Tài liệu Báo cáo khoa học: Structural and functional specificities of PDGF-C and PDGF-D, the novel members of the platelet-derived growth factors family docx

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 ( />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:
(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
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 rab-
bit. 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 contain-
ing the photoreceptor axons, but not in the mature
lens [39]. In monkey eyes, PDGF-D was detected
in the muscle layers of both ciliar body and iris,
and throughout the epithelial layers. Interestingly,
PDGFR-b is also expressed in the iris, indicating
possibilities of both autocrine and paracrine PDGF-D
activity. The PDGF-D expression pattern of both
rodent and primate eyes indicates a role for PDGF-D
in lens growth through secretion into the posterior eye
chamber. When PDGF-D was blocked with antibodies
in intact rat eye organ culture, the proliferation of lens
epithelial cell was reduced by 75%, suggesting a major
in vivo role for PDGF-D in the highly coordinated
growth of eye tissues. Most interestingly, the PDGF-D

protein was found in its full-length form in all eye
tissues and cells investigated without sign of activation
by proteolytic removal of the CUB domain.
In periodontal mineralization, PDGF-D ⁄ PDGFR-b
appear to constitute an autocrine pathway when perio-
dontal ligament cells differentiate to cementoblasts
during in vitro formation of mineralized nodules [86].
During the mineralization process, the levels of both
PDGFR-b and PDGF-D in the ligament cells increase
significantly, whereas PDGF-B was not detected in
these cells.
PDGF-D in angiogenesis and wound healing
PDGF-D has been shown to stimulate angiogenesis
and deposition of the extracellular matrix, and thus
plays a part in the wound healing process [87–89].
Overexpression of PDGF-D in otherwise PDGF-
D-negative skin increased both the amount of macro-
phages in the dermis and the interstitial fluid pressure
[87]. The PDGF-D-mediated elevation of interstitial
fluid pressure is consistent with PDGFR-b being essen-
tial for the maintenance of steady state pressure level.
As the keratin 14 promoter is strongly upregulated
during wound healing, abundant PDGF-D was deliv-
ered when skin punch biopsy wounds were made in
mice. In this initial inflammation phase of wound heal-
ing, activated platelets secrete PDGF-A, -B and -C,
but not PDGF-D [76]. No endogen PDGF-D mRNA
could be detected [87], indicating that normally
PDGF-D does not play a role in the inflammatory
phase. A recent report supports the involvement of

PDGF-D after vessel injury by showing PDGF-D to
contribute to neointimal hyperplasia by stimulation of
migration and proliferation of vascular smooth muscle
cells as well as stimulation of the matrix metallopro-
teinase-2 of these cells [90]. Most data suggest that
PDGF-D is involved in maturing of the blood vessels,
not being merely an angiogenic factor. However, one
recent report shows PDGF-D to be a potent angio-
genic factor inducing new blood vessels [88]. When
NIH ⁄ 3T3 cells overexpressing PDGF-D were injected
in nude mice, PDGF-D induced tumour formation.
Analysis of the blood vessel morphology and vascular
densities in these tumours revealed long and irregular
vessels resembling the phenotype of PDGF-B-induced
vessels, but the PDGF-D-expressing tumours had a
significantly higher total vessel number.
PDGF-C in malignancies
Abnormalities in the PDGF ⁄ PDGFR system exist,
such as constitutive activation of PDGFR kinases,
activating mutations of the PDGFR kinases, autocrine
signalling due to overexpression of PDGFs and the
PDGFRs, contributing to a number of human dis-
eases, especially malignancies including osteosarcomas
[91], lung carcinomas [92], gliomas, malignant astrocy-
tomas and medulloblastomas [82,93,94]. The oncogenic
transformation mediated by the autocrine PDGF loop
takes place through activation of the ras ⁄ MAPK path-
way that increases cellular proliferation, and activation
of the PI3K ⁄ Akt pathway promoting cell survival [95].
The discovery that PDGF-B is a v-sis homologue

made PDGF-B one of the earliest discovered onco-
genes, connecting PDGF ⁄ PDGFR to cellular transfor-
mation [96]. The finding that PDGF-C acts much like
PDGF-AB, the most mitogenic of all PDGFs [97], as
it activates both PDGFR-aa and -ab, indicates a role
for PDGF-C as an oncogene [41,82,98]. PDGF-C
induces tumours in nude mice, activates anchorage-
dependant growth, and is a potent transforming
growth factor of NIH ⁄ 3T3 cells [88]. The in vivo
tumourigenesis may partially be explained by PDGF-
C-mediated VEGF expression, promoting indirect sti-
mulation 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 was
provided when PDGF-C was identified as a secreted
transforming growth factor in Ewing family sarcomas
[99]. In these aggressive bone and soft tissue tumours,
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 5733
arising due to aberrant EWS ⁄ FLI transcription factors,
PDGF-C was identified as an upregulated downstream
target. The expression of a dominant negative PDGF-
C in Ewing sarcoma cells caused a marked reduction
in anchorage-independent growth [99] but PDGF-C
was unable to fully reconstitute all features of the
EWS ⁄ FLI phenotype [100]. These findings, in combi-
nation with observations that selective inhibitors of
PDGFR inhibited anchorage-independent growth, sug-

gest that in Ewing sarcoma cell lines an autocrine
PDGF-C ⁄ PDGFR loop may be important for neo-
plastic transformation [98]. However, a recent report
shows that only PDGFR-b is expressed in Ewing
family sarcoma cell lines and tissue, and as PDGF-C
does not bind PDGFR-b secreted PDGF-C must exert
its effect through another mechanism [101].
PDGF-C in brain tumours – glioblastoma and
medulloblastoma
PDGF and PDGFR autocrine signalling is both an
initial event as well as important in the progression
from astrocytoma to glioblastoma multiforme [102].
Most interestingly, PDGF-C mRNA was detected in
several glioblastoma cell lines and in five primary
glioblastoma multiforme tissues examined, whereas lit-
tle or no expression was found in normal foetal and
adult brain tissues [82]. The 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
autocrine loop has not been defined. Data on the
expression of PDGF-C in medulloblastoma tumours
have not been reported, but a study of five medullo-
blastoma cell lines showed high mRNA levels of all
four PDGFs [94]. Interestingly, cell lines with high
neuronal marker expression (NF-L and NP-Y) (indic-
ative of neuronal differentiation) and low c-myc
expression expressed the PDGFs and their cognate
receptors, whereas cells with high c-myc expression

