COMMUNICATION
Domain Structure of Pleiotrophin Required for
Transformation*
Nan
Zhang,
Rong
Zhong, and
Thomas F.
Deuel
From the Division of Growth Regulation, Beth Israel Deaconess
Medical Center, Harvard Medical School,
Boston, Massachusetts 02215
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ABSTRACT |
The pleiotrophin (PTN) gene (Ptn) is
a potent proto-oncogene that is highly expressed in many primary human
tumors and constitutively expressed in cell lines derived from these
tumors. The product of the Ptn gene is a secreted 136-amino
acid heparin binding cytokine with distinct lysine-rich clusters within
both the N- and C-terminal domains. To seek domains of PTN functionally
important in neoplastic transformation, we constructed a series of
mutants and tested their transforming potential by four independent
criteria. Our data establish that a domain within PTN residues 41 to 64 and either but not both the N- or C-terminal domains are required for
transformation; deletion of both the N and C termini abolishes the
transformation potential of PTN. Furthermore, deletion of two internal
5-amino acid residue repeats enhances the transformation potency of PTN
2-fold. Our data indicate that PTN residues 41-64 contain an essential
domain for transformation and suggest the hypothesis that this domain
requires an additional interaction of the highly basic clusters of the
N or C terminus of PTN with a negatively charged "docking" site to
enable the transforming domain itself to engage and initiate PTN
signaling through its cognate receptor.
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INTRODUCTION |
Pleitrophin is an 18-kDa heparin-binding cytokine that was
purified from bovine uterus as a weak mitogen for fibroblasts (1) and
as a neurite-outgrowth promoting factor from neonatal rat brain (2).
The Ptn cDNA encodes a highly basic protein of 168 amino
acids with a 32-amino acid signal peptide and clusters of lysine
residues of dissimilar amino acid sequence at the N and C termini (3,
4). PTN1 has been described
as a mitogen for endothelial (6-8) and epithelial cells (7, 8) and for
fibroblasts (1, 7). Expression of the Ptn gene is tightly
regulated in a temporally and cell-type-specific manner during
development (5). In contrast, Ptn gene expression in adults
is constitutive and limited to fewer cell types than in development,
such as selective populations of neurons and glia (5). The
Ptn gene is a proto-oncogene (11). Cells transformed by
Ptn develop into highly vasculized, aggressive tumors
when implanted into the nude mouse and characteristically have
significant disarray of cytoskeletal structure. Furthermore, the
Ptn mRNA is highly expressed in a significant proportion
of samples from different human tumors and in about one-fourth of over
40 human tumor cell lines of different origins (7, 9, 10). PTN is
highly expressed in MDA-MB-231 cells, a cell line derived from a highly
malignant human breast cancer that constitutively expresses high levels
of the endogenous Ptn gene. A truncated mutant of PTN
constitutively expressed in these cells reverted the transformed phenotype of the breast cancer cell (12), establishing the importance of endogenous PTN signaling in maintaining the malignant phenotype of
these cells. To pursue the molecular basis of PTN signaling in
transformation, we constructed and tested a series of mutant Ptn molecules to establish domains required for
transformation of NIH 3T3 cells. We now report that an internal domain
within PTN residues 41-64 is required to transform NIH 3T3 cells. We also report the interesting finding that either the N- or C-terminal lysine-rich domain is essential to enable residues 41-64 to initiate transformation and that the potency of PTN to transform is regulated by
two 5-amino acid internal "repeats."
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MATERIALS AND METHODS |
Expression Plasmids--
The human Ptn cDNA
encodes an ~18 kDa protein of 168 amino acids before cleavage of a
32-amino acid signal peptide (Fig. 1). We constructed 11 mutant human
Ptn genes using PCR and "transformer" site-directed
mutagenesis (CLONTECH, San Francisco, CA) (Fig. 1). The mutant genes were cloned into the
KpnI/EcoRI site downstream of the SV40 early
promoter in the eukaryotic expression vector pAGE103 (11), which
consists of basic elements including the origin of replication, a
polyadenylation splicing signal, and the neomycin-resistance gene
driven by the thymidine kinase promoter. The mutant PTN proteins, PTN
1-122 (
123-136), PTN 1-64 (
65-136), and PTN 1-40
(
41-136) are C-terminal deletions of the PTN 136 amino acid
protein. Two internal repeated amino acid sequences (GAECK) were
identified at residues 41-45 and 64-68 of PTN. PTN
65-68 and PTN
42-45/
65-68 were constructed to ablate one or both of the two
internal repeats. PTN Lys-91
Asn/Arg-92
Gln mutations were
introduced into WT Ptn by site-directed mutagenesis. PTN
40-136 (
1-40), PTN 69-136 (
1-68), and PTN 101-136
(
1-100) were constructed as N-terminal deletions of human PTN. PTN
13-122 (
1-12,
123-136) was created to delete both N- and
C-terminal polylysine clusters. All of the Ptn constructs
were confirmed by DNA sequencing (see Fig. 1).

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Fig. 1.
