Malignant transformation of human cells by constitutive expression of platelet-derived growth factor-BB

Platelet-derived growth factors (PDGFs) comprise a family of growth factors strongly implicated in human oncogenesis. A number of human tumors overexpress PDGF family members or have translocations activating PDGF receptors. Whereas the epidemiologic evidence implicating PDGF in human tumors is strong, malignant transformation of human cells by overexpression of PDGF has not been demonstrated. We have previously developed a human cell line by the sequential introduction of large T cells and telomerase, and we have demonstrated that these cells express functionally active PDGF receptor (PDGFR) beta. In order to determine whether growth factor-mediated transformation of human cells could occur, these cells were transduced with a retrovirus encoding PDGF-BB. Constitutive expression of PDGF-BB led to malignant transformation in nude mice. This is the first demonstration of constitutive signaling causing malignant transformation of human cells. Some of the changes that occur because of constitutive growth factor expression can be reversed by the clinically approved tyrosine kinase inhibitor Glivec, whereas other changes are not reversible by tyrosine kinase inhibitors. Our model allows the assessment of epigenetic changes that occur during human carcinogenesis. In addition, these studies provide insight into the clinical failure of tyrosine kinase inhibitors as monotherapy for advanced malignancy.     

Disruption of intermolecular disulfide bonds in PDGF-BB dimers by N-acetyl-L-cysteine does not prevent PDGF signaling in cultured hepatic stellate cells

Oxidative stress is important in the pathogenesis of liver fibrosis through its induction of hepatic stellate cell (HSC) proliferation and enhancement of collagen synthesis. Reactive oxygen species have been found to be essential second messengers in the signaling of both major fibrotic growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), in cultured HSC and liver fibrosis. The non-toxic aminothiol N-acetyl-L-cysteine (NAC) inhibits cellular activation and attenuates experimental fibrosis in liver. Prior reports show that NAC is capable of reducing the effects of TGF-beta in biological systems, in cultured endothelial cells, and HSC through its direct reducing activity upon TGF-beta molecules. We here analyzed the effects of NAC on PDGF integrity, receptor binding, and downstream signaling in culture-activated HSC. We found that NAC dose-dependently induces disintegration of PDGF in vitro. However, even high doses (>20mM) were not sufficient to prevent the phosphorylation of the PDGF receptor type beta, extracellular signal-regulated kinase, or protein kinase B (PKB/Akt). Therefore, we conclude that the PDGF monomer is still active. The described antifibrotic effects are therefore mainly attributable to the structural impairment of TGF-beta signaling components reported previously.     

Elucidating progesterone effects in breast cancer: cross talk with PDGF signaling pathway in smooth muscle cell

Several studies indicate that progesterone exerts relevant effects in breast tissue. However, the exact role of this steroid in breast cancer development and progression has not been elucidated. Here, we show that platelet-derived growth factor (PDGF)-A is one of the progesterone target genes on breast cancer MCF7 and T47D cells. A paracrine role for PDGF-A was investigated, since its receptor expression was down-regulated from breast cancer cells. Progesterone increased PDGF-A protein release as evaluated by Western blotting and ELISA. Medium from Progesterone-treated MCF7 cells resulted in phosphorylation of smooth muscle cells PDGF receptor alpha. This effect was not observed after treatment with PDGF inhibitor. MCF7 cells-secreted PDGF-A was able to increase smooth muscle cell viability and proliferation and decrease apoptosis, effects that were prevented by the use of a PDGF-A neutralizing antibody. Notably, cell invasion was not influenced by PDGF-A secreted by MCF7 cells. Our results elucidated for the first time the cross talk between progesterone and PDGF signaling pathway. The fact that MCF7-secreted PDGF elicited crucial roles in vascular wall smooth muscle cells, suggested a paracrine pathway for progesterone. Targeting these progesterone-induced processes may provide novel therapeutic strategies for hormone-dependent human breast cancer.     

The mitochondrial reactive oxygen species regulator p66Shc controls PDGF-induced signaling and migration through protein tyrosine phosphatase oxidation

Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFβR phosphorylation in two independent cell systems demonstrated a decrease in PDGFβR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFβR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFβR phosphorylation, showing that mitochondrial ROS in general promote PDGFβR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.     

