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REVIEW
1 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 171 77 Stockholm, Sweden; 2 Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institutet, SE 171 77 Stockholm, Sweden; 3 Department of Medicine, Karolinska Institutet, SE 171 77 Stockholm, Sweden
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Abstract |
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signaling in gastrulation and in the development of the cranial and cardiac neural crest, gonads, lung, intestine, skin, CNS, and skeleton. Similarly, roles for PDGFR-β signaling have been established in blood vessel formation and early hematopoiesis. PDGF signaling is implicated in a range of diseases. Autocrine activation of PDGF signaling pathways is involved in certain gliomas, sarcomas, and leukemias. Paracrine PDGF signaling is commonly observed in epithelial cancers, where it triggers stromal recruitment and may be involved in epithelial–mesenchymal transition, thereby affecting tumor growth, angiogenesis, invasion, and metastasis. PDGFs drive pathological mesenchymal responses in vascular disorders such as atherosclerosis, restenosis, pulmonary hypertension, and retinal diseases, as well as in fibrotic diseases, including pulmonary fibrosis, liver cirrhosis, scleroderma, glomerulosclerosis, and cardiac fibrosis. We review basic aspects of the PDGF ligands and receptors, their developmental and pathological functions, principles of their pharmacological inhibition, and results using PDGF pathway-inhibitory or stimulatory drugs in preclinical and clinical contexts.
[Keywords: PDGF receptor; cancer; development; fibrosis; platelet-derived growth factor]
; Ross et al. 1974; Westermark and Wasteson 1976). Human PDGF was originally identified as a disulfide-linked dimer of two different polypeptide chains, A and B, separable using reversed phase chromatography (Johnsson et al. 1982). The B-chain (PDGF-B) was characterized by amino acid sequencing, revealing a close homology between PDGF-B and the product of the retroviral oncogene v-sis of simian sarcoma virus (SSV) (Doolittle et al. 1983; Waterfield et al. 1983). Subsequent studies confirmed that the human cellular counterpart (c-sis) was identical to PDGF-B and that autocrine PDGF activity was sufficient for SSV transformation in vitro. This was a paradigm-shifting discovery about the relationship between neoplastic cell transformation and normal growth control. For the first time, the importance of autocrine growth stimulation in neoplastic transformation was demonstrated. As discussed below, it is now well established that autocrine PDGF stimulation plays a role also in some human cancers.
PDGF-A was characterized by cDNA cloning (Betsholtz et al. 1986). This resolved a paradoxical lack of correlation between secretion of PDGF-like growth factors from tumor cell lines and their expression of c-sis; it turned out that most such cell lines express PDGF-A and secrete PDGF-AA homodimers (Heldin et al. 1986). Together with the demonstration that PDGF-BB homodimers are produced by SSV-transformed or PDGF-B-expressing cells, these results showed that the PDGF family consisted of three proteins—PDGF-AA, PDGF-AB, and PDGF-BB—encoded by two genes, PDGF-A and PDGF-B (for review, see Heldin and Westermark 1999). This view lasted for more than 15 years until combinations of genomic and biochemical efforts identified two additional PDGF genes and proteins—PDGF-C (Li et al. 2000) and PDGF-D (Bergsten et al. 2001; LaRochelle et al. 2001). The currently known PDGF genes and polypeptides belong to a family of structurally and functionally related growth factors including also the vascular endothelial growth factors (VEGFs) (Fredriksson et al. 2004a). PDGF/VEGF growth factors are conserved throughout the animal kingdom (Fig. 1) and form part of a large superfamily of proteins containing cystine knots (McDonald and Hendrickson 1993).
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The present review summarizes current knowledge about PDGF functions in health and disease. We provide a brief background to PDGF biochemistry and cell biology and discuss how some of the cellular responses to PDGFs relate to functions in mammalian development and disease. In this context, we also discuss the recently established roles of PDGF/VEGF-like growth factors (PVFs) in invertebrates. We summarize how different mechanisms contribute to the regulation of bioavailability and tissue distribution of the PDGFs, which are key parameters during development. For detailed information on particular aspects of PDGF biology, such as signal transduction and the many reported effects of PDGFs in cell culture, the reader is referred to other reviews and original literature, some of which are cited below.
