REVIEW ARTICLE Structural and functional specificities of PDGF-C and PDGF-D, the novel members of the platelet-derived growth factors family Laila J. Reigstad 1,2 , Jan E. Varhaug 2,3 and Johan R. Lillehaug 1 1 Department of Molecular Biology, University of Bergen, Norway 2 Department of Surgical Sciences, University of Bergen, Norway 3 Haukeland University Hospital, Bergen, Norway Introduction The platelet-derived growth factors PDGF-A and -B have since the late 1970s been recognized as important factors regulating embryonic development, differenti- ation, cell growth and many diseases including malig- nancies. The PDGFs have been classified as members of the superfamily of growth factors characterized by the strongly conserved pattern of six cysteine residues making up intra- and intermonomer disulfide bridges, the cystine knot family of proteins [1–3]. Examples of cystine knot subfamilies are the glycoprotein hormone family [4], the cyclotide family [5,6], and the TGFb family and NGF family [2]. Extended information about subfamilies can be obtained in the Cystine Knot Database (http://hormone.stanford.edu/cystine-knot). This review focuses on the structure and function of the two novel members, PDGF-C and -D, of the PDGF subfamily of the cystine knot superfamily. The PDGFs show high sequence identity with the vascular Keywords PDGF; cystine knot; CUB; growth factor domain Correspondence J. R. Lillehaug, Department of Molecular Biology, University of Bergen, Post Box 7800, 5020 Bergen, Norway Fax: +47 55 58 96 83 Tel: +47 55 58 64 21 E-mail: johan.lillehaug@mbi.uib.no (Received 15 July 2005, revised 19 September 2005, accepted 22 September 2005) doi:10.1111/j.1742-4658.2005.04989.x 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 fam- ily 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 struc- tural (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. Abbreviations CNS, central nervous system; CUB, Clr ⁄ Cls, urchin EGF-like protein and bone morphogenic protein 1; CVB3, coxsackievirus B3; EGF, endothelial growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor. FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS 5723 endothelial growth factors (VEGF) and the family is therefore often referred to as the PDGF ⁄ VEGF family. The PDGF family of growth factors The PDGF family consists of PDGF-A, -B, -C and -D [7–12]. The cystine knot motif of the four PDGFs contains two disulfide bridges linking the antiparallel strands of the peptide chain forming a ring penetrated by the third bridge [3]. This forces the protein to adapt a three-dimensional arrangement that partly exposes hydrophobic residues to the aqueous surroundings, leading to the formation of either homo- or hetero- dimers (PDGF-AA, -AB, -BB, -CC, -DD) [13,14]. In addition to a conserved cystine knot motif, these four growth factors show a high sequence identity. The four PDGFs are inactive in their monomeric forms. They share the same receptors; the PDGF receptor-a and -b. These receptors dimerize when the dimeric PDGF binds. The receptors may combine to generate homo- or heterodimers, resulting in three possible combina- tions, PDGFR-aa,-ab and -bb, having different affinities towards the four PDGFs. All PDGFs play important roles in embryogenesis and adult maintenance, in addition to participating in the phenotypes of various diseases and malignancies. The novel PDGFs are both involved in progressive renal diseases, glioblastomas, medulloblastomas and fibrosis of a variety of tissues. PDGF-C appears to play an important role in Ewing family sarcomas, while PDGF-D is linked to lung, prostate and ovarian cancers. Discovery of the PDGFs PDGF-A and PDGF-B have been extensively studied since the 1970s, while PDGF-C and PDGF-D were discovered recently. The PDGF-c gene was published in 2000 by three independent groups and named PDGF-C [8], Fallotein [15] and SCDGF [7]. In each case, the discovery was based on the identification of a cDNA sequence showing similarity to members in the PDGF ⁄ VEGF family of growth factors. PDGF-c and PDGF-C are now the accepted name for this gene and protein, respectively. The last member of the PDGF family was published in 2001, again by three independent groups, and named PDGF-D and SCDGF-B [10–12]. The gene was identified by BLAST search in the EST database for homologues of the PDGF ⁄ VEGF family. PDGF-d and PDGF-D are now the accepted names for this gene and protein, respectively. The pdgf genes PDGF-c and PDGF-d were named and placed in the PDGF ⁄ VEGF family because they encode the highly conserved cystine knot motif characteristic of the growth factor family. While the classical PDGF-a and PDGF-b mainly encode the growth factor domain, PDGF-c and PDGF-d encode a unique two-domain structure with an N-terminal ‘Clr ⁄ Cls, urchin endo- thelial growth factor (EGF)-like protein and bone morphogenic protein 1’ (CUB) domain [16] in addition to the C-terminal growth factor domain (Fig. 1A). The pdgf genes are located on four different chromo- somes; PDGF-a and -b on chromosomes 7 and 22 [17,18], and PDGF-c and -d on chromosomes 4 and 11 [19], respectively. The genomic organization of the pdgf genes is quite similar, although PDGF-c and -d genes are significantly longer due to large intron sizes and cover about 200 kb compared to approximately 20 kb for PDGF-a and -b [19–21]. Each of the four pdgf genes contains a long 5¢ untranslated region and a verified (PDGF-a and -b)or putative (PDGF-c and -d) signal peptide in exon 1 (Fig. 1B). In the PDGF-c and -d genes, exons 2 and 3 encode the CUB domains, while in PDGF-a and -b these exons encode precursor sequences residing 5¢ to the cystine knot encoding