PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.     

Platelet-derived growth factor potentiates phorbol ester-induced neuronal differentiation of human neuroblastoma cells.

Previous studies have shown that platelet-derived growth factor (PDGF) and PDGF receptors are expressed in the mammalian central nervous system and that primary cultured neuroblasts from rat hindbrain have functional PDGF beta-receptors. Here, it is shown that cultured human neuroblastoma cells express PDGF alpha- and beta-receptors, but not PDGF-A and PDGF-B chain mRNA. In contrast to alpha-receptor expression, beta-receptor expression appears to be associated with a mature neuronal phenotype. Under serum-free growth conditions, PDGF-AA and -BB induce a trophic and weak mitogenic response in SH-SY5Y neuroblastoma cells, showing that the PDGF receptors in these cells are functional. In combination with 12-O-tetradecanoylphorbol-13-acetate, all three PDGF isoforms induce sympathetic neuronal differentiation of the SH-SY5Y cells, as shown by morphology and by increased expression of the genes coding for growth-associated protein 43 and neuropeptide tyrosine, respectively. This indicates a potential role for PDGF in the development of sympathetic neurons in particular and of the nervous system in general.     

Structural and functional specificities of PDGF-C and PDGF-D, the novel members of the platelet-derived growth factors family

The platelet-derived growth factor (PDGF) family was for more than 25 years assumed to consist of only PDGF-A and -B. The discovery of the novel family members PDGF-C and PDGF-D triggered a search for novel activities and complementary fine tuning between the members of this family of growth factors. Since the expansion of the PDGF family, more than 60 publications on the novel PDGF-C and PDGF-D have been presented, highlighting similarities and differences to the classical PDGFs. In this paper we review the published data on the PDGF family covering structural (gene and protein) similarities and differences among all four family members, with special focus on PDGF-C and PDGF-D expression and functions. Little information on the protein structures of PDGF-C and -D is currently available, but the PDGF-C protein may be structurally more similar to VEGF-A than to PDGF-B. PDGF-C contributes to normal development of the heart, ear, central nervous system (CNS), and kidney, while PDGF-D is active in the development of the kidney, eye and brain. In adults, PDGF-C is active in the kidney and the central nervous system. PDGF-D also plays a role in the lung and in periodontal mineralization. PDGF-C is expressed in Ewing family sarcoma and PDGF-D is linked to lung, prostate and ovarian cancers. Both PDGF-C and -D play a role in progressive renal disease, glioblastoma/medulloblastoma and fibrosis in several organs.     

Expression and function of PDGF-C in development and stem cells

Platelet-derived growth factor C (PDGF-C) is a relatively new member of the PDGF family, discovered nearly 20 years after the finding of platelet-derived growth factor A (PDGF-A) and platelet-derived growth factor B (PDGF-B). PDGF-C is generally expressed in most organs and cell types. Studies from the past 20 years have demonstrated critical roles of PDGF-C in numerous biological, physiological and pathological processes, such as development, angiogenesis, tumour growth, tissue remodelling, wound healing, atherosclerosis, fibrosis, stem/progenitor cell regulation and metabolism. Understanding PDGF-C expression and activities thus will be of great importance to various research disciplines. In this review, however, we mainly discuss the expression and functions of PDGF-C and its receptors in development and stem cells.     

Platelet-derived growth factor-C (PDGF-C) activation by serine proteases: implications for breast cancer progression

The PDGF (platelet-derived growth factor) family members are potent mitogens for cells of mesenchymal origin and serve as important regulators of cell migration, survival, apoptosis and transformation. Tumour-derived PDGF ligands are thought to function in both autocrine and paracrine manners, activating receptors on tumour and surrounding stromal cells. PDGF-C and -D are secreted as latent dimers, unlike PDGF-A and -B. Cleavage of the CUB domain from the PDGF-C and -D dimers is required for their biological activity. At present, little is known about the proteolytic processing of PDGF-C, the rate-limiting step in the regulation of PDGF-C activity. In the present study we show that the breast carcinoma cell line MCF7, engineered to overexpress PDGF-C, produces proteases capable of cleaving PDGF-C to its active form. Increased PDGF-C expression enhances cell proliferation, anchorage-independent cell growth and tumour cell motility by autocrine signalling. In addition, MCF7-produced PDGF-C induces fibroblast cell migration in a paracrine manner. Interestingly, PDGF-C enhances tumour cell invasion in the presence of fibroblasts, suggesting a role for tumour-derived PDGF-C in tumour-stromal interactions. In the present study, we identify tPA (tissue plasminogen activator) and matriptase as major proteases for processing of PDGF-C in MCF7 cells. In in vitro studies, we also show that uPA (urokinase-type plasminogen activator) is able to process PDGF-C. Furthermore, by site-directed mutagenesis, we identify the cleavage site for these proteases in PDGF-C. Lastly, we provide evidence suggesting a two-step proteolytic processing of PDGF-C involving creation of a hemidimer, followed by GFD-D (growth factor domain dimer) generation.     

PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor

The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.     

Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC)

The platelet-derived growth factor (PDGF) family, which regulates many physiological and pathophysiological processes has recently been enlarged by two new members, the isoforms PDGF-C and -D. Little is known about the expression levels of these new members in hepatic fibrosis. We therefore investigated by quantitative real time PCR (Taqman) the mRNA expression profiles of all four PDGF isoforms in transdifferentiating primary cultured hepatic stellate cells (HSC), an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-beta1. All four isoforms were expressed in HSC transdifferentiating to myofibroblast-like cells (MFB) albeit with different profiles: while PDGF-A mRNA exhibited minor fluctuations only, PDGF-B was rapidly down-regulated. In contrast, both PDGF-C and -D mRNA were strongly induced: PDGF-C up to 5 fold from day 2 to day 8 and PDGF-D up to 8 fold from day 2 to day 5 of culture. Presence of PDGF-DD in activated HSC was confirmed at the protein level by immunocytochemistry. Stimulation of HSC and MFB with PDGF-BB led to down-regulation of the new isoforms, whereas TGF-beta1 upregulated PDGF-A only. We further show that PDGF receptor-beta (PDGFR-beta) mRNA was rapidly upregulated within the first day of culture and was constantly expressed from day 2 on while the expression profile of PDGFR-alpha mRNA was very similar to that of PDGF-A during transdifferentiation. Given the dramatic changes in PDGF-C and -D expression, which may compensate for down-regulation of PDGF-B, we hypothesize that the new PDGF isoforms may fulfil specific functions in hepatic fibrogenesis.     

Characterization of platelet-derived growth factor-C (PDGF-C): expression in normal and tumor cells, biological activity and chromosomal localization

The predicted platelet-derived growth factor-C (PDGF-C) polypeptide contains an N-terminal CUB-like domain and a C-terminal domain with homology to members of the PDGF/vascular endothelial growth factor (VEGF) family. PDGF-C mRNA is widely expressed in normal tissues and does not appear to be up-regulated in the tumor cell lines tested. The PDGF-C gene was mapped to human chromosome 4q31-32. PDGF-C protein and the CUB domain of PDGF-C expressed in Escherichia coli, were able to stimulate proliferation of human artery smooth muscle cells, but were inactive on umbilical vein endothelial cells, osteoblasts, fibroblasts, skeletal muscle cells (SkMC), bovine chondrocytes, and rat myocardium cells. Although the mitogenic activity of PDGF-C and the CUB domain was only observed at concentrations ranging from 1 to 10 microg/ml, substitution of Cys(124) by Ser or deletion of Cys(124) significantly reduced the mitogenic activity. Our data suggest a possible role of the CUB domain of PDGF-C in addition to its role in maintaining latency of the PDGF domain.     

PDGF-D Expression Is Down-Regulated by TGFβ in Fibroblasts

Transforming growth factor-β (TGFβ) is a key mediator of fibrogenesis. TGFβ is overexpressed and activated in fibrotic diseases, regulates fibroblast differentiation into myofibroblasts and induces extracellular matrix deposition. Platelet-derived growth factor (PDGF) is also a regulator of fibrogenesis. Some studies showed a link between TGFβ and PDGF in certain fibrotic diseases. TGFβ induces PDGF receptor alpha expression in scleroderma fibroblasts. PDGF-C and -D are the most recently discovered ligands and also play a role in fibrosis. In this study, we report the first link between TGFβ and PDGF-D and -C ligands. In normal fibroblasts, TGFβ down-regulated PDGF-D expression and up-regulated PDGF-C expression at the mRNA and protein levels. This phenomenon is not limited to TGFβ since other growth factors implicated in fibrosis, such as FGF, EGF and PDGF-B, also regulated PDGF-D and PDGF-C expression. Among different kinase inhibitors, only TGFβ receptor inhibitors and the IκB kinase (IKK) inhibitor BMS-345541 blocked the effect of TGFβ. However, activation of the classical NF-κB pathway was not involved. Interestingly, in a model of lung fibrosis induced by either bleomycin or silica, PDGF-D was down-regulated, which correlates with the production of TGFβ and other fibrotic growth factors. In conclusion, the down-regulation of PDGF-D by TGFβ and other growth factors may serve as a negative feedback in the network of cytokines that control fibrosis.     

Modulation of PDGF-C and PDGF-D expression during bleomycin-induced lung fibrosis

PDGF isoforms are a family of polypeptides that bind to cell surface receptors and induce fibroblast proliferation and chemotaxis. The PDGF-A and -B chain isoforms have been implicated in fibroproliferative lung injury in animal models and in human disease. Two recently recognized PDGF polypeptides, PDGF-C and -D, differ from the PDGF-A and -B isoforms in that they require proteolytic cleavage before they can bind and activate the PDGF receptors. Our findings demonstrate that administration of bleomycin to murine lungs leads to a significant increase in PDGF-C mRNA expression and a significant decrease in PDGF-D mRNA expression. PDGF-C expression was localized to areas of lung injury by in situ hybridization, and PDGF-C expression was not upregulated in the lungs of BALB/c mice that are resistant to bleomycin-induced lung fibrosis. Moreover, there is in vivo phosphorylation of the PDGF-receptor that binds PDGF-C in response to bleomycin administration. These observations strongly suggest a role for PDGF-C in bleomycin-induced pulmonary fibrosis.     