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The PDGF/VEGF family of ligands and receptors |
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All PDGFs and VEGFs are dimers of disulfide-linked polypeptide chains (for review, see Heldin and Westermark 1999). In mammals, a total of nine different genes encode four different PDGF chains (PDGF-A, PDGF-B, PDGF-C, and PDGF-D) and five different VEGF chains (VEGF-A, VEGF-B, VEGF-C, VEGF-D; and placenta growth factor, PlGF) (for review, see Ferrara et al. 2003; Fredriksson et al. 2004a). One heterodimer (PDGF-AB) has been demonstrated in human platelets. Although the PDGF-AB heterodimer is endowed with somewhat different signaling properties from the homodimers (Ekman et al. 1999), its physiological importance remains unclear. PDGF-AB occurrence in platelets may be specific to humans (Stroobant and Waterfield 1984). Also, the endogenous expression patterns of PDGF-A and PDGF-B are generally nonoverlapping (Hoch and Soriano 2003), suggesting that heterodimers are infrequent in vivo. Presently, evidence for genetic interactions between pdgfa and pdgfb is also lacking (Li et al. 2000). Thus, although there may be special cases for heterodimer formation and function within the PDGF ligand family, homodimers appear to dominate, at least during development.
Mammalian PDGFs and VEGFs separate into four distinguishable classes of proteins (Fig. 1). All members carry a growth factor core domain containing a conserved set of cysteine residues. The core domain is necessary and sufficient for receptor binding and activation. Classification into PDGFs or VEGFs is based on receptor binding. It has been generally assumed that PDGFs and VEGFs are selective for their own receptors. This view was recently challenged by the demonstration that VEGF-A may bind to and activate PDGF receptors in bone-marrow-derived mesenchymal stem cells (Ball et al. 2007). This study also challenges the general view that VEGFs target mainly endothelial cells, whereas mesenchymal cells are targeted by PDGFs. Further challenge to the functional distinctions between PDGFs and VEGFs comes from findings that VEGF-C and PDGF-A both regulate oligodendrocyte development, however, through distinct receptors. VEGFs and PDGFs also both appear to function in hematopoietic development, neurogenesis, and neuroprotection. These functions are further discussed below.
Mammalian PDGF receptors
PDGFs act via two RTKs (PDGFR- and PDGFR-β) with common domain structures, including five extracellular immunoglobulin (Ig) loops and a split intracellular tyrosine kinase (TK) domain. This structure is shared with c-Fms, c-Kit, and Flt3, the receptors for CSF-1, SCF, and Flt3-ligand, respectively. The VEGFs act through a distinct but structurally related subfamily of RTKs— VEGFRs 1, 2, and 3. Ligand binding promotes receptor dimerization, which initiates signaling. Depending on ligand configuration and the pattern of receptor expression, different receptor dimers may form (Heldin and Westermark 1999). Theoretically (and generally based on cell culture experiments), the possible PDGF–PDGFR interactions are multiple and complex and include the formation of receptor heterodimers (Fig. 2). However, in vivo there is functional evidence only for a few interactions; i.e., those of PDGF-AA and PDGF-CC via PDGFR-, and PDGF-BB via PDGFR-β (Fig. 2).It is likely that also PDGF-DD acts through PDGFR-β in vivo, but evidence for this is currently lacking. PDGFR heterodimer formation occurs in vivo as seen by crossing mice carrying PDGFR- and PDGFR-β signaling mutants (Klinghoffer et al. 2002). The importance of the different PDGF–PDGFR interactions, and the signals elicited, are further discussed below. For a comprehensive overview of PDGF receptor signal transduction mechanisms, the reader is referred to previous reviews on this topic (Heldin and Westermark 1999; Rönnstrand and Heldin 2001; Tallquist and Kazlauskas 2004).
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PDGF/VEGF signaling is conserved throughout the animal kingdom. The fruit fly Drosophila melanogaster has three PVFs (PVF-1–3), and a single PDGF/VEGF receptor (PVR). The nematode Caenorhabditis elegans has four receptors (VER-1–4), and one putative ligand (PVF-1) (Hoch and Soriano 2003; Tarsitano et al. 2006), whose direct interaction with the VERs remains to be established. Invertebrate PVR/VERs resemble the mammalian VEGF receptors in that they have seven (rather than five) extracellular Ig loops. The PVFs also resemble the mammalian VEGFs in that they contain a C-terminal cysteine-rich motif, which is missing in the PDGFs (Fig. 1). Thus, it appears that an ancestral VEGF system has duplicated to generate the vertebrate VEGF and PDGF families through divergent evolution. C. elegans (Ce)PVF-1 was recently found to bind and activate human VEGFR-1 and VEGFR-2 and to induce angiogenic responses in human umbilical vein endothelial cells and chick chorioallantoic membranes. In contrast, CePVF-1 did not bind VEGFR-3 or PDGFR-β (Tarsitano et al. 2006). The invertebrate PFV-PVR/VER system therefore seems to be structurally and functionally orthologous to mammalian VEGF-A/B/PlGF–VEGFR1/2.