sequence. The hinge regions of PDGF-c and PDGF-d connecting the CUB and the cystine knot domains are encoded by exons 4 and 5, respectively. These hinge region sequences encode con- served basic motifs and similar motifs are found in PDGF-A and -B. The motifs are identified as proteo- lytic cleavage sites for proteases used in post-transla- tional protein processing. Exon 4 of PDGF-d encodes a unique sequence not present in the other PDGFs. So far, no function has been assigned to this sequence. The exons in PDGF-c (exons 5 and 6) and PDGF-d (exons 6 and 7) encoding the cystine knot motifs resemble the corresponding exons in the PDGF-a and -b (exons 4 and 5) genes. Both in PDGF-a and -b, exon 6 encodes a C-terminal basic retention sequence that may be removed during the maturing and release of the proteins. Analysis on the 3¢ untranslated mRNA region of PDGF-C identified five adenylate ⁄ uridylate- rich elements, these being the best characterized mam- malian determinant for highly unstable RNAs [15]. The pdgf promoters PDGF-A and -C share common mechanisms of gene regulation. Their expression is controlled by the zinc finger transcription factors Egr1 and Sp1, which have affinity for overlapping GC-rich binding sites in the PDGF-C and -D, structure and function L. J. Reigstad et al. 5724 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS proximal region of the PDGF-a and PDGF-b promot- ers [22,23]. So far, no information on the PDGF-d promoter has been published, but functional character- istics of the human PDGF-c promoter has been repor- ted [24]. Comparison of the PDGF-c promoter of human smooth muscle cells with the characterized human PDGF-a promoter identified a GC-rich sequence ()35 to )1) in PDGF-c, with high similarity to the )76 to )47 sequence of the PDGF-a promoter. Both Egr1 and Sp1 were shown to bind the 35 bp sequence of the PDGF-c promoter. FGF-2 stimulates Egr1 expression through the Erk ⁄ MAPK pathway, and Egr1 translocates to the nucleus where it binds to the proximal PDGF-c promoter resulting in increased PDGF-C expression. Alternative splicing of the PDGFs No alternative splicing of PDGF-c mRNA has been demonstrated. However, alternative splicing is sugges- ted because two shorter PDGF-C cDNAs have been obtained [25]. Based on the variant PDGF-c sequences isolated by PCR, the splice donor ⁄ acceptor sites are located to nucleotides 719 ⁄ 720 and 988 ⁄ 989, resulting in two alternative proteins; one short variant encom- passing almost only the CUB domain, and the longer variant containing the CUB domain and the final 30 residues in the C-terminal end of the growth factor domain. These splice variants are also present in human thyroid papillary carcinomas (L. J. Reigstad, J. E. Varhaug and J. R. Lillehaug, unpublished results). Based on mRNA analysis of PDGF-d, splice variants have been reported to be present in mouse heart, liver and kidney [26]. Interestingly, deletion of exon 6 cau- ses a frame shift and an early stop codon in exon 7, resulting in a protein lacking the growth factor domain and without mitogenic activity (Fig. 1B). The PDGF- D protein encoded by this splice variant could only be detected in mouse tissues and not in human cell lines or tissues. A second PDGF-d RNA splice variant lacks A B Fig. 1. PDGF protein and gene structure. (A) Schematic drawing of the four full-length PDGF proteins. In PDGF-C and -D, the hydrophobic putative N-terminal signal pep- tide (black) is separated from the N-terminal CUB domain (110 residues, red) by a short region (orange). A hinge region (blue) separ- ates the CUB domain from the C-terminal growth factor domain containing the cystine knot motif (115 residues, yellow). PDGF-A can be alternatively spliced and carries two stop codons resulting in proteins of 198 and 211 amino acid residues. The numbers show residue numbering in the PDGFs. (B) Schematic drawing of gene structures encoding the four PDGF polypeptide chains. Exons are coloured and numbered: CUB domain (red), hinge region (blue) and growth factor domain (yellow). The introns are shown in white. Start codons (ATG), stop codons (Stop) and the proteolytic cleavage sites (black arrows) are denoted. Exons and introns are not drawn in scale. The PDGF-A and -B genes cover approximately 20 kb and the PDGF-C and -D cover approximately 200 kb. Alternative splicing has been shown for PDGF-A and PDGF-D, in which exon 6 is missing (see text). L. J. Reigstad et al. PDGF-C and -D, structure and function FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS 5725 18 bp within the CUB domain of both mouse and human PDGF-D mRNA [26,27]. In the case of PDGF-a, exon 6 may be present or not resulting in two splice variants encoding a long and a short PDGF-A protein (Fig. 1B) [28]. PDGF-B mRNA has not been reported to be alternatively spliced. The PDGF proteins The PDGF-A and -B proteins contain only the growth factor domains whereas PDGF-C and -D have a unique two-domain structure containing the N-ter- minal CUB domain separated from the C-terminal growth factor domain by a hinge region (Fig. 1A). PDGF-C and -D share an overall sequence identity of 42% with highest similarity in the CUB and cystine knot-containing growth factor domain, whereas the hinge region and the N-terminal region show less identity [10,12]. While PDGF-A and -B can form both homo- and heterodimers (PDGF-AA, -AB, -BB), PDGF-C and -D exist only as homodimers (PDGF-CC and -DD). The full-length PDGF-C and -D monomers are 54–55 kDa and 49–56 kDa, respectively, differing from their theo- retical sizes of 39 and 43 kDa based on their amino acid sequences [8,12,29]. The divergence from the theoretical values indicates