showed low levels of PDGFs and receptors. PDGFR-a
was endogenously activated in three out of five cell
lines, while PDGFR-b was not expressed, suggesting
an autocrine loop in medulloblastoma involving
PDGFR-a only. In cell lines having endogenously acti-
vated PDGFR-a, PDGF-C was highly expressed while
PDGF-A could not be detected or detected at very low
levels, indicating PDGF-C to play a significant role in
an autocrine loop of medulloblastoma tumours.
PDGF-C in progressive renal diseases
Characteristics of progressive renal diseases are a high
proliferation of interstitial cells (hypercellularity),
increased matrix accumulation and influx of macro-
phages to the glomeruli [105]. The autocrine loop of
PDGF-B ⁄ PDGFR-b has been shown in many progres-
sive renal diseases. From recent findings, PDGF-D
appears to act either in concert with, or compete with,
PDGF-B in autocrine signalling within the mesangium
[80]. Usually, PDGF-C mRNA is constitutively
expressed in parietal epithelial cells and smooth muscle
cells of human kidneys, but in injured renal states
Ewing family sarcoma
Progressive renal disease Glioblastoma/Medulloblastoma Fibrosis
Lung cancer
Prostate cancer
Ovarian cancer
PDGF-D
(98,99,100,101)
(82,94)
(30,80,81)

(85,110)
(82,89)
Lung (67,84), Kidney (30,85)
Pancreas (108), Heart (8,38,67,109)
Liver (106,112)
Liver (80,110,112)
Kidney (85)
(89)
(89)
(44,66)
PDGF-C
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 is linked
to lung, prostate and ovarian cancers. See
text and references (numbers presented
together with the arrows) for detailed
descriptions.
PDGF-C and -D, structure and function L. J. Reigstad et al.
5734 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS
PDGF-C is significantly upregulated in the mesangial
and interstitial cells, suggesting that PDGF-C is
involved in renal pathogenesis [30,81]. PDGF-C is
expressed in the glomerular mesangium and surround-
ing interstitial cells in a diversity of renal diseases but
not in normal human kidneys. Most interestingly,
PDGFR-a is extensively expressed by interstital cells of

normal kidney and the expression is further increased in
the diseased kidney, suggesting that PDGF-C directly
interferes with the reported PDGF-A ⁄ PDGFR-a auto-
crine signalling present in the interstitium [80].
With respect to mesangioproliferative glomerulonep-
ritis in rats, PDGF-C is upregulated in the glomerul ar
mesangium but not in endothelial or epithelial glomer-
ular cells [30]. PDGF-C mRNA is markedly upregulated
in interstitial cells of rats suffering from severe tubulo-
interstitial fibrosis [85]. Although both PDGF-C
and PDGF-A are present in the interstitium, analyses
of rats with induced kidney disease show PDGF-A,
-B and -D mRNA levels to peak at days 7–9, while
PDGF-C mRNA has its maximum on day 28 after
disease induction. This suggests temporally different
levels of PDGF-C and PDGF-A possibly leading to a
dominating PDGF-C ⁄ PDGFR-a autocrine loop in later
stages of the disease.
PDGF-C in liver, lung, heart and pancreatic
fibrosis
A recent report shows that overexpression of PDGF-C
leads to liver fibrosis, steatosis (overproduction of mat-
rix) and hepatocellular carcinomas in transgene PDGF-
C mice [106]. PDGF-C is likely to be a hepatic fibrosis
inducer as the PDGF-C overexpression resulted in signi-
ficantly increased mRNA and protein levels of key pro-
fibrotic proteins like 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 fibro-
sis starts with activation and intense proliferation of the
hepatic stellate cells, producing robust pericellular and
perivenular collagen type I deposits [106]. PDGF-C
maintains the activation of the hepatic stellate cells.
Ageing transgenic PDGF-C mice developed more severe
fibrosis and after 12 months they developed hepatocellu-
lar carcinoma, with enlarged liver having multilocular
pseudocysts without epithelial lining and showing
increased angiogenesis.
Both PDGF-A and -B are expressed in animal mod-
els of lung fibrosis and fibrotic human lung [107].
When murine lung fibrosis was induced by bleomycin,
no change in the mRNA expressions of the classical
PDGFs were detected, while PDGF-C and -D mRNA
levels changed dramatically [84]. Concomitantly, the
level of phosphorylated PDGFR-a was markedly
upregulated in bleomycin-treated lungs. PDGF-C
mRNA could not be detected in bleomycin-resistant
mice in response to bleomycin-treatment and PDGF-C
induction was seen only in mouse 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 pan-
creatic carcinoma cells [108]. PDGF-B and several
other growth factors have previously been shown to
stimulate this desmoplastic reaction. Investigations of

surgically resected pancreatic cancerous lesions showed
PDGF-C mRNA to be upregulated compared to non-
neoplastic pancreatic tissue. The upregulation correla-
ted with collagen type I and III expression, indicating
that PDGF-C is a potent inducer of matrix overpro-
duction and thereby fibrosis in the pancreas.
Two reports show the effect of in vivo PDGF-C
overexpression using transgenic techniques in which
the full-length PDGF-c gene was targeted to the mouse
heart where PDGF-C was overexpressed [8,38]. The
overexpression induced hyperproliferation of myocar-
dial interstitial cells, giving expanded heart interstitium
and resulting in animals with progressive fibrosis and
cardiac hypertrophy [8]. The expanded heart intersti-
tium gave drastic disorganized myocardial fibres, pro-
gressive collagen accumulation and increased thickness
of the ventricular walls.
Additionally, recent reports on major histocompati-
bility complex class II knockout mice shows elevated
expression of PDGF-C in the hearts after infection
with coxsackievirus B3 (CVB3), but not in hearts of
immunocompetent mice infected with CVB3 [67,109].
In the immunodeficient mice, the CVB3 infection led
to chronic myocarditis characterized by marked cell
infiltration to the myocardium, and necrosis of cardio-
myocytes, resulting in heart fibrosis. In these hearts,
upregulation of both PDGF-C and PDGFR-a was
located to the areas of inflammatory cell invasion.
Taken together, in myocardial, liver, lung and pancre-
atic fibrosis, PDGF-C plays a role in fibrosis develop-