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Expression, processing, and bioavailability of the PDGF ligands |
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PDGFs act primarily as paracrine growth factors. PDGFs may also be engaged in autocrine loops in tumors (discussed below). Physiological autocrine roles, similar to those recently described for VEGF-A in endothelial cells (Lee et al. 2007), have not been demonstrated for the PDGFs. PDGFs are generally produced by discrete populations of cells and act locally to drive different cellular responses (for review, see Hoch and Soriano 2003; Betsholtz 2004). Both PDGF and PDGFR expression patterns are spatio-temporally regulated in vivo during development and in certain physiological hypertrophic responses. PDGF expression in cultured cells is dynamic and responsive to a variety of stimuli, including hypoxia, thrombin, cytokines, and growth factors, including PDGF itself (for review, see Heldin and Westermark 1999). Also, PDGFR expression is dynamic. General mesenchymal expression of PDGFRs is low in vivo, but increases dramatically during inflammation and in culture. Several factors induce PDGFR expression, including TGF-β, estrogen (probably linked to hypertrophic smooth muscle responses in the pregnant uterus), interleukin-1 (IL-1), basic fibroblast growth factor-2 (FGF-2), tumor necrosis factor-, and lipopolysaccharide (Heldin and Westermark 1999 and references therein).
The detailed expression patterns of the individual PDGF ligands and receptors are complex and have been reviewed elsewhere (Heldin and Westermark 1999; Hoch and Soriano 2003). There are some general patterns, however: PDGF-B is mainly expressed in vascular endothelial cells, megakaryocytes, and neurons. PDGF-A and PDGF-C are expressed in epithelial cells, muscle, and neuronal progenitors. PDGF-D expression is less well characterized, but it has been observed in fibroblasts and SMCs at certain locations (possibly suggesting autocrine functions via PDGFR-β). PDGFR- is expressed in mesenchymal cells. Particularly strong expression of PDGFR- has been noticed in subtypes of mesenchymal progenitors in lung, skin, and intestine and in oligodendrocyte progenitors (OPs) (discussed further below). PDGFR-β is expressed in mesenchyme, particularly in vascular SMCs (vSMCs) and pericytes.
The mammalian PDGF and PDGFR genes are located on different chromosomes, and their transcriptional regulation seems largely independent. It remains to be established if some of the overlapping expression patterns for PDGF-A and PDGF-C result from common transcription regulatory mechanisms. The transcriptional regulation of the PDGF-A and PDGF-B genes has been extensively studied and is reviewed elsewhere (Heldin and Westermark 1999; Kaetzel 2003). Little is still known about the transcriptional regulation of PDGF-C and PDGF-D and the PDGFRs.
PDGF biosynthesis and secretion
PDGF biosynthesis and processing are controlled at multiple levels and differ for the different PDGFs. There is currently no evidence for regulated secretion of the PDGFs, which instead appears to be constitutively released (Fruttiger et al. 2000). PDGF-A and PDGF-B become disulfide-linked into dimers already as propeptides. PDGF-C and PDGF-D have been less studied in this regard. PDGF-A and PDGF-B contain N-terminal pro-domains that are removed intracellularly by furin or related proprotein convertases (for review, see Fredriksson et al. 2004a). N-terminal processing is necessary for PDGF-A to acquire receptor-binding ability (for review, see Heldin and Westermark 1999; Fredriksson et al. 2004a). Likely, PDGF-B also requires N-terminal propeptide removal to become active.
In contrast, PDGF-C and PDGF-D are not processed intracellularly but are instead secreted as latent (conditionally inactive) ligands (for review, see Fredriksson et al. 2004a; Reigstad et al. 2005). Activation in the extracellular space requires dissociation of the growth factor domain from the CUB domain (Fig. 1). Plasmin and tissue plasminogen activator (tPA) have been demonstrated to proteolytically remove the CUB domain in PDGF-C, rendering it biologically active (Fredriksson et al. 2004b). Although the endogenous protease(s) responsible for PDGF-C activation in vivo remains to be identified, tPA endogenously expressed in cultured fibroblasts activates PDGF-CC expressed by the same cells. Plasmin can cleave and activate also PDGF-D, but tPA cannot (Fredriksson et al. 2004b). TPA needs to interact with both the CUB domain and the core domain in order for cleavage and activation of PDGF-C to occur, which likely explains this specificity.