that PDGF-C and -D may be post-translationally modified. In addition to being secreted to the extracellular space, the PDGF-C protein is shown to be constitutively expressed in the cytoplasm in rat smooth muscle cells residing in arteries and arterioles [30]. Additionally, our data (L. J. Reigstad, J. E. Varhaug and J. R. Lillehaug, unpublished results) show full-length PDGF-C to be present in both the cytoplasm and nucleus, a feature also described for PDGF-B [31,32]. The function of these two PDGFs in the nucleus is unclear. The region N-terminal of the CUB domain in PDGF-C and PDGF-D The N-terminal ends of both PDGF-C and -D contain a hydrophobic sequence predicted to be a signal pep- tide with a putative peptidase cleavage site between residues 22 and 23 [8,10,12]. When part of the putative PDGF-C signal sequence was deleted no mitogenic activity was detected, suggesting that PDGF-C is secre- ted by the aid of this N-terminal region [7]. The sequence between residues 23 and 50 of PDGF-C, and residues 23–56 of PDGF-D, contain no known motifs or domains (Fig. 1). The CUB domain of PDGF-C and PDGF-D The CUB domain was first identified in complement subcomponents Clr ⁄ Cls, urchin EGF-like protein and bone morphogenic protein 1 [16]. These proteins are often referred to as the prototype CUB domains. Like the prototype CUB domains, the PDGF-C and -D CUB domains span approximately 110 residues and show 27–37% and 29–32% sequence identity to the prototypic CUB domains, respectively (Fig. 2) [8]. The CUB domains of PDGF-C and -D share  55% sequence identity. CUB domains are assembled as a Fig. 2. The CUB domain. The CUB domains of PDGF-C and PDGF-D aligned with the prototype CUB domains of human neuropilin (acces- sion no CAI40251) and human bone morphogenic protein-1 (BMP-1, accession no CAA69974). Red, squared areas highlight sequence iden- tity among the sequences. The four cysteines conserved in the prototypical CUB domains are labelled in yellow. The two cysteines missing in the PDGF-C and PDGF-D are marked by red circles while the two cysteines present are marked in blue circles. The two cysteines of PDGF-C (accession no AAF80597) correspond to Cys104 and 124, whereas in PDGF-D (accession no AAK38840) these cysteines are Cys109 and 131. PDGF-C and -D, structure and function L. J. Reigstad et al. 5726 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS compact ellipsoidal b-sandwich, with a hydrophobic core essential for the overall domain folding. The b-sandwich is built up of two five-stranded b-sheets of antiparallel b-strands [33–36]. Most CUB domains are reported to contain four conserved cysteines that form two disulfide bridges between nearest-neighbour cyste- ines, resulting in disulfide bridges located on opposite edges of the domain. As both the PDGF-C and -D CUB domains contain only two cysteines [7] which, compared to the classical CUB domains, are the two most-C-terminally located cysteines, the CUB domains of PDGF-C and -D may have only one disulfide bridge. At present it is unclear how this may influence their 3D structure. In the two crystallised CUB domains of a serine protease associated with serum mannose-binding proteins (MAPS), the N-terminally located CUB domain contains only one disulfide bridge, while the second CUB domain of MAPS has two bridges. One disulfide bridge instead of two may result in a slightly less tight b-sandwich in the N-ter- minally located CUB domain, but the structural and functional significance of only one bridge remains unknown [35]. The CUB domain is found in several extracellular proteins, many involved in development, and they are thought to mediate protein–protein and protein–carbo- hydrate interactions, in addition to binding to low- molecular-mass ligands [16,37]. Several reports state that the CUB domains of PDGF-C and -D have to be cleaved extacellularly to make the C-terminal growth factor domains active [8,10,12]. The CUB domains are believed to prevent PDGF-C and -D binding to their receptors by structurally blocking receptor-binding res- idues of the growth factor domain. In contrast, two reports state that full-length PDGF-C and -D exhibit in vitro mitogenic activity towards coronary artery smooth muscle cells and fibroblasts [25,26]. Addition- ally, the CUB domain of PDGF-C exhibits mitogenic activity on human coronary artery cells independent of the presence of its growth factor domain, suggesting a possible biological activity of the CUB domains them- selves [25]. Interestingly, when Cys124 of the PDGF-C CUB domain was mutated to serine, the mitogenic activity of CUB was reduced by approximately 50%. The mitogenic CUB activity could not be confirmed in transgenic mouse hearts overexpressing CUB and over- expression gave no pathological effect in the heart [38]. CUB domains may facilitate unique, undiscovered functions of full-length PDGF-C and -D. This is reflec- ted in a report on full-length PDGF-D of eye lens tis- sue, in which the secreted PDGF-D does not appear to be proteolytically cleaved [39]. The CUB may mediate interactions between PDGF-C or PDGF-D and elements of the extracellular or pericellular matrix. Furthermore, a role for the PDGF CUB domains in receptor binding is suggested based on studies of the transmembrane receptor, neuropilin-1, which consists of two CUB domains and a coagulation factor domain, acting as coreceptors for VEGF-A and sem- aphorins (reviewed in [40]). Crystallography studies of the MAPS protein containing two CUB domains sug- gests that CUB domains may also participate in pro- tein heterodimer formations [35]. The hinge region and proteolytic cleavage for growth factor activation The hinge regions