGF. These observations are consistent with a role for LIF in promoting bone formation, both by influencing directional migration of osteoblasts and in laying down new matrix. © 1996 Wiley-Liss, Inc.     

Monoclonal antibody C3.1 is a platelet derived growth factor (PDGF) antagonist

A monoclonal antibody C3.1 raised against NR6 cells and partially purified PDGF receptor blocked PDGF stimulated [3H] thymidine incorporation in Swiss mouse 3T3 cells and immunoprecipitated a 180 kDa phosphoprotein from NR6 cells. The phosphoprotein bound to a C3.1 sepharose 4B affinity matrix and could be specifically eluted with PDGF but not by EGF or basic FGF. These preliminary results suggests that the ability of C3.1 to inhibit PDGF stimulated mitogenesis may be due to its direct or allosteric interaction at the PDGF receptor binding site.     

Five PDGF B amino acid substitutions convert PDGF A to a PDGF B-like transforming molecule.

We used site-directed mutagenesis to determine the minimum number of PDGF B residues needed to convert PDGF A to a potently transforming PDGF B-like molecule. Substitution of two PDGF B subdomains, 106-115 and 135-144, were found to be critical. These substitutions were sufficient to broaden the ability of PDGF A to activate beta as well as alpha platelet-derived growth factor (PDGF) receptors and increase its transforming efficiency to that of PDGF B. Within subdomain I, either PDGF B residues Arg-109 and Asn-115 or Arg-109, Leu-110, and Arg-113, in combination with subdomain II PDGF B residues Asn-136, Arg-137, and Arg-142 were identified as being essential. Those mutants with transforming ability comparable with PDGF B showed significantly lower efficiencies of beta receptor triggering. Thus, our studies identify a small number of PDGF B amino acids indispensable for beta PDGF receptor interaction and suggest that a low level of beta PDGF receptor activation is sufficient to dramatically increase PDGF transforming efficiency in NIH 3T3 cells.     

Differential Tumorigenic Potential and Matriptase Activation between PDGF B versus PDGF D in Prostate Cancer

The platelet-derived growth factors (PDGF A, B, C, and D) and their receptors (α-PDGFR and β-PDGFR) play an indispensible role in physiologic and pathologic conditions, including tumorigenesis. The transformative β-PDGFR is overexpressed and activated during prostate cancer progression, but the identification and functional significance of its complementary ligand have not been elucidated. This study examined potential oncogenic functions of β-PDGFR ligands PDGF B and PDGF D, using nonmalignant prostate epithelial cells engineered to overexpress these ligands. In our models, PDGF D induced cell migration and invasion more effectively than PDGF B in vitro. Importantly, PDGF D supported prostate epithelial cell tumorigenesis in vivo and showed increased tumor angiogenesis compared with PDGF B. Autocrine signaling analysis of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways found PDGF D-specific activation of the c-jun-NH(2)-kinase (JNK) signaling cascade. Using short hairpin RNA and pharmacologic inhibitors, we showed that PDGFD-mediated phenotypic transformation is β-PDGFR and JNK dependent. Importantly, we made a novel finding of PDGF D-specific increase in the shedding and activation of the serine protease matriptase in prostate epithelial cells. Our study, for the first time to our knowledge, showed ligand-specific β-PDGFR signaling as well as PDGF D-specific regulation of matriptase activity and its spatial distribution through shedding. Taken together with our previous finding that matriptase is a proteolytic activator of PDGF D, this study provides a molecular insight into signal amplification of the proteolytic network and PDGF signaling loop during cancer progression. Mol Cancer Res; 10(8); 1087-97. ?2012 AACR.     

The B chain of PDGF alone is sufficient for mitogenesis.