Extracellular retention and distribution of PDGFs
Spatially uneven distribution (gradients and depots) of growth factors, cytokines, and morphogens defines their biological activity and action range. Diffusion of PDGF in the tissue interstitium is regulated by binding to extracellular matrix components (Fig. 3). For PDGF-A and PDGF-B, such binding is accomplished in part by the positively charged C-terminal motifs (referred to as retention motifs) containing a high proportion of basic amino acid residues (Fig. 1). PDGF-C and PDGF-D lack basic retention motifs, but CUB domains are implicated in protein–protein and protein–carbohydrate interactions in other contexts and may regulate extracellular distribution of latent PDGF-C and PDGF-D. The presence of the retention motif is determined by alternative splicing in PDGF-A and by alternative C-terminal proteolytic processing in PDGF-B (Fig. 3). Alternative splicing has also been demonstrated for several of the members of the VEGF family, leading to the formation of multiple isoforms that differ in extracellular matrix binding (Fig. 1; for review, see Ferrara et al. 2003). Alternative splicing of the PDGF-A transcript is cell type-specific and differs both among tumor cell lines (Afrakhte et al. 1996) and in different organs during development (J. Andrae, H. Boström, and C. Betscholtz, unpubl.).
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). Certain cells transfected with PDGF-B expression vectors secrete soluble PDGF-BB into the conditioned medium. However, a major part of endogenously expressed PDGF-B becomes trapped on the cell surface or in the extracellular matrix, where it subsequently can be released by thrombin (Kelly et al. 1993; Soyombo and DiCorleto 1994).
Insights into the role of PDGF retention have come from studies of PDGF interaction with heparan sulfate proteoglycans (HSPGs) and phenotypic analysis of PDGF-B retention motif knockout mice. PDGFs bind to heparin and HSPGs similar to many other growth factors and morphogens with critical functions during development (e.g., hedgehogs, bone morphogenetic protieins [BMPs], and Wnts) (Feyzi et al. 1997; Lustig et al. 1999; Lin 2004; Häcker et al. 2005; Abramsson et al. 2007). Targeted deletion of the PDGF-B retention motif in mice leads to pericyte detachment from the microvessel wall (Abramsson et al. 2003; Lindblom et al. 2003). Reduced heparan sulfate (HS) N-sulfation (caused by lack of the enzyme N-deacetylase/N-sulfotransferase-1) similarly leads to pericyte detachment and delayed pericyte migration in vivo (Abramsson et al. 2007). This is probably caused by attenuated PDGF-BB binding to HS. PDGF-BB/HS interaction appears to depend on overall N- and O-sulfation of HS, whereas saccharide fine structure appears to be of lesser importance. Taken together, available evidence suggests a model in which PDGF-BB secreted from endothelial cells interacts with HS at the endothelial surface or in the periendothelial matrix. This would lead to local deposits of PDGF-BB, which, in turn, are critical for the correct investment of pericytes in the vessel wall. HS binding is also necessary for proper localization and function of VEGF-A (Ruhrberg et al. 2002). It was shown recently that HS may act in trans—i.e., from pericytes—to potentiate VEGF-receptor function in endothelial cells (Jakobsson et al. 2006). Similarly, HS expressed on endothelial cells may function to enhance PDGF-BB-mediated PDGFR-β signaling in neighboring pericytes.
PDGF binds also to certain non-HSPG extracellular proteins, but the physiological relevance of these interactions is unclear. Binding of PDGF-B has been demonstrated to -2-macroglobulin (Bonner and Osornio-Vargas 1995), possibly acting as a scavenger for PDGF-B through low-density lipoprotein (LDL) receptor-related protein (LRP) receptors on macrophages and other cells (Bonner et al. 1995). PDGF-B also binds to SPARC and adiponectin, which may trap the growth factor in the extracellular space (Raines et al. 1992; Arita et al. 2002).
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PDGF receptor signaling transduction and downstream events |
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). Autophosphorylation activates the receptor kinase and provides docking sites for downstream signaling molecules (Kazlauskas and Cooper 1989). Docking of receptor substrates and further signal propagation involves protein–protein interactions through specific domains; e.g., Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains recognizing phosphorylated tyrosines, SH3 domains recognizing proline-rich regions, pleckstrin homology (PH) domains recognizing membrane phospholipids, and PDZ domains recognizing C-terminal-specific sequences (for review, see Heldin et al. 1998). Most of the PDGFR effectors bind to specific sites on the phosphorylated receptors through their SH2 domains.
PDGFR-induced signaling pathways
Both PDGFR- and PDGFR-β engage several well-characterized signaling pathways—e.g. Ras-MAPK, PI3K, and PLC-
—which are known to be involved in multiple cellular and developmental responses (Fig. 4). PDGFRs connect to Ras-MAPK mainly through the adaptor proteins Grb2 and Shc. Grb2 binds the activated PDGFR through its SH2 domain and complexes Sos1 through its SH3 domains. Sos1 in turn activates Ras, leading to downstream activation of Raf-1 and the MAPK cascade. MAPK signaling activates gene transcription, leading to stimulation of cell growth, differentiation, and migration (for review, see Bar-Sagi and Feramisco 1986; Seger and Krebs 1995).