of PDGF-C and -D, separating the CUB and the growth factor domains (Fig. 1), show no homology to known sequences [41] but contain dibasic cleavage sites for proteolytic removal of the CUB domains and thereby activation of the growth factor domains. PDGF-C and -D contain both the CUB and growth factor domains when they are secreted and pro- teolytic cleavage is therefore suggested to take place extracellularly. Plasmin cleaves PDGF-C at RKSR234 [8,41], and PDGF-D at RKSK257 [12]. Tissue plasmi- nogen activatior (tPA) cleaved PDGF-C at RKSR234 in vivo [42,43] and urokinase plasminogen activator (uPA) was found to cleave PDGF-D at RGRS250, thereby activating this growth factor [44]. PDGF-A is cleaved by furin at RRKR86 [45], while PDGF-B is cleaved at RGRR81 by a still unidentified protease [46]. The PDGF growth factor domain The determination of the crystal structure of nerve growth factor [47], transforming growth factor b2 [48], PDGF-BB [49] and chorionic gonadotropin [4] revealed unexpected topological similarities among these four proteins belonging to separate families of growth factors. Despite very little sequence similarity, they all contain an unusual arrangement of cysteines linked in disulfide bridges to form a conserved cystine knot motif [1,2,50]. The cystine knot is located in a conserved b-sheet structure referred to as the growth factor domain. Although the four growth factor super- families have a common topology, they differ in the number of disulfide bonds, the interfaces used to form the dimers, and the way in which the monomers dimer- ize [1,2]. In the PDGF ⁄ VEGF family, the crystal structures of PDGF-BB [49], VEGF-AA [51,52], VEGF-AA together with elements of its Flt1 receptor [53], and PlGF-1 dimer [54] have been solved at 3.0, 1.9, 1.7 and 2.0 A ˚ , respectively. Characteristic for these growth L. J. Reigstad et al. PDGF-C and -D, structure and function FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS 5727 factor domains are two long, highly twisted antiparal- lel pairs of b-strands in an antiparallel side-by-side mode. They all contain eight (I–VIII) highly conserved cysteines. The monomeric antiparallel b-strands are connected by three loops, referred to as loops 1, 2 and 3 (Fig. 3A), and due to the head-to-tail arrangement, loop 2 of one monomer will be close to loops 1 and 3 of the other monomer when the dimer is formed (Fig. 3B). Six of the conserved cysteines are engaged in three intrachain disulfide bonds (Cys I-VI, III-VII, V-VIII) stabilizing the cystine knot structure, while two cysteines (Cys II and IV) are involved in inter- chain disulfide bonds (Fig. 3C,D) [55,56]. The three intrachain disulfide bonds makes the cystine knot very Fig. 3. The PDGF-C growth factor domain. Ribbon presentations of the proposed PDGF-C model [57] displaying the twisted b-sheets and the N-terminal a-helix of the PDGF-C monomer (A) and the PDGF-CC dimer (B). The N-terminal (N) and C-terminal (C) ends for the monomer are marked. The three loops (loop 1-2-3) connecting the b-strands are labelled. (C, D) Sequence alignments of the growth factor domains of PDGF-A (accession no P15692), PDGF-B (accession no. 1109245 A), PDGF-C (acces- sion no AAF80597), PDGF-D (accession no AAK38840), VEGF-A (accession no NP003367) and PIGF-1 (accession no. 1FZV). Red, squared areas show sequence identity among the sequences. The eight conserved cysteines are shown in yellow. The extra cysteines of PDGF-C and -D are labelled in blue. The green squares highlight the area of disagreement in sequence align- ment (see text). (C) Sequence alignment of PDGF ⁄ VEGF family members where the green square highlights the area containing the insert of three residues between con- served cysteines III and IV in both PDGF-C and -D, and that PDGF-D is missing the con- served cysteine V. (D) Sequence alignment of PDGF ⁄ VEGF family members showing PDGF-C and -D to contain all eight con- served cysteines and have an insert of three residue between cysteine V and VI (green area). PDGF-C and -D, structure and function L. J. Reigstad et al. 5728 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS stable as the first (Cys I-VI) and second (Cys III-VII) disulfide bonds link two adjacent b-strands, making a ring which is penetrated by the third (Cys V-VIII) disulfide bond, covalently connecting two further b-strands [49,54]. Additional stability to the dimer structure is the extensive hydrophobic core formed by residues from both monomers. The PDGF-C growth factor domain shares 27–35% sequence identity with the rest of the PDGF ⁄ VEGF family [8], and with specific reference to the other three PDGFs the identity is nearly 25% [20]. The growth factor domains of PDGF-A and -B show approxi- mately 50% sequence identity, while the identity between the domains of PDGF-C and -D is also nearly 50%. Compared to the eight conserved cysteines in PDGF- A and -B, PDGF-C contains four and PDGF-D two additional cysteines. These extra cysteines and the lack of solved PDGF-C and -D 3D structures makes identification of the cysteines that participate in the conserved disulfide bridges difficult. Because of this, two different sequence alignments of PDGF-C and -D covering the area of conserved cysteines III to VI are included here (Fig. 3C,D). Figure 3C shows the align- ment of PDGF-C and -D to allow three residues (NCA and NCG, respectively) between conserved cysteines III and IV, an insert not present in the other members of the PDGF ⁄ VEGF family. This alignment also indicates that PDGF-D lacks the conserved cys- teine V [7,8,10,12,15,25,57]. The alignment in Fig. 3D has a different three-residue insert, which is located between conserved