ment. In those tissue areas with upregulated PDGF-C,
receptor-downstream activation of Akt was detected.
PDGF-D in malignancies
PDGF-D induces tumours in nude mice, activates
anchorage-dependant growth in vitro and is a potent
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 5735
transforming growth factor of NIH ⁄ 3T3 cells [88]. The
in vivo tumorigenesis may partially be explained by
PDGF-D up-regulating VEGF expression.
PDGF-D in progressive renal diseases
An autocrine loop of PDGF-B and PDGFR-b appears
to be active when normal kidney is transformed to
renal interstitial fibrosis, characterized by high prolifer-
ation of interstitial cells and increased matrix accumu-
lation [105]. In normal rat and human glomeruli the
expression of PDGF-D mRNA is low, but when
mesangioproliferative nephritis was induced in rats the
mRNA and protein expression increased significantly,
with peak mRNA expression two days delayed com-
pared to PDGF-B mRNA [85]. A dramatic upregula-
tion of PDGF-D in serum of the nephritic rats was
observed, indicating that PDGF-D participates in
more than localized autocrine or paracrine stimulation.
This high level of circulating PDGF-D was also detec-
ted after the infection of healthy mice with adeno-
virus containing a PDGF-D construct. These mice
developed mesangioproliferative nephritis, indicating
PDGF-D to be the most potent stimulus for murine
mesangial cell proliferation identified to date [110].

The glomerular and systemic in vivo overexpression
of PDGF-D in the pathogenesis of mesangio-prolifera-
tive changes is responsible for a large glomerular macro-
phage influx, for significant increase in glomerular
size, and an increased proliferation of the mesangial
cells that have gained a myofibroblast phenotype
(expression of a-smooth muscle actin) which is respon-
sible for the increased accumulation of collagen type I
and IV matrix [80,85,110]. Furthermore, PDGF-D is
a likely candidate for controlling the progression of
metastatic renal cell carcinoma since its overexpression
enhanced tumour progression and metastasis in an
orthotropic tumour model in SCID mice [111].
PDGF-D in fibrosis
Progressive renal diseases often end as interstitial fibro-
sis, and data on human and murine PDGF-D protein
expression at this endpoint have been reported [80].
After induction of interstitial fibrosis in mice, expres-
sion of PDGF-D, -B and the PDGFRs was detected in
the interstitial fibroblasts, in contrast to the interstitial
cells of normal kidney. The PDGFR-b protein was dis-
tributed in the tubointerstitial fibrosis areas with an
immunostaining pattern corresponding to the sites of
PDGF-D but less so to the PDGF-B positive sites.
Similar results were obtained in humans suffering from
chronic renal nephropathy, suggesting that PDGF-D
exerts its fibrogenic effect by activating PDGFR-b,
and furthermore to be as significant as PDGF-B in
progressive renal diseases. PDGF-D may act in concert
with, or compete with, PDGF-B in autocrine signalling

within the mesangium. PDGF-D mRNA is constitu-
tively expressed in the normal mouse lung but by indu-
cing lung fibrosis, PDGF-D expression was reduced
[84]. Mice receiving adenovirus-carried PDGF-D devel-
oped fibrosis of the liver [110]. Furthermore, in an
in vitro model system of hepatic fibrogenesis, both
PDGF-D mRNA and protein levels increased signifi-
cantly [112].
PDGF-D in malignant tumours
The PDGF-D expression in normal brain is low, but
in human glioblastoma and medulloblastoma PDGF-
D appears to participate in a previously unrecognized
PDGF autocrine loop with PDGFR-b, as PDGF-B
could not be detected in the majority of the cases
investigated [82,89].
PDGF-D accelerates the early onset of prostate
tumour nodules when prostate cancer was induced in
mice, revealing a remarkable increase in contact
between the prostate carcinoma cells and the stromal
cells, which is critical for prostate cancer progression
and metastases [66]. As human prostate cancer tissue
expresses significant levels of PDGF-D ⁄ PDGFR-b,
PDGF-D can be suggested to participate in both auto-
crine and paracrine loops involved in prostate cancer
development and ⁄ or progression. PDGF-D was further
linked to prostate cancer progression by the cellular
colocalization of PDGF-D and its activator uPA in
prostate carcinoma cells [44].
PDGF-D is upregulated in human lung and ovarian
cancers compared with the normal tissues. Increased

PDGF-D levels were also detected in sera of lung and
ovarian cancer patients [89]. This high level of circula-
ting PDGF-D is also detected in mice infected with
adenovirus-containing PDGF-D, resulting in peri-
vascular lymphoid cell infiltrates of 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 potent cytokines important for normal embryogen-
esis and maintenance of adult tissues in several species.
Both PDGF-C and -D are involved in various malfunc-
tions such as progressive renal diseases, fibrosis in
many organs, with specific functions (PDGF-C) in
Ewing family sarcomas, and (PDGF-D) in lung,
PDGF-C and -D, structure and function L. J. Reigstad et al.
5736 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS
prostate, and ovarian cancer. Structure models of
PDGF-C suggest it to resemble VEGF-A but conclu-
sive determination of the 3D structures of both PDGF-
C 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.
References
1 McDonald NQ & Hendrickson WA (1993) A structural
superfamily of growth factors containing a cystine knot
motif. Cell 73, 421–424.