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; Franke et al. 1995), some members of the PKC family including atypical isoforms (Nakanishi et al. 1993; Akimoto et al. 1996), p70 S6 kinase (Chung et al. 1994), JNK (Lopez-Ilasaca et al. 1997), and small GTPases of the Rho family (Hawkins et al. 1995). Activation of the PI3K pathway by PDGFRs promotes actin reorganization, directed cell movements, stimulation of cell growth, and inhibition of apoptosis (Hu et al. 1995).
PLC- binds PDGFRs, which results in its activation through phosphorylation (for review, see Tallquist and Kazlauskas 2004). PLC- activation leads to mobilization of intracellular calcium ions and the activation of PKC (Berridge 1993). The effects of PDGFR-mediated PLC- activation include stimulation of cell growth and motility (Kundra et al. 1994).
Several additional signaling molecules are engaged by PDGFRs, including enzymes, adaptors, and transcription factors. Activation of the Src TK promotes Myc transcription and mitogenic responses (for review, see Erpel and Courtneidge 1995). Also, members of the Fer and Fes TK family bind to PDGFRs (Kim and Wong 1995). PKC-
is phosphorylated by PDGFR-β, leading to its activation and translocation to the cell membrane (Li et al. 1996). This signal may be involved in cell differentiation. The adaptors Nck and Crk bind to PDGFRs through their SH2 domain and are involved in activation of JNK (Nishimura et al. 1993; Su et al. 1997). The adaptor Grb7 contains a SH2 domain and binds PDGFR-β (Yokote et al. 1996). STAT transcription factors may bind to PDGF receptors, leading to their phosphorylation and activation (Darnell 1997).
PDGF receptors interact also with integrins, which enhances cell proliferation, migration, and survival (for review, see Assoian 1997; Frisch and Ruoslahti 1997). PDGFR interaction with integrins helps localizing PDGFRs and interacting molecules at focal adhesions, which are sites where several signaling pathways initiate and cross-talk (Clark and Brugge 1995). Recently, Na+/H+ exchanger regulatory factors (NHERFs) were shown to bind PDGFR-β and link it with focal adherence kinase and the cortical actin cytoskeleton (James et al. 2004), as well as to N-cadherin (Theisen et al. 2007) and the phosphatase PTEN (Fig. 4; Takahashi et al. 2006). Additional evidence for compartmentalization of PDGFRs and their downstream signals within cells comes from the intriguing observation that PDGFR- (but not PDGFR-β) localizes to the primary cilium of fibroblasts (Schneider et al. 2005). Mutants that fail to form cilia do not activate PDGFR- but maintain PDGFR-β signaling. Primary cilia are also essential for hedgehog signaling (Eggenschwiler and Anderson 2007). Hedgehog receptors (patched) and PDGFR- show a conspicuous overlap in their expression pattern during development (Karlsson et al. 1999, 2000), and it is thus possible that this correlation extends to signaling at the subcellular level as well.
Negative control of PDGFR signaling
PDGF signaling is carefully regulated by feedback control mechanisms. Stimulatory and inhibitory signals arise in parallel, and the ultimate response depends on the balance between these signals (for review, see Heldin et al. 1998). The SHP-2 tyrosine phosphatase binds PDGFR through its SH2 domain and dephosphorylates the receptor and its substrates (Lechleider et al. 1993). Ras-GAP, which negatively regulates Ras, also binds PDGFR-β through its SH2 domain (Fantl et al. 1992).
Ligand occupancy of PDGFRs promotes endocytotic receptor internalization. The major destiny of internalized PDGFRs seems to be lysosomal degradation, thereby limiting the duration of PDGFR signaling (Heldin et al. 1982; Sorkin et al. 1991; Mori et al. 1995). Recycling of PDGFR-β, but not PDGFR-, was recently observed in cells deficient for the phosphatase TC-PTP (Karlsson et al. 2006), which is a negative regulator of PDGFR-β phosphorylation (Persson et al. 2004). Trafficking toward lysosomal degradation of PDGFR-β depends on interactions with c-Cbl and receptor ubiquitination. The adapter protein Alix, which interacts with the C-terminal domain of PDGFR-β, facilitates ubiquitination and degradation of c-Cbl, thereby inhibiting PDGFR-β down-regulation (Lennartsson et al. 2006).