Wild type and mutant PTN constructs. As
shown in the top panel, the complete amino acid sequence of
PTN contains 168 amino acids, including 32 amino acid residues of
signal peptides. The full-length human Ptn cDNA fragment
is shown in the middle panel and contains 5 exons. In the
bottom panel, the structures of 11 human Ptn
cDNA mutant gene products are present.
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Cells and DNA Transfections--
NIH 3T3 cells were maintained
in Dulbecco's modified Eagle's medium with 10% calf serum. The cells
were plated at a density of 1 × 106 per 100-mm dish
and transfected 24 h later by calcium phosphate precipitation
(11). Transfectants selected with G418 at an active strength of 700 µg/ml media were changed every three days until colony appeared, and
clonal cell lines were established from expansion of single colonies.
Colonies with high levels of PTN expression were pooled and analyzed.
Northern Blot Analysis--
Total cellular RNAs were isolated by
the guanidinium thiocyanate method and analyzed as described previously
(12).
Cell Growth, Focus Formation, and Soft Agar Assay--
To
establish rates of cell growth, 1 × 105 of clonally
selected cells were seeded in triplicate onto 35-mm dishes,
trypsinized, and counted using a hemocytometer at 24 and 48 h. For
focus formation assays, 2 × 105 cells for each
construct were plated in triplicate onto 60-mm dishes, stained with
crystal violet, photographed after 16 days, and counted.
Anchorage-independent growth in soft agar was carried out as described
previously (11). Briefly, 5 × 104 of cells
representative of each of the stably transfected Ptn constructs were suspended in 3 ml of (0.35% w/v) agar containing Dulbecco's modified Eagle's medium, 10% calf serum and overlaid onto
a 0.7% (w/v) agar solution in two 60-mm dishes. After 16 days,
colonies of >20 cells were scored as positive using an inverted microscope equipped with a measuring grid.
Tumor Formation in Nude Mice--
Tumor formation in 6-week-old
female athymic nude mice (strain nu/nu; Harlan Sprague-Dawley,
Indianapolis, IN) was tested by injecting subcutaneously 2 × 106 cells suspended in 200 µl of sterile
phosphate-buffered saline into each flank. Animals with tumors were
monitored daily starting at 10 days. After 6 weeks, selected animals
were sacrificed, and tumor size was measured in two perpendicular diameters.
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RESULTS |
The amino acid sequences of WT PTN and mutant proteins are
illustrated in Fig. 1. NIH 3T3 cells were transfected with WT or mutant
Ptn expression plasmids (11), and the expression levels of
each stably transfected cell line were determined by Northern blot
analysis (Fig. 2). Each of the mutant and
WT cDNA transcripts was readily detected. However, the levels of
expression of PTN 1-40 and PTN 101-136 were low compared with others,
perhaps resulting from mRNA instability. Exogenous PTN and PTN
mutants expressed in the transfected cells lines were also detected in
cell lysates and conditioned media by Western blot analysis (data not
shown).

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Fig. 2.
The top panel shows the exogenous WT and
mutant Ptn mRNA expression patterns of clonally
selected, stably transfected NIH 3T3 cells. As shown in the
bottom panel, the 28 and 18 S rRNAs are indicated by
arrows to the right of ethidium bromide gel as
loading amount controls.
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The growth rate of each of the stably transfected NIH 3T3 cells were
then determined. The rate of growth of cells transfected with PTN
1-122, PTN 1-64, PTN
65-68, PTN
42-45/
65-68, PTN Lys-91
Asn/Arg-92
Gln, PTN 41-136, and WT PTN increased nearly
1.5-fold at 24 h and >2-fold at 48 h relative to the control
NIH 3T3 cells (transfected with the empty vector). Cells lines
transfected with PTN 1-40, PTN 69-136, PTN 101-136, and PTN 13-122
grew at a rate similar to that of the control cells.
In focus forming assays, cell lines derived from WT PTN, PTN 1-122,
PTN 1-64, PTN 41-136, PTN
42-45, PTN
65-68, PTN
42-45/
65-68, and PTN Lys-9
Asn/Arg-92
Gln grew more
rapidly than control NIH 3T3 cells. At confluence, they had grown to a
density ~2-fold higher than that of control or NIH3T3 cells stably
expressing the mutant cDNAs PTN 1-40, PTN 69-136, PTN 101-136,
and PTN 13-122. However, the more rapidly growing cells grew in
clusters, and cell numbers were difficult to quantitate after 3 days.
The cells were highly refractile and spindled-shaped in appearance
(data not shown). After 16 days, foci were readily observed in cultures of these cells (Fig. 3), whereas foci
were not detected in cultures of NIH 3T3 cells expressing PTN 1-40,
PTN 69-136, PTN 101-136, or PTN 13-122 or in control cells. The
number of foci on each dish were counted (Table
I) and compared with the control cells. The foci in cells expressing PTN 1-122, PTN
65-
68, PTN Lys-91
Asn/Arg-92
Gln, and PTN 41-136 were readily detected but
somewhat reduced in number in comparison with WT PTN 1-136. Foci were
not found in cultures expressing PTN 1-40, PTN 69-136, PTN 101-136, and PTN 13-122. Interestingly, an increase in numbers of foci was
observed in cells expressing PTN 1-64 and PTN
42-45/
65-68, indicating that loss of the C-terminal residues 65-136 or deletion of
the two internal repeat sequences enhance the transformation potency of
PTN ~2-fold in focus forming assays.