Src family kinase-dependent phosphorylation of a 29-kDa caveolin-associated protein

PDGF receptors and Src family kinases are concentrated in caveolae, where signal transduction cascades involving these molecules are thought to be organized. The Src family tyrosine kinases are cotransducers of signals emanating from the activated PDGF receptor. However, the Src family kinase substrates that are involved in PDGF-induced signaling remain to be fully elucidated. We have identified a 29-kDa protein in caveolae that was phosphorylated in response to PDGF stimulation. This protein, pp29, was tightly bound to the caveolar coat protein caveolin-1. pp29 was among the most prominent phosphoproteins observed in cells overexpressing Fyn, suggesting that it may be a Fyn substrate. Consistent with this, pp29 was among a specific subset of proteins whose PDGF-stimulated phosphorylation was blocked by expression of kinase inactive Fyn. These data indicate that pp29 lies downstream of Fyn activation in a PDGF-stimulated signaling pathway, and that pp29 is an abundant site for nucleation of signal transduction cascades.     

Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis

Hepatic stellate cells (HSCs) and transdifferentiated myofibroblasts are the principal producers of excessive extracellular matrix in liver fibrosis and cirrhosis. Activation of HSC is regulated by several cytokines and growth factors, including platelet-derived growth factor B-chain (PDGF-B), a potent mitogen for HSC, and overexpressed during hepatic fibrogenesis. Previous studies showed that MAPK and phosphatidylinositol 3' kinase are key signaling pathways involved in PDGF-induced stimulation of HSC. Based on the involvement of PDGF-B in fibrogenesis, reducing ligand stimulation of proliferative cytokine- or growth factor receptors interfering with receptor signaling therefore presents an interesting strategy for hepatic fibrosis prevention or interruption. We therefore generated an adenoviral vector serotype 5 (Ad5) expressing an antisense mRNA of the PDGF B-chain (Ad5-CMV-asPDGF) for application in an experimentally induced liver fibrogenesis model. The transgene clearly showed the ability to down-regulate endogenous PDGF B-chain and PDGFRbeta mRNA in culture-activated HSC and rat livers. The asPDGF mRNA also attenuates experimental liver fibrogenesis indicated by reduced levels of alpha-SMA and collagen type I expression.     

Novel PDGF family members: PDGF-C and PDGF-D

Platelet-derived growth factors (PDGFs) were discovered almost two decades ago. The classical PDGF polypeptide chains, PDGF-A and PDGF-B, are well studied and they regulate a number of physiological and pathophysiological processes in many types of mesenchymal cells via two receptor tyrosine kinases, PDGF receptors alpha and beta. Recently, two additional PDGF polypeptide chains were discovered, namely PDGF-C and PDGF-D. The discovery of two additional ligands for the two PDGF receptors suggests that PDGF-mediated signaling is more complex than previously anticipated.     

PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma

PDGFs and their cognate tyrosine kinase alpha- and beta-receptors are involved in multiple tumor-associated processes including autocrine growth stimulation of tumor cells, stimulation of tumor angiogenesis and recruitment and regulation of tumor fibroblasts. The recent development of clinically useful PDGF antagonists, like STI571/Glivec, has increased the interest in PDGF receptors as cancer drug targets. Autocrine PDGF receptor signaling occurs in certain malignancies characterized by mutational activation of PDGF or PDGF receptors, for instance, dermatofibrosaracoma protuberans, gastrointestinal stromal tumors, and hypereosinophilic syndrome. The roles of PDGF in regulation of tumor angiogenesis and tumor fibroblasts are more general, and probably occur in most common solid tumors. Concerning tumor angiogenesis recent studies have predominantly focused on the importance of PDGF receptor signaling for tumor pericyte recruitment. PDGF receptors in the tumor stroma have also attracted attention as interesting drug targets because of their function as regulators of tumor interstitial fluid pressure, tumor transvascular transport and tumor drug uptake. In summary, the improved understanding of the role of PDGF signaling in tumor biology, and the introduction of PDGF antagonists, has set the stage for a continued development of PDGF antagonists as novel cancer drugs.     