Cellular responses to PDGFR signaling
Some of the cellular responses to PDGFs take place within seconds to minutes after PDGFR activation and are independent of gene expression and protein synthesis. PDGFR- and PDGFR-β mediate similar but not identical rapid responses. Both receptors stimulate rearrangement of actin filaments, but only PDGFR-β promotes formation of circular ruffles. PDGFR-β also mobilizes calcium ions more efficiently than PDGFR- (Diliberto et al. 1992; Fatatis and Miller 1997). PDGFR-β inhibits gap junctional communication between cells through phosphorylation of the gap junction protein connexin 43 (Hossain et al. 1998). It is unclear whether this ability is shared with PDGFR-.
In addition to the rapid post-transcriptional responses, PDGFRs (like other RTKs) induce fast transcriptional changes involving so-called immediate early genes (IEGs) (Cochran et al. 1983). IEGs are direct targets of the transcription factors that get activated (by post-translational modification) through various signaling pathways. The IEG responses are likely necessary for many of the long-term effects of PDGFs in vitro and in vivo. To what extent the IEG responses contribute specificity to PDGFR signaling is, however, unclear. Different RTKs induce virtually identical sets of IEGs (Fambrough et al. 1999). Also, different signaling pathways activated by the same receptor (PDGFR-β) induce largely overlapping sets of IEGs in vitro (Fambrough et al. 1999). From these studies, it would appear that quantitative rather than qualitative differences in the IEG responses mediate the specific responses to different RTKs and signaling pathways. This view is supported by in vivo analysis of an allelic series of pdgfrb tyrosine-to-phenylalanine mutations disrupting connection to different substrates and signaling pathways (Tallquist et al. 2003). Mutations in single or multiple tyrosine residues led to quantitatively different but qualitatively similar developmental effects (Tallquist et al. 2003). In contrast, similar analyses of PDGFR- revealed strikingly different roles of the different downstream signaling pathways. For PDGFR-β, the disruption of signaling through PI3K alone has no, or only minor, developmental consequences (Heuchel et al. 1999; Tallquist et al. 2000a). In contrast, PI3K is indispensable for PDGFR- function during development (Klinghoffer et al. 2002). Intriguingly (and in further contrast to PDGFR-β), deficient coupling to Src from PDGFR- resulted in specific problems with the oligodendrocyte lineage, whereas other processes, such as skeletal development, remained normal.
The different IEGs appear to cooperate in their regulation of downstream cellular and developmental events. By analyzing gene trap mutants for >20 IEGs downstream from PDGFR signaling, a striking degree of phenotypic overlap and genetic interaction was revealed. Different mutated IEGs produced qualitatively similar responses. Moreover, the combination of mutations in several genes strengthened specific phenotypic outcomes known to depend on PDGFR signaling, such as craniofacial, cardiovascular, or kidney developmental processes (Schmahl et al. 2007). The large overlap between the signaling pathways, IEGs, and biological processes argue in favor of a model in which specificity is generated through a combination of quantitative differences in magnitude and duration of the responses occurring at different levels in the signaling cascade. One shall not forget, however, that a major part of the specificity of the PDGFRs in developmental functions depends on cell type- and context-specific PDGFR expression. The expression patterns of the PDGFRs correlate with the major sites of their functions; e.g., PDGFR- is strongly expressed in OPs and PDGFR-β in pericyte progenitors. Lack of redundancy is therefore dependent in part on the different transcriptional regulation of the two pdgfr genes. By knocking in a PDGFR-β intracellular domain into the pdgfra gene, phenotypically normal animals were obtained, showing that PDGFR-β signaling can fully substitute for PDGFR- signaling if it is expressed at the right place and time (Klinghoffer et al. 2001). The reverse replacement, however, indicates that PDGFR- signaling can only partially compensate for the loss of PDGFR-β signaling. Targeted replacements in PDGFR- with other RTK signaling domains have also demonstrated partial rescue, at best (Klinghoffer et al. 2001; Hamilton et al. 2003). Taken together, these data suggest that specificity of PDGFR signaling is achieved through a combination of cell type-specific expression and differential engagement of downstream signaling pathways.