The platelet-derived growth factor (PDGF) is a mitogen derived from human platelets consisting of two related polypeptides termed A and B chains. The entire B chain of PDGF is highly (96%) homologous to a portion of p28sis, the transforming protein of simian sarcoma virus. It has been suggested that p28sis exerts its transforming potential by mimicking the growth promoting activity of PDGF and stimulating the cell in an autocrine manner. We have directly examined the mitogenic potential of p28sis and the B chain homologous region by expressing these heterologous sequences in the yeast Saccharomyces cerevisiae. In our constructions, these proteins are encoded by portions of the v-sis gene. Expression and secretion from the yeast cell is achieved by using a yeast promoter and the alpha-factor pheromone secretory leader. The sis proteins thus expressed and secreted are immunoreactive with anti-PDGF antisera and are mitogenic for cultured fibroblasts. Furthermore, they mediate this mitogenic activity by specific binding to the PDGF cell surface receptor. Gel electrophoresis and cell binding analysis indicates that the mitogenic species is primarily a disulphide-bonded dimer. We are able to conclude that p28sis is a mitogen and that a polypeptide corresponding to the B chain alone is sufficient to account for the mitogenic activity attributed to PDGF.     

PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.     

Identification of three amino acids in the platelet-derived growth factor (PDGF) B-chain that are important for binding to the PDGF beta-receptor.

Platelet-derived growth factor (PDGF) is a disulfide-linked dimeric protein composed of two homologous polypeptide chains denoted A and B. Two types of PDGF receptors, alpha and beta, have been characterized. Whereas PDGF-AA binds only to PDGF alpha-receptors, PDGF-BB binds to both receptor types with high affinity. To map the regions of the PDGF B-chain that confer its ability to bind with high affinity to the PDGF beta-receptor, we expressed PDGF A/B-chain chimeras in COS cells and analyzed them with regard to PDGF alpha- and beta-receptor binding. A systematic analysis revealed that replacement of Asn-115, Arg-154, and Ile-158 of the PDGF B-chain with the corresponding A-chain amino acids led to a dramatic decrease in the affinity for the beta-receptor. Conversely, introduction of B-chain amino acids into the A-chain in the region spanning from Asn-115 to Ile-158 yielded a product with high affinity for the beta-receptor. These data thus indicate that Asn-115, Arg-154, and Ile-158 are likely to be part of the active site of the PDGF B-chain.     

PDGF stimulation induces phosphorylation of talin and cytoskeletal reorganization in skeletal muscle

Modifications in the interactions of the muscle cytoskeleton with the cell membrane occur during cell growth and adaptation, although the mechanisms regulating these interactions are unknown. We have observed that myotendinous junctions (MTJs), which are the primary sites of turnover of the thin filament-membrane associations in skeletal muscle, are greatly enriched in receptors for PDGF. The high concentration of PDGF receptors at MTJs suggested to us that receptor binding may initiate cytoskeletal remodeling in skeletal muscle. We tested this possibility by examining the organization and phosphorylation of cytoskeletal components of L6 myocytes after PDGF stimulation. We have found that 10 min after PDGF stimulation, L6 myoblasts exhibit no stress fibers discernible by phalloidin binding, and that vinculin relocates from focal contacts into a diffuse cytoplasmic distribution. After 60 min of incubation, these changes are largely reversed. Indirect immunofluorescence shows that at 10-min PDGF stimulation, there are no changes in the distribution of talin, the beta 1 subunit of integrin, pp125FAK or desmin. Phosphotyrosine distribution changes upon stimulation from focal contacts to being located both in focal contacts and granules concentrated in perinuclear regions. These granules also immunolabel with anti-PDGF receptor Immunoprecipitations with anti-phosphotyrosine show that polypeptides at 180 and 230 kD show the greatest increase in tyrosine phosphorylation after PDGF stimulation. Immunoblots of anti-phosphotyrosine precipitates show that these polypeptides are the PDGF receptor and talin. We also examined the possibility that the cytoskeletal reorganization observed may result from calpain activation caused by elevated intracellular calcium induced by PDGF stimulation. However, immunoblots of control and stimulated cells show no decrease in the inactive calpain proenzyme or increase in the proteolytic, autolyzed forms of calpain pursuant to stimulation. Furthermore, stimulation produces no increase in the proportion of the 190-kD talin fragment characteristic of calpain- mediated cleavage. The retention of talin and integrin at focal contacts after talin phosphorylation, while vinculin is redistributed, indicate that phosphorylation of talin in PDGF-stimulated cells leads to separation of talin-vinculin associations but not talin-integrin associations. We propose that PDGF binding to PDGF receptors at MTJs may provide one means of regulating myofibril associations with the muscle cell membrane.     