2 Sun PD & Davies DR (1995) The cystine-knot growth-
factor superfamily. Annu Rev Biophys Biomol Struct 24,
269–291.
3 Vitt UA, Hsu SY & Hsueh AJ (2001) Evolution and
classification of cystine knot-containing hormones and
related extracellular signaling molecules. Mol Endocri-
nol 15, 681–694.
4 Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW,
Canfield RE, Machin KJ, Morgan FJ & Isaacs NW
(1994) Crystal structure of human chorionic gonado-
tropin. Nature 369, 455–461.
5 Craik DJ & Daly NL (2005) Oxidative folding of the
cystine knot motif in cyclotide proteins. Protein Pept
Lett 12, 147–152.
6 Craik DJ, Daly NL, Mulvenna J, Plan MR & Trabi M
(2004) Discovery, structure and biological activities of
the cyclotides. Curr Protein Pept Sci 5, 297–315.
7 Hamada T, Ui-Tei K & Miyata Y (2000) A novel gene
derived from developing spinal cords, SCDGF, is a
unique member of the PDGF ⁄ VEGF family. FEBS
Lett 475, 97–102.
8 Li X, Ponten A, Aase K, Karlsson L, Abramsson A,
Uutela M, Backstrom G, Hellstrom M, Bostrom H,
Li H, Soriano P, Betsholtz C, Heldin CH, Alitalo K,
Ostman A & Eriksson U (2000) PDGF-C is a new
protease-activated ligand for the PDGF alpha-receptor.
Nat Cell Biol 2, 302–309.
9 Ding H, Wu X, Kim I, Tam PP, Koh GY & Nagy A
(2000) The mouse Pdgfc gene: dynamic expression in
embryonic tissues during organogenesis. Mech Dev 96,

209–213.
10 LaRochelle WJ, Jeffers M, McDonald WF, Chillakuru
RA, Giese NA, Lokker NA, Sullivan C, Boldog FL,
Yang M, Vernet C, Burgess CE, Fernandes E, Deegler
LL, Rittman B, Shimkets J, Shimkets RA, Rothberg
JM & Lichenstein HS (2001) PDGF-D, a new pro-
tease-activated growth factor. Nat Cell Biol 3, 517–521.
11 Hamada T, Ui-Tei K, Imaki J & Miyata Y (2001)
Molecular cloning of SCDGF-B, a novel growth factor
homologous to SCDGF ⁄ PDGF-C ⁄ fallotein. Biochem
Biophys Res Commun 280, 733–737.
12 Bergsten E, Uutela M, Li X, Pietras K, Ostman A,
Heldin CH, Alitalo K & Eriksson U (2001) PDGF-D
is a specific, protease-activated ligand for the PDGF
beta-receptor. Nat Cell Biol 3, 512–516.
13 Heldin CH & Westermark B (1990) Signal transduction
by the receptors for platelet-derived growth factor.
J Cell Sci 96, 193–196.
14 Heldin CH, Eriksson U & Ostman A (2002) New
members of the platelet-derived growth factor family
of mitogens. Arch Biochem Biophys 398, 284–290.
15 Tsai YJ, Lee RK, Lin SP & Chen YH (2000) Identifi-
cation of a novel platelet-derived growth factor-like
gene, fallotein, in the human reproductive tract.
Biochim Biophys Acta 1492, 196–202.
16 Bork P & Beckmann G (1993) The CUB domain.
A widespread module in developmentally regulated
proteins. J Mol Biol 231, 539–545.
17 Dalla-Favera R, Gallo RC, Giallongo A & Croce CM
(1982) Chromosomal localization of the human homo-

log (c-sis) of the simian sarcoma virus onc gene.
Science 218, 686–688.
18 Betsholtz C, Johnsson A, Heldin CH, Westermark B,
Lind P, Urdea MS, Eddy R, Shows TB, Philpott K,
Mellor AL et al. (1986) cDNA sequence and chromo-
somal localization of human platelet-derived growth
factor A-chain and its expression in tumour cell lines.
Nature 320, 695–699.
19 Uutela M, Lauren J, Bergsten E, Li X, Horelli-
Kuitunen N, Eriksson U & Alitalo K (2001) Chromo-
somal location, exon structure, and vascular expression
patterns of the human PDGFC and PDGFC genes.
Circulation 103, 2242–2247.
20 Fredriksson L, Li H & Eriksson U (2004) The PDGF
family: four gene products form five dimeric isoforms.
Cytokine Growth Factor Rev 15, 197–204.
21 Li X & Eriksson U (2003) Novel PDGF family mem-
bers: PDGF-C and PDGF-D. Cytokine Growth Factor
Rev 14, 91–98.
22 Khachigian LM, Williams AJ & Collins T (1995) Inter-
play of Sp1 and Egr-1 in the proximal platelet-derived
growth factor A-chain promoter in cultured vascular
endothelial cells. J Biol Chem 270, 27679–27686.
23 Khachigian LM, Lindner V, Williams AJ & Collins T
(1996) Egr-1-induced endothelial gene expression: a
common theme in vascular injury. Science 271, 1427–
1431.
24 Midgley VC & Khachigian LM (2004) Fibroblast
growth factor-2 induction of platelet-derived growth
factor-C chain transcription in vascular smooth

muscle cells is ERK-dependent but not JNK-depen-
dent and mediated by Egr-1, J Biol Chem. 279,
40289–40295.
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 5737
25 Dijkmans J, Xu J, Masure S, Dhanaraj S, Gosiewska
A, Geesin J, Sprengel J, Harris S, Verhasselt P,
Gordon R & Yon J (2002) Characterization of platelet-
derived growth factor-C (PDGF-C): expression in
normal and tumor cells, biological activity and chromo-
somal localization. Int J Biochem Cell Biol 34, 414–426.
26 Zhuo Y, Hoyle GW, Zhang J, Morris G & Lasky JA
(2003) A novel murine PDGF-D splicing variant results
in significant differences in peptide expression and
function. Biochem Biophys Res Commun 308, 126–132.
27 Hamada T, Ui-Tei K, Imaki J, Takahashi F, Onodera
H, Mishima T & Miyata Y (2002) The expression of
SCDGF ⁄ PDGF-C ⁄ fallotein and SCDGF-B ⁄ PDGF-D
in the rat central nervous system. Mech Dev 112, 161–
164.
28 Collins T, Bonthron DT & Orkin SH (1987) Alterna-
tive RNA splicing affects function of encoded platelet-
derived growth factor A chain. Nature 328, 621–624.
29 Changsirikulchai S, Hudkins KL, Goodpaster TA,
Volpone J, Topouzis S, Gilbertson DG & Alpers CE
(2002) Platelet-derived growth factor-D expression in
developing and mature human kidneys. Kidney Int 62,
2043–2054.
30 Eitner F, Ostendorf T, Van Roeyen C, Kitahara M, Li
X, Aase K, Grone HJ, Eriksson U & Floege J (2002)