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Principles of genetic and pharmacological PDGF and PDGFR inhibition |
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and PDGFR-β, the bcr–abl fusion protein (toward which it was developed) (Carroll et al. 1997), c-kit, and Flt3. Imatinib has been approved by the US Food and Drug Administration for treatment of patients with Philadelphia chromosome-positive chronic myelogenous leukemia (carrying the bcl–abl fusion protein) (Druker et al. 2001) and gastrointestinal stromal tumors (GIST) carrying activating mutations in c-kit or PDGFR- (Demetri et al. 2002). Imatinib has also demonstrated clinical efficacy against myeloproliferative diseases involving rearrangements of the PDGFRB gene (Apperley et al. 2002) and dermatofibrosarcoma protuberans (DP), a tumor carrying translocated and activated PDGF-B genes (McArthur et al. 2005). Several other PDGFR kinase inhibitors have been developed (for review, see Levitzki 2004) and tested in clinical trials (Homsi and Daud 2007). All PDGFR kinase inhibitors used so far also block c-Kit and Flt3, two of the other three receptors with similar domain organization as PDGFRs. This specificity problem must be kept in mind when kinase inhibitors are used in order to evaluate the role of PDGF signaling in (patho)physiological processes. Imatinib nevertheless shows promise as a drug for a number of clinical conditions in which PDGFR signaling is activated (discussed in subsequent sections). The side effects of imatinib also appear to be minor from a general perspective as well as in comparison with other PDGFR inhibitors tested so far (for review, see Homsi and Daud 2007).
Currently, other strategies for small-molecule-mediated inhibition of PDGFR signaling are scarce. A recent study identified a nuclease-hypersensitive element in the PDGF-A promoter as a putative target for small molecule pharmacologicals, however. The guanine-rich strand of the DNA in this region forms a dynamic G-quadruplex, which upon binding of the small molecule TMPyP4 becomes stabilized, silencing the PDGF-A promoter (Qin et al. 2007). How specific this inhibition is and whether this can be used to shut down PDGF-A expression in vivo remain to be established.
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Developmental functions of PDGFs/PDGFRs |
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), lung alveolar SMCs (Boström et al. 1996), and gastrointestinal villus cluster cells (Karlsson et al. 2000) were all pinpointed for the first time through the analysis of PDGF-B and PDGF-A knockout mice, respectively. Important developmental roles for PDGF/PDGFR-like proteins have also been demonstrated in nonmammalian vertebrates. As discussed below, some of these appear to be analogous to the roles of the mammalian PDGFs and VEGFs. |
Role of PDGF signaling in gastrulation and formation of cranial and cardiac neural crest |
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have early developmental functions. Xenopus embryos injected with dominant-negative PDGF receptor mRNA show aberrant gastrulation, in which the involuting prospective head mesodermal cells fail to migrate beneath the overlying PDGF-A-positive blastocoel roof ectoderm (Ataliotis et al. 1995). Upon PDGFR inhibition, the mesodermal cells detach from the ectoderm and undergo apoptosis. Rescue from apoptosis did not rescue the migration defect, suggesting that PDGFR(s) independently controls both processes (Van Stry et al. 2004). In contrast to these effects, inhibition of PDGFR signaling did not affect the migration and survival of axial mesoderm in Xenopus. Axial mesoderm develops through a different morphogenetic process (mediolateral intercalation) compared with head mesoderm and at a distance from (and therefore probably independent of) the PDGF-A-expressing ectoderm (Van Stry et al. 2004).
A role for PDGF signaling during gastrulation is also suggested from experiments in sea urchins. PDGFR expression occurs in the developing mesoderm, and different strategies to inhibit PDGF or PDGFR function result in gastrulation defects (Ramachandran et al. 1995, 1997). A role for PDGF signaling in the mesoderm during zebrafish gastrulation has also been proposed (Montero and Heisenberg 2004). The PDGFR-specific kinase inhibitor AG1296 inhibited formation of mesendodermal cell protrusions and cell polarization in vivo in fish embryos. In vitro, PDGF-A stimulated these activities (Montero and Heisenberg 2004). Taken together, data from frog, sea urchin, and fish suggest an evolutionary conserved role for PDGF signaling during gastrulation in deuterostomes.
In mouse embryo development, PDGF-A expression onsets early and can be detected already in the preimplantation embryo (Rappolee et al. 1988; Mercola et al. 1990; Palmieri et al. 1992). At this stage, PDGF-A is coexpressed with PDGFR- in the blastocyst inner cell mass. In spite of these early expression patterns, a role for PDGFs during mouse gastrulation is not apparent (Hoch and Soriano 2003). PDGF-A and PDGFR--null mouse embryos nevertheless show severe impairment of early mesenchymal derivatives in both embryo and extraembryonic tissues (Hamilton et al. 2003). A proportion of these embryos die before or at embryonic day 10.5 (E10.5), which is, however, variable and genetic background-dependent (Hoch and Soriano 2003).