The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.     

PDGF-BB多肽刺激肺动脉平滑肌细胞PDGFmRNA表达

应用地高辛（Ｄｉｇｏｘｉｇｅｎｉｎ）标记的血小板源性生长因子（ＰＤＧＦ）Ａ和ＰＤＧＦＢ链ｃＤＮＡ探针,原位检测了ＰＤＧＦＢＢ多肽对单层培养的肺动脉平滑肌细胞ＰＤＧＦ基因表达的影响。结果表明,无血清培养的肺动脉平滑肌细胞内ＰＤＧＦＡ和ＰＤＧＦＢ链ｍＲＮＡ阳性表达颗粒稀少,ＰＤＧＦＢＢ多肽培养的肺动脉平滑肌细胞ＰＤＧＦＡ和ＰＤＧＦＢ链ｍＲＮＡ阳性表达颗粒增多,较密集的分布于整个细胞内。全自动图像分析结果显示,ＰＤＧＦＡ和ＰＤＧＦＢ链ｍＲＮＡ表达,ＰＤＧＦＢＢ培养组分别为无血清培养组的１７４倍和１７倍,差异显著（Ｐ＜００１）。本研究结果提示,ＰＤＧＦＢＢ多肽能刺激肺动脉平滑肌细胞ＰＤＧＦｍＲＮＡ表达,促进其分泌,在慢性低氧性肺动脉高压血管平滑肌细胞增殖中具有重要作用     

Extracellular matrix is a source of mitogenically active platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A_s (short splice version) is secreted, PDGF A_L (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and η-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions. © 1996 Wiley-Liss, Inc.     

PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling

Neurons and oligodendrocytes are produced in the adult brain subventricular zone (SVZ) from neural stem cells (B cells), which express GFAP and have morphological properties of astrocytes. We report here on the identification B cells expressing the PDGFRalpha in the adult SVZ. Specifically labeled PDGFRalpha expressing B cells in vivo generate neurons and oligodendrocytes. Conditional ablation of PDGFRalpha in a subpopulation of postnatal stem cells showed that this receptor is required for oligodendrogenesis, but not neurogenesis. Infusion of PDGF alone was sufficient to arrest neuroblast production and induce SVZ B cell proliferation contributing to the generation of large hyperplasias with some features of gliomas. The work demonstrates that PDGFRalpha signaling occurs early in the adult stem cell lineage and may help regulate the balance between oligodendrocyte and neuron production. Excessive PDGF activation in the SVZ in stem cells is sufficient to induce hallmarks associated with early stages of tumor formation.     

Targeting the PDGF signaling pathway in tumor treatment

Platelet-derived growth factor (PDGF) isoforms and PDGF receptors have important functions in the regulation of growth and survival of certain cell types during embryonal development and e.g. tissue repair in the adult. Overactivity of PDGF receptor signaling, by overexpression or mutational events, may drive tumor cell growth. In addition, pericytes of the vasculature and fibroblasts and myofibroblasts of the stroma of solid tumors express PDGF receptors, and PDGF stimulation of such cells promotes tumorigenesis. Inhibition of PDGF receptor signaling has proven to useful for the treatment of patients with certain rare tumors. Whether treatment with PDGF/PDGF receptor antagonists will be beneficial for more common malignancies is the subject for ongoing studies.     