Expression of a novel PDGF isoform, PDGF-C, in
normal and diseased rat kidney. J Am Soc Nephrol 13,
910–917.
31 Yeh HJ, Pierce GF & Deuel TF (1987) Ultrastructural
localization of a platelet-derived growth factor ⁄ v-sis-
related protein(s) in cytoplasm and nucleus of simian
sarcoma virus-transformed cells. Proc Natl Acad Sci
USA 84, 2317–2321.
32 Rakowicz-Szulczynska EM, Rodeck U, Herlyn M &
Koprowski H (1986) Chromatin binding of epidermal
growth factor, nerve growth factor, and platelet-derived
growth factor in cells bearing the appropriate surface
receptors. Proc Natl Acad Sci USA 83, 3728–3732.
33 Romao MJ, Kolln I, Dias JM, Carvalho AL, Romero
A, Varela PF, Sanz L, Topfer-Petersen E & Calvete JJ
(1997) Crystal structure of acidic seminal fluid protein
(aSFP) at 1.9 A resolution: a bovine polypeptide of the
spermadhesin family. J Mol Biol 274, 650–660.
34 Varela PF, Romero A, Sanz L, Romao MJ, Topfer-
Petersen E & Calvete JJ (1997) The 2.4 A resolution
crystal structure of boar seminal plasma PSP-I ⁄ PSP-II:
a zona pellucida-binding glycoprotein heterodimer of
the spermadhesin family built by a CUB domain archi-
tecture. J Mol Biol 274, 635–649.
35 Feinberg H, Uitdehaag JC, Davies JM, Wallis R, Drick-
amer K & Weis WI (2003) Crystal structure of the
CUB1-EGF-CUB2 region of mannose-binding protein
associated serine protease-2. EMBO J 22, 2348–2359.
36 Romero A, Romao MJ, Varela PF, Kolln I, Dias JM,
Carvalho AL, Sanz L, Topfer-Petersen E & Calvete JJ

(1997) The crystal structures of two spermadhesins
reveal the CUB domain fold. Nat Struct Biol 4, 783–
788.
37 Kristiansen M, Kozyraki R, Jacobsen C, Nexo E, Verr-
oust PJ & Moestrup SK (1999) Molecular dissection of
the intrinsic factor-vitamin B12 receptor, cubilin, dis-
closes regions important for membrane association and
ligand binding. J Biol Chem 274, 20540–20544.
38 Ponten A, Li X, Thoren P, Aase K, Sjoblom T, Ost-
man A & Eriksson U (2003) Transgenic overexpression
of platelet-derived growth factor-C in the mouse heart
induces cardiac fibrosis, hypertrophy, and dilated
cardiomyopathy. Am J Pathol 163, 673–682.
39 Ray S, Gao C, Wyatt K, Fariss RN, Bundek A,
Zelenka P & Wistow G (2005) Platelet-derived growth
factor D, tissue-specific expression in the eye, and a
key role in control of lens epithelial cell proliferation.
J Biol Chem 280, 8494–8502.
40 Nakamura F & Goshima Y (2002) Structural and func-
tional relation of neuropilins. Adv Exp Medical Biol
515, 55–69.
41 Gilbertson DG, Duff ME, West JW, Kelly JD,
Sheppard PO, Hofstrand PD, Gao Z, Shoemaker K,
Bukowski TR, Moore M, Feldhaus AL, Humes JM,
Palmer TE & Hart CE (2001) Platelet-derived growth
factor C (PDGF-C), a novel growth factor that binds
to PDGF alpha and beta receptor. J Biol Chem 276,
27406–27414.
42 Fredriksson L, Li H, Fieber C, Li X & Eriksson U
(2004) Tissue plasminogen activator is a potent activa-

tor of PDGF-CC. EMBO J 23, 3793–3802.
43 Fredriksson L, Ehnman M, Fieber C & Eriksson U
(2005) Structural requirements for activation of latent
platelet-derived growth factor CC by tissue plasmino-
gen activator. J Biol Chem 280, 26856–26862.
44 Ustach CV & Kim HR (2005) Platelet-derived growth
factor D is activated by urokinase plasminogen activa-
tor in prostate carcinoma cells. Mol Cell Biol 25, 6279–
6288.
45 Siegfried G, Khatib AM, Benjannet S, Chretien M &
Seidah NG (2003) The proteolytic processing of pro-
platelet-derived growth factor-A at RRKR (86) by
members of the proprotein convertase family is func-
tionally correlated to platelet-derived growth factor-
A-induced functions and tumorigenicity. Cancer Res
63, 1458–1463.
46 Ostman A, Thyberg J, Westermark B & Heldin CH
(1992) PDGF-AA and PDGF-BB biosynthesis: propro-
tein processing in the Golgi complex and lysosomal
degradation of PDGF-BB retained intracellularly.
J Cell Biol 118, 509–519.
47 McDonald NQ, Lapatto R, Murray-Rust J, Gunning
J, Wlodawer A & Blundell TL (1991) New protein fold
revealed by a 2.3-A resolution crystal structure of nerve
growth factor. Nature 354, 411–414.
PDGF-C and -D, structure and function L. J. Reigstad et al.
5738 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS
48 Daopin S, Piez KA, Ogawa Y & Davies DR (1992)
Crystal structure of transforming growth factor-beta 2:
an unusual fold for the superfamily. Science 257, 369–