Over-expression of PDGF-C using a lung specific promoter results in abnormal lung development

PDGF isoforms are a family of polypeptides that bind to cell surface receptors and induce fibroblast proliferation and chemotaxis. PDGF-A and -B chain isoforms have previously been shown to be involved in murine lung development. A new PDGF polypeptide, PDGF-C, was recently recognized and differs from the PDGF-A and -B isoforms in that it requires proteolytic cleavage before it can bind and activate the PDGF alpha receptor. In these studies PDGF-C was over-expressed during embryogenesis using the lung specific surfactant protein C promoter. PDGF-C transgenic pups died from respiratory insufficiency within minutes following birth. At E18.5, nontransgenic lungs exhibited lung morphology consistent with the saccular stage of lung development. In contrast, E18.5 transgenic lungs retained many features of the canalicular stage of lung development and had abundant numbers of large poorly differentiated mesenchymal cells. These results suggest that PDGF-C is activated during lung development and is a potent growth factor for mesenchymal cells in vivo.     

Expression of a Cx43 Deletion Mutant in 3T3 A31 Fibroblasts Prevents PDGF-Induced Inhibition of Cell Communication and Suppresses Cell Growth

Communication through gap junctions was first suggested to have a role in the social control of cell growth over 30 years ago. However, despite extensive experimentation, the importance of gap junctions as a general mechanism of growth control remains to be established. A number of different studies have shown that a common early response of cells in culture to polypeptide growth factors such as PDGF is a rapid and transient inhibition of cell communication suggesting that a cell may have to lose communication with its neighbors before it can undergo cell division. Here we show that 3T3 A31 fibroblasts exposed to PDGF exhibit a 50% decrease in cell communication as measured by dye transfer in the absence of significant changes in the cellular content and distribution of Cx43. Likewise, PDGF inhibited cell communication in cells transfected either with a vector which did not contain a cDNA or with an expression vector encoding full-length Cx43 fused to a c-myc tag (Cx43-M). In contrast, 3T3 A31 fibroblasts transfected with an expression construct encoding a deletion mutant of Cx43 (Cx43-256M) consisting of amino acids 1-256 of Cx43 fused to a c-myc tag maintain high levels of gap junction activity following exposure to PDGF. These results suggest that sites which trigger loss of cell communication in response to PDGF are located within amino acids 257 to 382 of the Cx43 molecule. Cells transfected with an expression vector encoding full-length Cx43 fused to a c-myc tail exhibited a reduced basal growth rate compared to both parent cells and cells transfected with a control vector but maintained a strong mitogenic response to PDGF. In contrast, both the basal growth rate and the mitogenic response to PDGF was markedly reduced in cells which expressed Cx43-256M consistent with the hypothesis that loss of cell communication is required before a cell can respond to mitogenic stimuli.     

The Xenopus platelet-derived growth factor α receptor: cDNA Cloning and demonstration that mesoderm induction establishes the lineage-specific pattern of ligand and receptor gene expression

We have cloned the Xenopus PDGF α receptor cDNA and have used this clone, along with cDNA encoding PDGF A, to examine their expression pattern in Xenopus embryos and to determine the factors responsible for lineage specificity. Recombinant Xenopus α receptor expressed in COS cells exhibits PDGF-A-dependent tyrosine kinase activity. We find that receptor mRNA is present in cultured marginal zone tissue explants and in animal cap tissue induced to form mesoderm either by grafting to vegetal tissue or by treatment with recombinant activin A. In contrast, PDGF A mRNA is expressed in cultured, untreated animal cap tissue and is suppressed by mesoderm induction. These results suggest that ectodermally produced PDGF A may act on the mesoderm during gastrulation and that mesoderm induction establishes the tissue pattern of ligand and receptor expression. © 1993Wiley-Liss, Inc.     

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.     

A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways.

The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF beta receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF beta receptor activated the PDGF beta receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF beta receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF beta receptor- and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF beta receptor. Activation of PDGF beta receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation.     

The PDGF family: four gene products form five dimeric isoforms

Platelet-derived growth factors (PDGFs) were discovered more than two decades ago. Today the PDGF family of growth factors consists of five different disulphide-linked dimers built up of four different polypeptide chains encoded by four different genes. These isoforms, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, act via two receptor tyrosine kinases, PDGF receptors alpha and beta. The classic PDGFs, PDGF-A and PDGF-B, undergo intracellular activation during transport in the exocytic pathway for subsequent secretion, while the novel PDGFs, PDGF-C and PDGF-D, are secreted as latent factors that require activation by extracellular proteases. The classical PDGF polypeptide chains, PDGF-A and PDGF-B, are well studied and they regulate several physiological and pathophysiological processes, mainly using cells of mesenchymal or neuroectodermal origin as their targets. The discovery of two additional ligands for the two PDGF receptors suggests that PDGF-mediated cellular signaling is more complex than previously thought.     