cysteines V and VI. In this align- ment, PDGF-D contains all eight conserved cysteines of the cystine knot motif [20,21,41]. Crystallization or NMR studies of PDGF-C and -D proteins will resolve this debate, but our published 3D model of the PDGF-C growth factor domain indicates the disulfide bridges in PDGF-C to consist of Cys250 and 294, Cys280 and 335, and Cys287 and 337, and the inter- monomeric bonds to consist of Cys274 and 286 [57]. At present, analysis of PDGF ⁄ VEGF domains show that PDGF-C is more similar to VEGFs than PDGFs [8,15,57], all in all favouring the alignment in Fig. 3C. The region C-terminal of the growth factor domain In PDGF-A and -B, the C-terminal regions contain a basic sequence with a dual function. First, the sequence mediates electrostatic interactions with com- ponents of the extracellular matrix such as heparin [58] and collagens [59]. Second, the basic sequence may cause retention of the growth factors within the produ- cer cell [60]. PDGF-C is shown to have a heparin- binding domain in the C-terminal region of the growth factor domain but the exact residues responsible for the binding have not been identified [25]. PDGF-D has not been shown to bind heparin. Post-translational modifications and regulations of PDGFs Several members of the PDGF family are predicted to have potential N-glycosylation sites. PDGF-C has three predicted N-glycosylation sites (N25, 55, 254), the last residing in the growth factor domain [8]. Due to the difference in expected (39 kDa) and observed (55 kDa) relative molecular mass as determined by SDS ⁄ PAGE electrophoretic mobility, it has been sug- gested that PDGF-C may be glycosylated. One report gives experimental indication of glycosylation, recom- binant full-length PDGF-C protein, secreted from insect cells, slightly changed mobility when treated with N-glycosidase F, but the mobility change did not result in the expected 39 kDa protein size [25]. PDGF- D has one predicted N-glycosylation site at N276 located in the growth factor domain [12] and PDGF-B has a verified N-glycosylation site at N63 [61]. PDGF- A has one predicted N-glycosylation site at N134 but is not reported to be N-glycosylated [62], despite a report from 1981 in which PDGF was stated to be glycosylated [63]. In the case of PDGF-C, the post- translational modification remains unidentified but SUMOylation or ubiquitinylation may be candidates. Receptor binding of PDGFs The PDGFs bind to the protein tyrosine kinase receptors PDGF receptor-a and -b. These two recep- tor isoforms dimerize upon binding the PDGF dimer, leading to three possible receptor combina- tions, namely -aa,-bb and -ab. The extracellular region of the receptor consists of five immunoglo- bulin-like domains while the intracellular part is a tyrosine kinase domain. The ligand-binding sites of the receptors are located to the three first immuno- globulin-like domains (reviewed in [64]). The residues in PDGF-A and -B responsible for receptor binding reside in loop 2, in addition to RKK161 in PDGF- AA and R27 and I30 in PDGF-BB. The residues involved in PDGF-CC and -DD receptor binding remain to be identified, but our published 3D model of PDGF-C suggests, when compared to the crystal structure of VEGF-AA complexed to domain 2 of its receptor, that the region containing residues W271 and LR312 might be involved [57]. L. J. Reigstad et al. PDGF-C and -D, structure and function FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS 5729 PDGF-CC specifically interacts with PDGFR-aa and -ab, but not with -bb, and thereby resembles PDGF- AB [8,41]. PDGF-DD binds to PDGFR-bb with high affinity, and to PDGFR-ab to a markedly lower extent and is therefore regarded as PDGFR-bb specific [10,12]. PDGF-AA binds only to PDGFR-aa, while PDGF-BB is the only PDGF that can bind all three receptor combinations with high affinity [65]. Both PDGF-CC and -DD activate PDGFRs resulting in downstream phosphorylation of extracellular signal-regulated pro- tein kinase ⁄ mitogen-activated protein kinase (Erk⁄ MAPK) and Akt ⁄ PKB pathways [57,66,67]. Fine tuning of PDGF and PDGFR isoform expression and regulation Expression of both receptors and each of the four PDGFs is under independent control, giving the PDGF ⁄ PDGFR system a high degree of combinatorial flexibility. To understand how the four PDGFs may generate different biological signals, five observations may be relevant. First, different cell types vary greatly in the ratio of PDGF isoforms and PDGFRs expressed. Second, the PDGFR expression levels are not constant. Different external stimuli such as inflammation, embry- onic development or differentiation modulate cellular receptor expression allowing binding of some PDGFs but not others. Additionally, some cells display only one of the PDGFR isoforms while other cells express both isoforms, simultaneously or separately. Third, different splice forms of the PDGFs appear to be expressed dif- ferently, as shown for the two PDGF-A proteins in rest- ing and active monocytes [68] and as indicated for the two PDGF-D proteins identified in mouse but not humans [26]. Fourth, regulation of the classical PDGFs after secretion includes covalent binding to the extracel- lular secreted protein, acidic and rich in cysteine (SPARC), which only binds PDGF-AB and -BB, decreasing their reactive concentrations and favouring PDGF-AA signalling [69]. Data on possible PDGF-CC or -DD binding to SPARC have not been reported. The major reversible PDGF-A and -B binding to extracellu- lar protein is a 2 -macroglobulin [70]. The PDGF–a 2 - macroglobulin complex serves multiple functions. It makes PDGF-AA, -AB and -BB unable to bind their receptors, it