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.     

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.     

PDGF-D, a new protease-activated growth factor

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.     

Generation of new Notch2 mutant alleles

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. We previously described construction and analysis of a hypomorphic allele of the Notch2 gene. Homozygosity for this allele leads to embryonic and perinatal lethality due to cardiovascular and kidney defects. We report here novel Notch2 mutant alleles generated by gene targeting in embryonic stem cells, including a conditional null allele in which exon 3 of the Notch2 gene is flanked by loxP sequences. These new Notch2 mutant alleles expand the set of tools available for studying the myriad roles of the Notch pathway during mammalian development and will enable analysis of Notch2 function at additional stages of embryogenesis and in adult mice.     

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.     

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 the SMADIP1 gene during early human development

There are four members of the platelet-derived growth factor (PDGF) family; PDGF-A, PDGF-B, PDGF-C and PDGF-D. Their biological effects are mediated via two tyrosine kinase receptors, PDGFR-alpha and PDGFR-beta, and PDGF-mediated signaling is critical for development of many organ systems. Analysis in adult tissues showed that PDGF-C was mainly expressed in kidney, testis, liver, heart and brain. During development, PDGF-C expression was widespread and dynamic, and found in somites and their derivatives, in kidney, lung, brain, and in several other tissues, particularly at sites of developing epidermal openings. PDGF-C may therefore have unique functions during tissue development and maintenance.     

Generation of mice with a conditional allele for the transforming growth factor beta3 gene

The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFβ3 (Tgfb3) encodes one of the three ligands for TGFβ receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3-/- mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3-/- mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3cko allele will now enable studies of TGFβ3 function in different cell or tissue types in embryonic development and during adulthood.     

PDGF-C controls proliferation and is down-regulated by retinoic acid in mouse embryonic palatal mesenchymal cells

BACKGROUND: Platelet-derived growth factor C (PDGF-C) was recently identified as a member of the PDGF ligand family. Some observation suggests that PDGF-C could play an important role in palatogenesis highlighted by the Pdgfc(-/-) mouse with cleft palate, which led us to examine the mechanism of PDGF-C signaling in palatogenesis. It is well known that retinoic acid (RA) is a teratogen that can effectively induce cleft palate in the mouse. Due to the critical roles of PDGF-C and RA in cleft palate, the link between cleft palate induced by RA and loss of PDGF-C was investigated. METHODS: Retarded mesenchymal proliferation is an important cause for cleft palate. To clarify the mechanism of PDGF-C in palatogenesis, we evaluated the effects of PDGF-C and anti-PDGF-C neutralizing antibody on proliferation activity in mouse embryonic palatal mesenchymal (MEPM) cells. RESULTS: Briefly, our results show PDGF-C promotes proliferation, anti-PDGF-C antibody inhibits it in MEPM cells, and RA downregulates the PDGF-C expression both at the mRNA and protein levels. CONCLUSIONS: These demonstrate that PDGF-C is a potent mitogen for MEPM cells, implying that inactivated PDGF-C by gene-targeting or reduced PDGF-C by RA may both cause inhibition of proliferation in palatal shelves, which might account for the pathogenesis of cleft palate in Pdgfc(-/-) mouse or RA-treated mouse. In conclusion, our results suggest that PDGF-C signaling is a new mechanism of cleft palate induced by RA.     

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.     

Generation of an Frs2alpha conditional null allele

The fibroblast growth factor (FGF) signaling family controls a broad spectrum of cellular processes in development and adult tissue homeostasis and function, which is expressed in almost all tissues at all stages. FGF receptor substrate 2 alpha (FRS2alpha) is an adaptor protein that recruits downstream substrates to the FGF receptor (FGFR) tyrosine kinase. Disruption of Frs2alpha gene in mice abrogates activation of the mitogen-activated protein kinase pathway by the FGFR and leads to embryonic lethality at day E7.5 post copulation. To circumvent the embryonic lethality resulting from disruption of the Frs2alpha gene, which hinders further characterization of the role of FRS2alpha in adult tissue function and homeostasis, we generated an Frs2alpha conditional null allele for temporally- and tissue-specific disruption of the Frs2alpha gene. Using gene targeting in mouse embryonic stem cells, we introduced two loxP sites flanking the largest coding exon, exon 5, in the Frs2alpha allele. Our results indicate that the floxed Frs2alpha (Frs2alpha(flox)) allele is a true conditional null allele that encodes wildtype activity and is converted to a null allele after Cre recombinase mediated recombination.     