373.
49 Oefner C, D’Arcy A, Winkler FK, Eggimann B &
Hosang M (1992) Crystal structure of human platelet-
derived growth factor BB. EMBO J 11, 3921–3926.
50 Murray-Rust J, McDonald NQ, Blundell TL, Hosang
M, Oefner C, Winkler F & Bradshaw RA (1993) Topo-
logical similarities in TGF-beta 2, PDGF-BB and
NGF define a superfamily of polypeptide growth fac-
tors. Structure 1, 153–159.
51 Muller YA, Christinger HW, Keyt BA & de Vos AM
(1997) The crystal structure of vascular endothelial
growth factor (VEGF) refined to 1.93 A resolution:
multiple copy flexibility and receptor binding. Structure
5, 1325–1338.
52 Muller YA, Li B, Christinger HW, Wells JA, Cunning-
ham BC & de Vos AM (1997) Vascular endothelial
growth factor: crystal structure and functional mapping
of the kinase domain receptor binding site. Proc Natl
Acad Sci USA 94, 7192–7197.
53 Wiesmann C, Fuh G, Christinger HW, Eigenbrot C,
Wells JA & de Vos AM (1997) Crystal structure at 1.7
A resolution of VEGF in complex with domain 2 of
the Flt-1 receptor. Cell 91, 695–704.
54 Iyer S, Leonidas DD, Swaminathan GJ, Maglione D,
Battisti M, Tucci M, Persico MG & Acharya KR
(2001) The crystal structure of human placenta growth
factor-1 (PlGF-1), an angiogenic protein, at 2.0 A reso-
lution. J Biol Chem 276, 12153–12161.
55 Haniu M, Hsieh P, Rohde MF & Kenney WC (1994)
Characterization of disulfide linkages in platelet-derived

growth factor AA. Arch Biochem Biophys 310, 433–437.
56 Haniu M, Rohde MF & Kenney WC (1993) Disulfide
bonds in recombinant human platelet-derived growth
factor BB dimer: characterization of intermolecular
and intramolecular disulfide linkages. Biochemistry 32,
2431–2437.
57 Reigstad LJ, Sande HM, Fluge O, Bruland O, Muga
A, Varhaug JE, Martinez A & Lillehaug JR (2003)
Platelet-derived growth factor (PDGF) -C, a PDGF
family member with a vascular endothelial growth
factor-like structure. J Biol Chem 278, 17114–17120.
58 Lustig F, Hoebeke J, Ostergren-Lunden G, Velge-
Roussel F, Bondjers G, Olsson U, Ruetschi U & Fager
G (1996) Alternative splicing determines the binding of
platelet-derived growth factor (PDGF-AA) to glycos-
aminoglycans. Biochemistry 35, 12077–12085.
59 Somasundaram R & Schuppan D (1996) Type I, II,
III, IV, V, and VI collagens serve as extracellular
ligands for the isoforms of platelet-derived growth fac-
tor (AA, BB, and AB). J Biol Chem 271, 26884–26891.
60 Ostman A, Andersson M, Betsholtz C, Westermark B
& Heldin CH (1991) Identification of a cell retention
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.
61 Kaetzel DM Jr, Morgan D, 3rd Reid JDT & Fenster-
maker RA (1996) Site-directed mutagenesis of the
N-linked glycosylation site in platelet-derived growth
factor B-chain results in diminished intracellular
retention. Biochim Biophys Acta 1298, 250–260.

62 Heldin CH & Westermark B (1999) Mechanism of
action and in vivo role of platelet-derived growth fac-
tor. Physiol Rev 79, 1283–1316.
63 Deuel TF, Huang JS, Proffitt RT, Baenziger JU, Chang
D & Kennedy BB (1981) Human platelet-derived growth
factor. Purification and resolution into two active pro-
tein fractions. J Biol Chem 256, 8896–8899.
64 Heldin CH, Ostman A & Ronnstrand L (1998) Signal
transduction via platelet-derived growth factor recep-
tors. Biochim Biophys Acta 1378, F79–F113.
65 Hart CE, Forstrom JW, Kelly JD, Seifert RA, Smith
RA, Ross R, Murray MJ & Bowen-Pope DF (1988)
Two classes of PDGF receptor recognize different iso-
forms of PDGF. Science 240, 1529–1531.
66 Ustach CV, Taube ME, Hurst NJ Jr, Bhagat S, Bonfil
RD, Cher ML, Schuger L & Kim HR (2004) A poten-
tial oncogenic activity of platelet-derived growth factor
d in prostate cancer progression. Cancer Res 64, 1722–
1729.
67 Grun K, Markova B, Bohmer FD, Berndt A, Kosmehl
H & Leipner C (2005) Elevated expression of PDGF-C
in coxsackievirus B3-induced chronic myocarditis. Eur
Heart J 26, 728–739.
68 Nagaoka I, Honma S, Someya A, Iwabuchi K &
Yamashita T (1992) Differential expression of the
platelet-derived growth factor-A and – B genes during
maturation of monocytes to macrophages. Comp
Biochem Physiol B 103, 349–356.
69 Raines EW, Lane TF, Iruela-Arispe ML, Ross R &
Sage EH (1992) The extracellular glycoprotein SPARC