Sera and conditioned media contain different isoforms of platelet-derived growth factor (PDGF) which bind to different classes of PDGF receptor

Platelet-derived growth factor (PDGF) is encoded by separate genes for two possible subunit chains (A-chain and B-chain) which can form three possible dimers (AA, AB, and BB). We have recently presented evidence that multiple forms of PDGF receptor exist which distinguish between these isoforms (Hart, C. H., Forstrom, J. W., Kelley, J. D., Smith, R. A., Ross, R., Murray, M. J., and Bowen-Pope, D. F. (1988) Science 240, 1529-1531). We used this specificity to determine the amount of PDGF from different sources which is able to bind to each class of receptor and found that each source had a characteristic isoform composition. Levels of total PDGF activity in sera from different species ranged more than 15-fold, from less than 1 ng/ml in dog, chicken, pig, and calf, to greater than 13 ng/ml in mouse and human. Despite these differences in PDGF content, the total mitogenic activities of the sera were comparable indicating that the relative importance of PDGF as a serum mitogen may vary considerably between species. Analysis of the total PDGF into the amounts of each isoform revealed great differences in composition. PDGF-BB constitutes only about 15% of the total binding activity in human PDGF purified by the method of Raines and Ross (Raines, E. W., and Ross, R. (1982) J. Biol. Chem. 257, 5154-5160) but is the predominant isoform in whole blood serum from all other species. In contrast to serum, medium conditioned by cultured PDGF-secreting cell types contained no detectable PDGF-BB except in two cases: medium conditioned by vascular endothelial cells and by cells transformed by simian sarcoma virus. The existence of isoform-specific PDGF receptors and the large variation in PDGF isoform composition dependent upon source may provide an important mechanism through which the effects of PDGF can be targeted to different cell types and/or toward eliciting different cell responses.     

The influence of blood cells and PDGF on porcine theca cell function in vitro

The role of red and white blood cells in the regulation of porcine theca cell function is poorly understood. Interactions between these cell types and a potential mediator of any interaction, PDGF, were investigated using a serum-free culture system. Theca cells were collected from 6-9mm antral follicles and plated at 50x10(3) viable cells/well. In the first experiment, macrophages were removed and theca cells+/-macrophages were cultured with a range of PDGF doses (0.1, 1, and 10ng/ml)+/-IGF-1. In the second experiment, red blood cells were removed with lysing buffer. In both experiments the effect of treatment on steroidogenesis and viable cell number was examined. Macrophage removal decreased oestradiol production but increased androstenedione output irrespective of the presence of IGF-1 (oestradiol+/-IGF-1, P<0.001; androstenedione P=0.02 without IGF-1, P<0.001 with IGF-1). PDGF increased oestradiol synthesis by whole and macrophage-free theca cell preparations but only in the presence of IGF-1 (P<0.001). In contrast, androstenedione production was unaffected by PDGF dose in the presence of IGF-1 (P=0.67). Without IGF-1, 10ng/ml PDGF tended to decrease androstenedione levels (P=0.06). Macrophage removal increased viable cell number at 144h (P<0.001+/-IGF-1) as did PDGF (P<0.001+/-IGF-1). In the absence of IGF-1, there was a PDGF x cell type interaction (P=0.02). Macrophage-free cultures with 10ng/ml PDGF had twice as many viable cells as whole preparations with no PDGF. In the second experiment, red blood cell removal did not affect steroidogenesis or the number of viable cells present at 144h when cells were cultured with IGF-1. The data show that theca cell/macrophages interactions do occur, and influence both steroidogenesis and viable cell number during culture. The macrophage product(s) enhanced oestradiol synthesis but reduced androstenedione production and the number of viable cells. As all these interactions were not mimicked by PDGF, PDGF cannot be the only factor mediating the theca/macrophage interaction. When cultured under optimised conditions the presence of red blood cells was not detrimental to theca cell steroidogenesis or the number of viable cells.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.