Although early developmental routes may differ between vertebrate species, some of the later consequences of gastrulation defects in Xenopus resemble the phenotypes observed in certain PDGF/R mutant mice. PDGFR signaling-inhibited Xenopus embryos develop reduced anterior structures and defective vertebral arch closure (spina bifida). Similar defects are observed in mouse PDGFR- knockouts and PDGF-A/C double knockouts due to problems with the developing cranial neural crest and axial skeleton. Neural crest mesenchyme expresses PDGFR- strongly in mouse (Morrison-Graham et al. 1992), zebrafish (Liu et al. 2002a), and Xenopus (Ho et al. 1994). Complete or neural crest-specific PDGFR- knockout in mice leads to severe developmental defects in neural crest mesenchyme derivatives, such as the cardiac outflow tract, the thymus, and the skeletal components of the facial region (Morrison-Graham et al. 1992; Soriano 1997; Tallquist and Soriano 2003). Both PDGF-A and PDGF-C are expressed at locations toward which neural crest cells migrate, such as the branchial arch epithelium (Orr-Urtreger and Lonai 1992; Ding et al. 2000; Aase et al. 2002; Liu et al. 2002b). For cardiac neural crest development, roles for both PDGFR- and PDGFR-β have been demonstrated (Richarte et al. 2007). PDGFR- is important mainly for nonneuronal cardiac neural crest development (Morrison-Graham et al. 1992). PDGFR-β and PDGF-B appear to play a role in neuronal cardiac neural crest development, as both PDGFR-β and PDGF-B knockouts display abnormal cardiac innervation (Van den Akker et al. 2008). Thus, PDGFR- and PDGFR-β signaling appear to have complementary roles in cardiac neural crest development.
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Directed cell migration—a conserved morphogenetic function of PDGFs |
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ablation in mice, the cellular processes that depend on PDGFR- signaling as well as the critical signaling pathways engaged appear to be similar (Soriano 1997; Van Stry et al. 2004). For example, signaling through PI3K downstream from PDGFR- appears to be critical during early embryonic development in mouse (Klinghoffer et al. 2002), Xenopus (Symes and Mercola 1996), and zebrafish (Montero et al. 2003).
Cell migration is a well-documented effect of PDGF stimulation in vitro (Heldin and Westermark 1999), but our understanding of PDGF-dependent cell movements in vivo is limited. PDGF signaling is undoubtedly required for the spreading of various populations of cells in developmental processes. This includes spreading of oligodendrocyte precursors in the spinal cord, of neural crest mesenchymal cells toward the branchial pouches and cardiac outflow tract, and of pericytes along newly formed angiogenic sprouts. The mechanisms by which the cells spread, and how PDGF regulates this process, remain obscure for most of the situations, however. Recent studies in Drosophila, Xenopus, and mouse have demonstrated a role for PDGF/VEGF family members in directed cell migration in at least some developmental processes in vivo.
The mechanism of PDGF-A/PDGFR--mediated mesodermal cell migration has been analyzed in some detail in Xenopus (Nagel et al. 2004). Using an in vitro explant model and a range of genetic tools, it was demonstrated that the blastocoel roof establishes a matrix-bound gradient of PDGF-A (long splice version) along which the PDGFR--positive mesodermal cells migrate directionally (Fig. 6). When graded signaling was disrupted experimentally, directional migration by mesodermal cells toward the animal pole was replaced by random migration (Nagel et al. 2004). This role for PDGF-A during Xenopus gastrulation resembles that of Drosophila PVF1 in the guidance of border cell migration in the egg chamber (Duchek et al. 2001). Also, there signaling is paracrine, with PVF1 expressed in the oocyte and PVR in the border cells. The border cells move as an aggregate from which a single cell takes a lead position by extending a long cellular protrusion in the direction of the migration of the cell aggregate. The directionality of this process depends on spatially graded signaling exerted by PVF1 (Fulga and Rørth 2002) in concert with the Drosophila EGFR ligands Keren and Spitz (McDonald et al. 2006). Specific misexpression of PVF1 misdirects the border cells to new locations (McDonald et al. 2003). However, uniform misexpression of PVF1 abolishes formation of the cellular protrusion and disrupts directionality of the migration, similar to the effect of uniform PDGF-A misexpression in the Xenopus blastocoel roof. These two examples of PDGF/PVF-induced cell migration are analogous to the role of VEGF-A in endothelial sprout guidance during angiogenesis. There, graded distribution of VEGF-A directs filopodial extensions and migratory behavior of endothelial tip cells, resulting in proper orientation of the angiogenic sprouts (Ruhrberg et al. 2002; Gerhardt et al. 2003). Thus, the ability to guide cell migration through the formation of growth factor gradients in the extracellular space appears to be common to several members of the PDGF/VEGF family, and conserved between vertebrates and invertebrates (Fig. 6).
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PDGF signaling in organogenesis |
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 TopAbstractThe PDGF/VEGF family of... |
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Abstract
The PDGF/VEGF family of...