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.     

Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor

We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-alpha homodimers and PDGF receptor-alpha/beta heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB.     

Suppressors of hedgehog signaling

Subversion of signals that physiologically suppress Hedgehog pathway results in aberrant neural progenitor development and medulloblastoma, a malignancy of the cerebellum. The Hedgehog antagonist RENKCTD11 maps to chromosome 17p13.2 and is involved in the withdrawal of the Hedgehog signaling at the granule cell progenitor transition from the outer to the inner external germinal layers, thus promoting growth arrest and differentiation. Deletion of chromosome 17p, the most frequent genetic lesion observed in this tumor, is responsible for the loss of function of RENKCTD11, resulting in upregulated Hedgehog signaling and medulloblastoma. Persistence of signals that limit Hedgehog activity is also associated with malignancy. Hedgehog signaling- induced downregulation of ErbB4 receptor expression is attenuated in medulloblastoma subsets in which the extent of Hedgehog pathway activity is limited, thus favoring the accumulation of ErbB4 with imbalanced alternative splice CYT-1 isoform over the CYT-2. This is responsible for both Neuregulin ligand-induced CYT-1-dependent prosurvival activity and loss of CYT-2-mediated growth arrest.     

Expression of mouse PDGF-A and PDGF alpha-receptor genes during pre- and post-implantation development: evidence for a developmental shift from an autocrine to a paracrine mode of action.

We examined the expression of platelet-derived growth factor (PDGF)-A and the PDGF alpha-receptor in pre-implantation and early post-implantation mouse embryos. At two-cell and blastocyst stages, all cells express mRNA and protein for both ligand and receptor. In contrast, early post-implantation embryos express PDGF-A chain mRNA in both embryonic ectoderm and in the ectoderm lining the ectoplacental cavity, while mRNA for PDGF alpha-receptor is localized to the mesoderm layers of both embryonic and extra-embryonic membranes. At days 3.5 and 7.5, receptors are demonstrably functional in response to exogenous PDGF-AA. We propose that chronic autostimulation of PDGF alpha-receptors occurs in pre-implantation embryos, whereas, following implantation, early mesoderm development is dependent on stimulation by ectodermally produced PDGF-A.     

Structural requirements for activation of latent platelet-derived growth factor CC by tissue plasminogen activator

Platelet-derived growth factor C (PDGF-C) is one of four members in the PDGF family of growth factors, which are known mitogens and survival factors for cells of mesenchymal origin. PDGF-C has a unique two-domain structure consisting of an N-terminal CUB and a conserved C-terminal growth factor domain that are separated by a hinge region. PDGF-C is secreted as a latent dimeric factor (PDGF-CC), which undergoes extracellular removal of the CUB domains to become a PDGF receptor alpha agonist. Recently, the multidomain serine protease tissue plasminogen activator (tPA), a thrombolytic agent used for treatment of acute ischemic stroke, was shown to cleave and activate PDGF-CC. In this study we determine the molecular mechanism of tPA-mediated activation of PDGF-CC. Using various PDGF-CC and tPA mutants, we were able to demonstrate that both the CUB and the growth factor domains of PDGF-C, as well as the kringle-2 domain of tPA, are required for the interaction and cleavage to occur. We also show that Arg231 in PDGF-C is essential for tPA-mediated proteolysis and that the released "free" CUB domain of PDGF-C can act as a competitive inhibitor of the cleavage reaction. Furthermore, we studied how the PDGF-C/tPA axis is regulated in primary fibroblasts and found that PDGF-C expression is down-regulated by hypoxia but induced by transforming growth factor (TGF)-beta1 treatment. Elucidating the regulation and the mechanism of tPA-mediated activation of PDGF-CC will advance our knowledge of the physiological function of PDGF-CC and tPA and may provide new therapeutic opportunities for thrombolytic and cardiovascular therapies.     

Molecular cloning of SCDGF-B, a novel growth factor homologous to SCDGF/PDGF-C/fallotein

Spinal cord-derived growth factor (SCDGF)/platelet-derived growth factor (PDGF)-C/fallotein has a unique two-domain structure, as it contains two regions homologousto CUB and PDGF/vascular endothelial growth factor (VEGF) domains. In this study, we isolateda novel gene homologous to SCDGF/PDGF-C/fallotein, and named SCDGF-B. The culture supernatant of CHO-K1 cells stably transfected with SCDGF-B showed mitogenic activity as SCDGF/PDGF-C/fallotein did. Although SCDGF-B and SCDGF/PDGF-C/fallotein might be the members of the PDGF/VEGF superfamily of growth factors, they were categorized into a new subfamily in addition to PDGF and VEGF subfamilies.