protects the PDGFs against proteolytic degradation, and may remove the PDGFs from circula- tion via a 2 -macroglobulin receptors. There are no data currently available about interactions between the novel PDGFs and a 2 -macroglobulin, but several other growth factors, such as FGF-2, TGF-b and TNF-a, also bind a 2 -macroglobulin. Fifth, the expression of highly speci- fic proteases that proteolytically activate the PDGFs will also influence the availability and activity of the dif- ferent isoforms. This can be exemplified by the proteo- lytic cleavage of PDGF-D. While the human prostate carcinoma cell line LNCaP produces a specific protease to process the full-length PDGF-D [66], there is no pro- tease capable of cleaving the full-length PDGF-D secre- ted by cells and tissues in the eye [39]. Gene knockout studies on the PDGFs For the PDGF-a gene knockout mouse, there are two restriction points concerning animal survival; one pren- atally at E10 and one postnatally at about two weeks [71]. The postnatally surviving mice had a symmetrical reduction of the size of most organs, developed lung emphysema due to lack of alveolar myofibroblasts, resulting in the loss of parenchymal elastin fibres and no formation of alveolar septa. The mice died about two weeks old due to respiratory problems. The phe- notype reveals a role for PDGF-A in embryonic devel- opment, as well as a highly specific and critical role for PDGF-A in lung alveolar myofibroblast differentiation and lung development. When the PDGF-b gene is knocked out the mice die perinatally, displaying several anatomical and histolog- ical abnormalities [72]. The glomerular tufts of the kidneys do not form as there is complete absence of mesangial cells, and instead one single or a few disten- ded capillary loops fill the glomerular space. Further- more, the heart and some large arteries dilate in late-stage embryos and fatal haemorrhages occur just prior to birth. Based on these findings, PDGF-B is assigned a crucial role in establishing certain renal and circulatory functions. Comparing the PDGF-a and PDGF-b knockout mice, there are similarities in the resulting phenotypes. Both the alveolar myofibroblasts and the mesangial cells express a-smooth muscle actin and have a con- tractile phenotype, functioning as anchors for an involuted epithelial sheet, the alveolar sac or the glom- erulus. By losing this anchor in the knockout mice, there is a failure of involution and the physiological functions are impaired, as a result of decreased surface area for gas exchange or glomerular filtration, in PDGF-A and PDGF-B mutants, respectively. Knockout studies on PDGF-c in mice clearly dem- onstrate a role for PDGF-C in embryonal development [73]. The knockout of PDGF-c results in mice dying perinatally owing to difficulties in feeding and brea- thing, as they have a complete cleft of the secondary palate because the palate bones do not meet. Addition- ally, the dorsal spinal cord was deformed in the lower spine. The null mutant PDGF-C embryos had PDGF-C and -D, structure and function L. J. Reigstad et al. 5730 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS subcutaneous oedema in the flank of the body between the limbs lacking connective tissue, and showed several blood-filled blisters in frontnasal and lateral forehead. In the early embryo development, the features of the knockout PDGF-c mice largely overlap with knockout PDGF-a mice. PDGF-c ⁄ PDGF-a mice showed growth retardation, pericardial effusion, a wavy neural tube and subepidermal blisters, dying before E17. In total, PDGF-C has specific roles in palatogenesis and in morphogenesis of the skin tissue. PDGF-d knockouts have not been reported. Functions of the PDGF-C and PDGF-D proteins The role of PDGF-A and -B proteins in normal pro- cesses, malignancies and diseases have been character- ized in a wide diversity of cells, organs and species (reviewed in [62]). This part of the review will therefore focus on the PDGF-C and PDGF-D proteins, as their functions are starting to be revealed. PDGF-C in normal processes The expression of PDGF-C mRNA in embryonic mouse tissue is located in the kidney, lung, brain, heart, spinal cord and several other tissues, and partic- ularly at sites of developing epidermal openings such as the mouth, nostrils, ears and eyelids [7–9,74]. In the adult mouse, PDGF-C is mainly expressed in kidney, testis, liver, brain and heart. Adult humans addition- ally express PDGF-C in the pancreas, adrenal gland, skeletal muscles, ovary, prostate, uterus and placenta [8,10,15,20,27,74]. PDGF-C mRNA and protein expression is also detected in normal human thyroid tissue (L. J. Reigstad, J. E. Varhaug and J. R. Lilleh- aug, unpublished results). PDGF-C in tissue remodelling PDGF-C appears to be involved in all three phases (inflammation, proliferation and remodelling ⁄ matur- ing) of wound healing [75]. Extensive expression and secretion of full-length PDGF-C from a-granules of isolated platelets indicate that it plays a role in the inflammatory phase [76]. In the proliferative phase of wound healing capillary growth is triggered by low oxygen, and PDGF-C was recently shown to revascu- larize ischemic mouse heart and limb in vivo as effi- ciently as VEGF and PlGF-1 [77]. PDGF-C mediates increased mRNA and protein levels of metalloprotein- ase-1 (MMP-1) and its inhibitor (TIMP-1), both being important in the remodelling phase of tissues [78]. These results are further verified by in vivo experiments showing that PDGF-C enhanced the repair of a full- thickness skin excision in a delayed diabetic wound healing mouse model by stimulation of fibroblast pro- liferation, epithelial