interacts with platelet-derived growth factor (PDGF) -
AB and – BB and inhibits the binding of PDGF to its
receptors. Proc Natl Acad Sci USA 89, 1281–1285.
70 Bonner JC, Goodell AL, Lasky JA & Hoffman MR
(1992) Reversible binding of platelet-derived growth
factor-AA-AB, and – BB isoforms to a similar site on
the ‘slow’ and ‘fast’ conformations of alpha 2-macro-
globulin. J Biol Chem 267, 12837–12844.
71 Bostrom H, Willetts K, Pekny M, Leveen P, Lindahl
P, Hedstrand H, Pekna M, Hellstrom M, Gebre-Med-
hin S, Schalling M et al. (1996) PDGF-A signaling is a
critical event in lung alveolar myofibroblast develop-
ment and alveogenesis. Cell 85, 863–873.
72 Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Lars-
son E & Betsholtz C (1994) Mice deficient for PDGF
B show renal, cardiovascular, and hematological
abnormalities. Genes Dev 8, 1875–1887.
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 5739
73 Ding H, Wu X, Bostrom H, Kim I, Wong N, Tsoi B,
O’Rourke M, Koh GY, Soriano P, Betsholtz C et al.
(2004) A specific requirement for PDGF-C in palate
formation and PDGFR-alpha signaling. Nat Genet 36,
1111–1116.
74 Aase K, Abramsson A, Karlsson L, Betsholtz C &
Eriksson U (2002) Expression analysis of PDGF-C in
adult and developing mouse tissues. Mech Dev 110,
187–191.
75 Grazul-Bilska AT, Johnson ML, Bilski JJ, Redmer
DA, Reynolds LP, Abdullah A & Abdullah KM

(2003) Wound healing: the role of growth factors.
Drugs Today (Barc) 39, 787–800.
76 Fang L, Yan Y, Komuves LG, Yonkovich S, Sullivan
CM, Stringer B, Galbraith S, Lokker NA, Hwang SS,
Nurden P, Phillips DR & Giese NA (2004) PDGF C is
a selective alpha platelet-derived growth factor receptor
agonist that is highly expressed in platelet alpha gran-
ules and vascular smooth muscle. Arterioscler Thromb
Vasc Biol 24, 787–792.
77 Li X, Tjwa M, Moons L, Fons P, Noel A, Ny A, Zhou
JM, Lennartsson J, Li H, Luttun A et al. (2005) Re-
vascularization of ischemic tissues by PDGF-CC via
effects on endothelial cells and their progenitors. J Clin
Invest 115, 118–127.
78 Jinnin M, Ihn H, Mimura Y, Asano Y, Yamane K &
Tamaki K (2005) Regulation of fibrogenic ⁄ fibrolytic
genes by platelet-derived growth factor C, a novel
growth factor, in human dermal fibroblasts. J Cell
Physiol 202, 510–517.
79 Cao R, Brakenhielm E, Li X, Pietras K, Widenfalk J,
Ostman A, Eriksson U & Cao Y (2002) Angiogenesis
stimulated by PDGF-CC, a novel member in the PDGF
family, involves activation of PDGFR-alphaalpha and –
alphabeta receptors. FASEB J 16, 1575–1583.
80 Taneda S, Hudkins KL, Topouzis S, Gilbertson DG,
Ophascharoensuk V, Truong L, Johnson RJ & Alpers
CE (2003) Obstructive uropathy in mice and humans:
potential role for PDGF-D in the progression of tubu-
lointerstitial injury. J Am Soc Nephrol 14, 2544–2555.
81 Eitner F, Ostendorf T, Kretzler M, Cohen CD,

Eriksson U, Grone HJ & Floege J (2003) PDGF-C
expression in the developing and normal adult
human kidney and in glomerular diseases. J Am Soc
Nephrol 14, 1145–1153.
82 Lokker NA, Sullivan CM, Hollenbach SJ, Israel MA
& Giese NA (2002) Platelet-derived growth factor
(PDGF) autocrine signaling regulates survival and
mitogenic pathways in glioblastoma cells: evidence that
the novel PDGF-C and PDGF-D ligands may play a
role in the development of brain tumors. Cancer Res
62, 3729–3735.
83 Lee YW, Ozeki M, Juhn SK & Lin J (2004) Expression
of platelet-derived growth factor in the developing
cochlea of rats. Acta Otolaryngol 124, 558–562.
84 Zhuo Y, Zhang J, Laboy M & Lasky JA (2004) Modu-
lation of PDGF-C and PDGF-D expression during
bleomycin-induced lung fibrosis. Am J Physiol Lung
Cell Mol Physiol 286, L182–L188.
85 Ostendorf T, van Roeyen CR, Peterson JD, Kunter U,
Eitner F, Hamad AJ, Chan G, Jia XC, Macaluso J,
Gazit-Bornstein G, Keyt BA, Lichenstein HS, LaRoc-
helle WJ & Floege J (2003) A fully human monoclonal
antibody (CR002) identifies PDGF-D as a novel
mediator of mesangioproliferative glomerulonephritis.
J Am Soc Nephrol 14, 2237–2247.
86 Nemoto E, Shimonishi M, Nitta Y & Shimauchi H
(2004) The involvement of platelet-derived growth fac-
tor receptors and insulin-like growth factor-I receptors
signaling during mineralized nodule formation by
human periodontal ligament cells. J Periodontal Res

39, 388–397.
87 Uutela M, Wirzenius M, Paavonen K, Rajantie I,
He Y, Karpanen T, Lohela M, Wiig H, Salven P,
Pajusola K, Eriksson U & Alitalo K (2004) PDGF-D
induces macrophage recruitment, increased interstitial
pressure, and blood vessel maturation during angio-
genesis. Blood 104, 3198–3204.
88 Li H, Fredriksson L, Li X & Eriksson U (2003)
PDGF-D is a potent transforming and angiogenic
growth factor. Oncogene 22, 1501–1510.
89 LaRochelle WJ, Jeffers M, Corvalan JR, Jia XC, Feng
X, Vanegas S, Vickroy JD, Yang XD, Chen F, Gazit
G et al. (2002) Platelet-derived growth factor D: tum-
origenicity in mice and dysregulated expression in
human cancer. Cancer Res 62, 2468–2473.
90 Chen J, Han Y, Lin C, Zhen Y, Song X, Teng S,
Chen C, Chen Y, Zhang Y & Hui R (2005)
PDGF-D contributes to neointimal hyperplasia in rat
model of vessel injury, Biochem Biophys Res Commun.
329, 976–983.
91 Sulzbacher I, Traxler M, Mosberger I, Lang S & Chott
A (2000) Platelet-derived growth factor-AA and – alpha
receptor expression suggests an autocrine and ⁄ or para-
crine loop in osteosarcoma. Mod Pathol 13, 632–637.
92 Antoniades HN, Galanopoulos T, Neville-Golden J &
O’Hara CJ (1992) Malignant epithelial cells in primary
human lung carcinomas coexpress in vivo platelet-
derived growth factor (PDGF) and PDGF receptor
mRNAs and their protein products. Proc Natl Acad
Sci USA 89, 3942–3946.