The PDGF system and its antagonists in liver fibrosis

Platelet derived growth factor (PDGF) signaling plays an important role in activated hepatic stellate cells and portal fibroblast proliferation, chemotaxis, migration and cell survival. PDGF receptors and ligands are upregulated in experimental liver fibrotic models as well as in human liver fibrotic diseases. Blocking of PDGF signaling ameliorates experimental liver fibrogenesis. The plurality of molecular and cellular activities of PDGF and its involvement in initiation, progression and resolution of hepatic fibrogenesis offers an infinite number of therapeutic possibilities. These include the application of therapeutic antibodies (e.g. AbyD3263, MOR8457) which specifically sequester individual PDGF isoforms or the inhibition of PDGF isoforms by synthetic aptamers. In particular, the isolation of innovative slow off-rate modified aptamers (e.g., SOMAmer SL1 and SL5) that carry functional groups absent in natural nucleic acids by the Systematic Evolution of Ligands by EXponential (SELEX) enrichment technique offers the possibility to design high affinity aptamers that target PDGF isoforms for clinical purposes. Dominant-negative soluble PDGF receptors are also effective in attenuation of hepatic stellate cell proliferation and hepatic fibrogenesis. Moreover, some multikinase inhibitors targeting PDGF signaling have been intensively tested during the last decade and are on the way into advanced preclinical studies and clinical trials. This narrative review aims to gauge the recent progression of research into PDGF systems and liver fibrosis.     

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.     

Synergy in ERK activation by cytokine receptors and tyrosine kinase growth factor receptors

Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) signal through EGF and PDGF receptors, which are important receptor tyrosine kinases (RTKs). Growth hormone (GH) and prolactin (PRL) are four helical bundle peptide hormones that signal via GHR and PRLR, members of the cytokine receptor superfamily. In this study, we examine crosstalk between signaling pathways emanating from these disparate receptor groups (RTKs and cytokine receptors). We find that GH and EGF specifically synergize for activation of ERK in murine preadipocytes. The locus of this synergy resides at the level of MEK activation, but not above this level (i.e., not at the level of EGFR, SHC, or Raf activation). Furthermore, dephosphorylation of the scaffold protein, KSR, at a critical serine residue is also synergistically promoted by GH and EGF, suggesting that GH sensitizes these cells to EGF-induced ERK activation by augmenting the actions of KSR in facilitating MEK-ERK activation. Similarly specific synergy in ERK activation is also detected in human T47D breast cancer cells by cotreatment with PRL and PDGF. This synergy also resides at the level of MEK activation. Consistent with this synergy, PRL and PDGF also synergized for c-fos-dependent transactivation of a luciferase reporter gene in T47D cells, indicating that events downstream of ERK activation reflect this signaling synergy. Important conceptual and physiological implications of these findings are discussed.     

Phosphospecific Antibodies Reveal Temporal Regulation of Platelet-Derived Growth Factor β Receptor Signaling

The platelet-derived growth factor β receptor (βPDGFR) is a receptor tyrosine kinase involved in multiple aspects of cell growth and differentiation. Upon activation, βPDGFR is phosphorylated at up to nine different tyrosine residues. Phosphorylation of the receptor results in at least two different outcomes: recruitment of signaling molecules and activation of intrinsic receptor kinase activity. In order to evaluate the phosphorylation state of the receptor, phosphospecific antibodies were generated against peptides encompassing βPDGFR phospho-Y751, phospho-Y771, or phospho-Y857. When phosphorylated, these sites enable the receptor to recruit signaling molecules PI3K or RasGAP, or enhance the receptor's kinase activity, respectively. We found that receptors phosphorylated at Y751, Y771, and Y857 display distinct temporal and spatial distribution by immunofluorescence. Subsequent biochemical studies revealed that receptor function corresponding to each of the phosphorylated sites was regulated as a function of time. Within the first 10 min, PDGF enhanced the receptor's kinase activity and initiated recruitment of PI3K and RasGAP. After prolonged exposure to PDGF, PI3K binding persisted to approximately 85% of the amount bound at 10 min, whereas binding of RasGAP and the exogenous kinase activity of the receptor diminished to less than 15% of the levels displayed at 10 min. We conclude that the phosphorylation state of the receptor, as well as its signaling capacity, is dynamic and changes as cells are continuously exposed to PDGF.     

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.