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.     

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.     

Generation of an Fgf9 conditional null allele

The fibroblast growth factor (FGF) family consists of 22 widely expressed regulatory polypeptides and controls a broad spectrum of cellular processes. Accumulating data show that FGF9 plays important roles both in embryogenesis and in adult tissue homeostasis. Ablation of Fgf9 alleles leads to lethality at the neonatal stage mainly due to malformations of the lung, as well as causing male-to-female sex reversal. To circumvent the neonatal lethality resulting from disruption of the Fgf9 gene, which hinders further characterization of the role of FGF9 in adult tissue function and homeostasis, we generated an Fgf9 conditional null allele for spatiotemporal- and tissue-specific disruption of Fgf9. Using gene targeting in mouse embryonic stem (ES) cells, we introduced two loxP sites flanking exon 1 in the Fgf9 allele, which encodes 93 amino acid residues at the N-terminal of FGF9. Our results indicate that the Fgf9 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after recombination mediated by the Cre recombinase.     

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.     

Establishment of conditional knockout alleles for the gene encoding the regulator of telomere length (RTEL)

Regulator of telomere length (RTEL) is a DNA helicase-like protein that has recently been demonstrated to be required for the maintenance of telomere length and genomic stability. Rtel null mice are embryonic lethal with the defects in the nervous system, the heart, the vasculature, and extra-embryonic tissues. Rtel could also be important for the postnatal development as its expression is strongly induced in the proliferating adult cells. To further characterize the role of RTEL in adult tissue function and homeostasis, we have generated the floxed (loxP-flanked) alleles allowing to inactivate RTEL through Cre-mediated recombination in a cell- or tissue-specific manner and also to circumvent the embryonic lethality of the Rtel null allele. Mice heterozygous or homozygous for these alleles are viable and fertile. Crossing the floxed Rtel allele with a ubiquitous Cre transgenic line resulted in embryonic defects identical to those previously described for the Rtel null embryos. These conditional alleles will therefore be the important genetic tools for dissecting the spatial and temporal roles of RTEL in the regulation of telomere length and genomic stability during postnatal development and tumorigenesis.     

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.     

Generation of Ppp2Ca and Ppp2Cb conditional null alleles in mouse

Protein phosphatase 2A (PP2A) is one of the most abundant serine/threonine phosphatases, with a critical role in embryonic development and human disease. There are two isoforms of the catalytic subunit of PP2A, Ppp2ca and Ppp2cb. Null mutation of Ppp2ca leads to early embryonic lethality at E6.5, hindering functional study of PP2A beyond this stage. We generated conditional null alleles of Ppp2ca and Ppp2cb by flanking with loxP sites exons 3 to 5 of Ppp2ca and exon 3 of Ppp2cb. Ppp2ca(fl/fl) mice did not display any visible phenotype. Homozygous mutants in which Cre-mediated excision resulted in global deletion of Ppp2ca displayed embryonic lethality and developmental defects similar to those previously reported. Ppp2cb(Δ/Δ) mice generated by the same strategy did not display any obvious morphological or physiological defects. These mouse strains can serve as important genetic tools to study the roles of PP2A during development and disease in a spatial- or temporal-specific manner.     

Generation of conditional alleles of the murine Iron Regulatory Protein (IRP)-1 and -2 genes

Central aspects of cellular iron metabolism are controlled by IRP1 and IRP2, which are ubiquitously expressed in mouse organs and cells. Total and constitutive deficiency of both IRPs causes embryonic lethality in the mouse. To bypass the early lethality and to study organ-specific and/or temporal functions of IRP1 and/or IRP2 we generated Irp1 and Irp2 conditional alleles. We used mouse lines where a betaGeo gene trap construct was inserted into the second intron of the Irp1 and the Irp2 gene, generating hypomorphic alleles by interrupting the corresponding open reading frame near the amino-termini. The gene trap cassettes are flanked by Frt sites and were co-inserted with LoxP sites flanking exon 3. Flp-mediated removal of the gene trap construct generates floxed alleles with wildtype functions. For both Irp genes, Cre-assisted deletion of exon 3 generates complete null alleles that, in the case of IRP2, are associated with altered body iron distribution and compromised hematopoiesis. If not removed, the gene trap construct causes partially penetrant embryonic lethality unrelated to IRP deficiency when inserted within the Irp1 but not the Irp2 locus. We discuss the implications for functional genomics in the mouse.