migration, extensive vasculariza- tion and neutrophil infiltration [41]. PDGF-C in angiogenesis The high PDGF-C expression in the angiogenic tissues of placenta, ovary and embryo has led to several in vitro and in vivo experiments defining PDGF-C as a potent angiogenic factor, similar to VEGF and the classical PDGFs. The underlying mechanisms are still to be understood. In the aortic ring outgrowth assay, PDGF- C mediated significant increased outgrowth of fibro- blasts and smooth muscle cells, to a degree comparable to that of VEGF, PDGF-AA and -BB [41]. PDGF-C efficiently stimulated the formation of new blood vessels with high vessel density growing towards the implanted dish of the chorioallantoic membrane (CAM) assay [79]. In addition, PDGF-C stimulated formation of new branches and vessel sprouts from those initially formed. Several reports show in vivo angiogenic PDGF-C effects. When PDGF-C-coated micropellets were added to mouse corneal micropockets, PDGF-C potently induced neovascularization of the avascular corneal tis- sue. In these experiments, PDGF-C was as potent as PDGF-BB and more potent than PDGF-AA. PDGF- C affects the endothelial cells lining the blood vessels by mobilizing the endothelial progenitor cells, promo- ting the differentiation of bone marrow progenitor cells into mature endothelial cells, and by stimulating the chemotaxis of different mature endothelial cells in ischemic heart and limb muscles. In these experiments, PDGF-C also gave enhanced vessel maturing (arterio- genesis) by inducing the differentiation of bone mar- row cells into smooth muscle cells which coat the endothelial cell layer of the vessels. PDGF-C in embryonic development and adults: kidney, central nervous system (CNS) and ears PDGF-C has important functions both in embryonic development and in adult tissues (Fig. 4) and there appears to be expression differences between species. High constitutive PDGF-C expression is present in the adult kidneys of mouse, rat and humans [8,30,80,81]. In human adult kidneys, PDGF-C is detected in vascu- lar endothelial cells and smooth muscle cells of arter- ies, in parietal glomerular cells but not in the glomerular tuft. In the tubulointerstitium, PDGF-C is located in collecting ducts and the loop of Henle [81]. PDGF-C protein localization in adult rodent kidneys L. J. Reigstad et al. PDGF-C and -D, structure and function FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS 5731 was similar to the adult human kidney, but the rodent kidneys did not contain PDGF-C protein in the pari- etal glomeruli cells [30,74,76]. In contrast to kidney development in rodents, the developing human glo- meruli express PDGF-C in the metanephric epithelial mesenchyme and in the parietal epithelial cells [30,81]. During kidney development, PDGFR-a is expressed in the glomerular epithelial mesenchyme, suggesting a paracrine signalling pathway for both PDGF-A and -C in kidney vascular and interstitial development [74]. In the embryonal rat CNS, PDGF-C mRNA was expressed in the notochord (prestage of the spinal cord) and subsequently in the maturing spinal cord, while the adult spinal cord does not express PDGF- C [27]. The presence of PDGF-C in the developing spinal cord has also been shown in chicken [7]. PDGF-C mRNA is detected in the floor plate and the ventricular zones of cortex and adjacent to the floor plate of the embryonic brain, whereas in the adult brain weak PDGF-C expression was observed only in the olfactory nucleus and pontine nuclei [27]. Quantitative RT-PCR analysis did not detect PDGF- C in human embryonic or adult brain tissues [82], although this has been shown through northern blot analyses [8,74]. PDGF-C mRNA has been detected in the develop- ing ears of mouse and rat [9,74,83]. During rat embryonic development, significant mRNA levels of PDGF-C, PDGF-A and both PDGFRs are expressed in cochlear progenitor hair cells of the inner ear [83]. PDGF-D in normal processes Since its discovery four years ago, PDGF-D has been linked to important functions both in embryogenesis and in adult tissues (Fig. 4). In human adult tissue, PDGF-D is highly expressed in heart, kidney, pan- creas, ovary, adipose tissue, stomach, bladder, trachea, testis and mammary gland [10,12,84]. In organs such as the kidney and lung, there are several differences in expression patterns between species. PDGF-D in embryonic development and adults: kidney, lung, CNS and eye Most information on PDGF-D biology has been obtained from studies using the kidney as a model. Starting with embryogenesis, PDGF-D protein in the human kidney is expressed mainly in visceral glomer- ular epithelial cells and in smooth muscle cells in renal arteries and also in some fibroblast-like intersti- tial cells but not in the fibrous capsule surrounding the embryonic kidney [29]. In mouse, PDGF-D is expressed in the highly vascularized fibrous capsule, the most peripheral part of the cortex metanephric mesenchyme, and in the basal aspect of the branch- ing ureter [12]. PDGF-D colocalizes with the PDGFR-b in the differentiating metanephric mesen- chyme, whereas PDGF-B expression is restricted to endothelial cells, indicating the possibility of PDGF- D ⁄ PDGFR-b constituting an autocrine loop, and PDGF-B acting in a paracrine manner to promote proliferation and migration of mesangial and intersti- tial cells in the kidney. In the developing human kidney, PDGF-D expression does not colocalize with PDGFR-b, as PDGF-D is expressed in the visceral epithelial cells and PDGFR-b in the mesangial cells. Thus here a paracrine role for PDGF-D in prolifer- ation and migration of the mesangial cells can be indicated [29]. In the human adult