93 Guha A, Dashner K, Black PM, Wagner JA & Stiles
CD (1995) Expression of PDGF and PDGF receptors
in human astrocytoma operation specimens supports
the existence of an autocrine loop. Int J Cancer 60,
168–173.
94 Andrae J, Molander C, Smits A, Funa K & Nister M
(2002) Platelet-derived growth factor-B and – C and
active alpha-receptors in medulloblastoma cells.
Biochem Biophys Res Commun 296, 604–611.
PDGF-C and -D, structure and function L. J. Reigstad et al.
5740 FEBS Journal 272 (2005) 5723–5741 ª 2005 The Authors Journal compilation ª 2005 FEBS
95 Rosenmuller T, Rydh K & Nanberg E (2001) Role of
phosphoinositide 3OH-kinase in autocrine transforma-
tion by PDGF-BB. J Cell Physiol 188, 369–382.
96 Doolittle RF, Hunkapiller MW, Hood LE, Devare SG,
Robbins KC, Aaronson SA & Antoniades HN (1983)
Simian sarcoma virus onc gene, v-sis, is derived from
the gene (or genes) encoding a platelet-derived growth
factor. Science 221, 275–277.
97 Ekman S, Thuresson ER, Heldin CH & Ronnstrand L
(1999) Increased mitogenicity of an alphabeta
heterodimeric PDGF receptor complex correlates
with lack of RasGAP binding. Oncogene 18, 2481–
2488.
98 Zwerner JP & May WA (2002) Dominant negative
PDGF-C inhibits growth of Ewing family tumor cell
lines. Oncogene 21, 3847–3854.
99 Zwerner JP & May WA (2001) PDGF-C is an
EWS ⁄ FLI induced transforming growth factor in
Ewing family tumors. Oncogene 20, 626–633.

100 Zwerner JP, Guimbellot J & May WA (2003) EWS ⁄
FLI function varies in different cellular backgrounds.
Exp Cell Res 290, 414–419.
101 Uren A, Merchant MS, Sun CJ, Vitolo MI, Sun Y,
Tsokos M, Illei PB, Ladanyi M, Passaniti A, Mackall
C & Toretsky JA (2003) Beta-platelet-derived growth
factor receptor mediates motility and growth of
Ewing’s sarcoma cells. Oncogene 22, 2334–2342.
102 Maher EA, Furnari FB, Bachoo RM, Rowitch DH,
Louis DN, Cavenee WK & DePinho RA (2001) Malig-
nant glioma: genetics and biology of a grave matter.
Genes Dev 15, 1311–1333.
103 Smits A, van Grieken D, Hartman M, Lendahl U,
Funa K & Nister M (1996) Coexpression of platelet-
derived growth factor alpha and beta receptors on
medulloblastomas and other primitive neuroecto-
dermal tumors is consistent with an immature stem
cell and neuronal derivation. Laboratory Invest 74,
188–198.
104 Mapstone T, McMichael M & Goldthwait D (1991)
Expression of platelet-derived growth factors, trans-
forming growth factors, and the ros gene in a variety
of primary human brain tumors. Neurosurgery 28,
216–222.
105 Ranieri E, Gesualdo L, Grandaliano G, Maiorano E &
Schena FP (2001) The role of alpha-smooth muscle
actin and platelet-derived growth factor-beta receptor
in the progression of renal damage in human IgA
nephropathy. J Nephrol 14, 253–262.
106 Campbell JS, Hughes SD, Gilbertson DG, Palmer TE,

Holdren MS, Haran AC, Odell MM, Bauer RL, Ren
HP, Haugen HS, Yeh MM & Fausto N (2005) Plate-
let-derived growth factor C induces liver fibrosis, stea-
tosis, and hepatocellular carcinoma. Proc Natl Acad
Sci USA 102, 3389–3394.
107 Lasky JA & Brody AR (2000) Interstitial fibrosis and
growth factors. Environ Health Perspect 108 (Suppl. 4),
751–762.
108 Aoyagi Y, Oda T, Kinoshita T, Nakahashi C, Hasebe
T, Ohkohchi N & Ochiai A (2004) Overexpression of
TGF-beta by infiltrated granulocytes correlates with
the expression of collagen mRNA in pancreatic cancer.
Br J Cancer 91, 1316–1326.
109 Yang D & Qui D (2005) Linkage between elevated
PDGF-C expression and myocardial fibrogenesis in
coxsackie-virus B3-induced chronic myocarditis. Eur
Heart J 26, 642–643.
110 Hudkins KL, Gilbertson DG, Carling M, Taneda S,
Hughes SD, Holdren MS, Palmer TE, Topouzis S,
Haran AC, Feldhaus AL & Alpers CE (2004) Exogen-
ous PDGF-D is a potent mesangial cell mitogen and
causes a severe mesangial proliferative glomerulopathy.
J Am Soc Nephrol 15, 286–298.
111 Xu L, Tong R, Cochran DM & Jain RK (2005) Block-
ing platelet-derived growth factor-D ⁄ platelet-derived
growth factor receptor beta signaling inhibits human
renal cell carcinoma progression in an orthotopic
mouse model. Cancer Res 65, 5711–5719.
112 Breitkopf K, Roeyen C, Sawitza I, Wickert L, Floege J
& Gressner AM (2005) Expression patterns of PDGF-

A-B-C and – D and the PDGF-receptors alpha and
beta in activated rat hepatic stellate cells (HSC). Cyto-
kine 31, 349–357.
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 5741