kidney, PDGF-D protein expression was also detected in smooth mus- cle cells of arteries, arterioles and vasa rectae. In contrast to human and mouse adult kidney, the rat adult kidney shows no PDGF-D protein in the glomeruli [85]. As in the kidneys, lungs show spe- cies-different PDGF-D expression. Cells in normal human lungs do not express PDGF-D protein at detectable levels [10], while in murine lungs PDGF-D mRNA is constitutively expressed [84]. In the embryo, PDGF-D mRNA is hardly detect- able in the spinal cord, but in the adult spinal cord prominent expression is located to the motor neurons [27]. In the brain, PDGF-D mRNA was registered in the thalamus and in a ventricular zone of the Kidney (9,30,74,81) CNS (5,27,74) Heart (74,100) Ear (9,74,83) Kidney (30,80,81) CNS (27,74) Embryonic development Adult tissue PDGF-C PDGF-D Kidney (27,29,80,85) Eye (27,39) CNS and brain (27) Lung (84,101) Peri. mineral. (86) Kidney (27,29) Eye (27) Brain (27,110) Fig. 4. Defined functions of PDGF-C and PDGF-D in specific organs during embryonic development and adult tissue. See text and refer- ences (numbers given) for detailed descriptions. Peri. mineral, peri- odontal mineralization. PDGF-C and -D, structure and function L. J. Reigstad et al. 5732 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS [...]... determination of the 3D structures of both PDGFC and -D is lacking The current information on their activities points to a wide scope of biological effects; however further understanding of how these factors interplay with other members of the cystine knot family and in particular the PDGF-A, -B, and VEGF growth factors, must be the focus of future investigations PDGF-C and -D, structure and function... This high level of circulating PDGF-D is also detected in mice infected with adenovirus-containing PDGF-D, resulting in perivascular lymphoid cell infiltrates of the lung and fibrosis in the liver [110] Conclusions The novel members, PDGF-C and -D, of the PDGF subfamily of the cystine knot family of growth factors are potent cytokines important for normal embryogenesis and maintenance of adult tissues... strains developing fibrosis These findings suggest a role for the PDGF-C in the development of lung fibrosis One of the characteristics of pancreatic cancer is the overproduction of extracellular matrix by interstitial cells This results in a tumour mass usually consisting of 60–80% interstitial cells and only 20–40% of pancreatic carcinoma cells [108] PDGF-B and several other growth factors have previously... embryonic brain and in the adult brain it was expressed in several anatomical nuclei PDGF-D mRNA is expressed in the developing rat eyes and also adult eyes of rat, cow, monkey and rabbit In the adult rat eye, PDGF-D is localized to the stroma of iris and ciliar body in the anterior segment, and to the outer plexiform layer of the retina containing the photoreceptor axons, but not in the mature lens... PDGFR-a and -b, TGFb1, and TIMP-1 and -2 in the liver of transgenic mice PDGF-C treatment increased both mRNA and protein levels of the metalloproteinase inhibitor, TIMP-1, in dermal fibroblasts in vitro [78] The PDGF-C induction of fibrosis starts with activation and intense proliferation of the hepatic stellate cells, producing robust pericellular and perivenular collagen type I deposits [106] PDGF-C. .. anchoragedependant growth, and is a potent transforming growth factor of NIH ⁄ 3T3 cells [88] The in vivo tumourigenesis may partially be explained by PDGFC-mediated VEGF expression, promoting indirect stimulation of tumour angiogenesis The apparent roles of PDGF-C in diseases and malignancies are discussed below and are summarized in Fig 5 PDGF-C in Ewing family sarcomas The first link between PDGF-C and malignancy... same pattern was detected for the mRNA expression of both PDGF-A and PDGFR-a Medulloblastoma tumours express both PDGFRs [103] and the PDGF-A, -B and -D [89,104] but an 5734 Fig 5 Involvement of PDGF-C and PDGF-D in diseases and malignancies Both PDGF-C and PDGF-D are linked to progressive renal diseases, fibrosis and brain tumours PDGF-C is furthermore involved in Ewing family sarcoma whereas PDGF-D... protease-activated ligand for the PDGF beta-receptor Nat Cell Biol 3, 512–516 Heldin CH & Westermark B (1990) Signal transduction by the receptors for platelet-derived growth factor J Cell Sci 96, 193–196 Heldin CH, Eriksson U & Ostman A (2002) New members of the platelet-derived growth factor family of mitogens Arch Biochem Biophys 398, 284–290 Tsai YJ, Lee RK, Lin SP & Chen YH (2000) Identification of a novel platelet-derived. .. Eriksson U (2004) The PDGF family: four gene products form five dimeric isoforms Cytokine Growth Factor Rev 15, 197–204 Li X & Eriksson U (2003) Novel PDGF family members: PDGF-C and PDGF-D Cytokine Growth Factor Rev 14, 91–98 Khachigian LM, Williams AJ & Collins T (1995) Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells J Biol... Identification of a cell retention PDGF-C and -D, structure and function 61 62 63 64 65 66 67 68 69 70 71 72 signal in the B-chain of platelet-derived growth factor and in the long splice version of the A-chain Cell Regul 2, 503–512 Kaetzel DM Jr, Morgan D, 3rd Reid JDT & Fenstermaker RA (1996) Site-directed mutagenesis of the N-linked glycosylation site in platelet-derived growth factor B-chain results . REVIEW ARTICLE Structural and functional specificities of PDGF-C and PDGF-D, the novel members of the platelet-derived growth factors family Laila J. Reigstad 1,2 ,. the lung and fibro- sis in the liver [110]. Conclusions The novel members, PDGF-C and -D, of the PDGF subfamily of the cystine knot family of growth factors are