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Fig. 3.
Focus formation of the pooled stably
transfected NIH 3T3 cells with WT and mutant Ptn genes
as well as pAGE 103 vector alone as a control. A high number of
foci were formed in the stably transfected NIH 3T3 cells with WT PTN
(B), PTN 1-122 (C), PTN 1-64 (D),
PTN 65-68 (F), PTN 42-45/ 65-68 (G),
PTN Lys-91 Asn/Arg-92 Gln (H), and PTN 41-136
(I), whereas few foci were observed in the stably
transfected NIH 3T3 cells with PTN 1-40 (E), PTN 69-1369
(J), PTN 101-136 (K), PTN 13-122
(L), and pAGE 103 vector alone (A).
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Table I
Focus formation and growth in soft agar of NIH 3T3 cells transfected
with Ptn or its mutants
NIH 3T3 cells were stably transfected with human PTN (WT), PTN 1-122,
PTN 1-64, PTN 65-68, PTN 42-45/ 65-68, PTN Lys-91 Asn/Arg-92 Gln, PTN 41-136, PTN 69-136, PTN101-136, and
PTN13-122 constructs or pAGE 103 vector alone as a control as
described under "Materials and Methods" and grown in the presence
of Geneticin (G418) (0.7 mg/ml) for 3 weeks. For focus formation
assays, 2 × 105 NIH 3T3 stably transfected cells
harboring WT Ptn or Ptn mutant constructs were
plated in triplicate onto 60-mm dishes, and after 16 days, the number
of foci on each dish were counted. For assays of anchorage-independent
growth, 5 × 104 cells were plated in soft agar onto two
60-mm dishes. After 16 days, colonies with more than 20 cells were
scored as positive.
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The stably transfected NIH 3T3 cells were then tested for colony
formation in soft agar. After 16 days, colonies with more than 20 cells
were scored as positive. NIH3T3 cells expressing WT PTN, PTN 1-122,
PTN 1-64, PTN
65-68, PTN
42-45/
65-68, PTN Lys-9
Asn/Arg-92
Gln, and PTN 41-136 formed large colonies (Fig.
4), whereas the control cells and cells
expressing PTN 1-40, PTN 69-136, PTN 101-136, or PTN 13-136 did not
(Table I).

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Fig. 4.
Colony formation of the pooled stably
transfected NIH 3T3 cells with WT and mutant Ptn genes
as well as pAGE 103 vector alone as a control in soft agar assay.
A high number of colonies were formed in the stably transfected NIH 3T3
cells with WT PTN (B), PTN 1-122 (C), PTN 1-64
(D), PTN 65-68 (F), PTN 42-45/ 65-68
(G), PTN Lys-91 Asn/Arg-92 Gln (H), and
PTN 41-136 (I), whereas few colonies were observed in the
stably transfected NIH 3T3 cells with PTN 1-40 (E), PTN
69-136 (J), PTN 101-136 (K), PTN13-122
(L), and pAGE 103 vector alone (A).
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To test tumor formation in nude mice, cells were implanted in flanks of
athymic nude mice. Mice were examined daily, starting 10 days after
injections, and tumors were measured at 6 weeks. Of the cells tested,
NIH 3T3 cells expressing PTN 1-122, PTN 1-64, PTN
42-45/
65-68, and WT PTN developed readily detectable tumors within 2 weeks (Table II). NIH 3T3 cells
transfected with the empty vector alone or cDNAs encoding PTN
69-136 or PTN 13-122 did not. After 6 weeks, the tumors observed in
the animals injected with the NIH 3T3 cells expressing PTN or its
mutants were examined. Surprisingly, significantly larger tumors were
found at sites of injection of NIH 3T3 cells expressing WT PTN and PTN
1-122 when compared with PTN, PTN 1-64, and the largest tumors were observed in PTN from which the two internal repeats were
deleted.
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Table II
Tumor formation in athymic nude mice of clonal NIH 3T3 cell lines
transfected with WT Ptn or its mutant gene constructs or pAGE 103 vector alone
Four animals per group were injected with 2 × 106
cells/site. Tumors were measured 6 weeks after the injection of cells.
The tumor size was calculated from the product of perpendicular
diameters of tumors.
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DISCUSSION |
In this study, we tested different domains of PTN to determine
which domains are required for transformation of NIH 3T3 cells. It was
established that amino acid residues 41-64 of PTN are required for
transformation; none of the mutant PTN proteins that lacked PTN
residues 41-64 transformed NIH 3T3 cells. Although the PTN receptor
has not been identified, the results suggest that residues 41-64
contain a critical domain for signaling. A surprising finding was the
requirement of either but not both the N- or C-terminal lysine-rich
domains together with PTN 41-64 to transform NIH 3T3 cells, indicating
that these domains support a similar functional role in transformation
by PTN. These two domains may function in a different way than PTN
residues 41-64 because the amino acid sequence of these two domains
differs significantly. However, both domains have a strong net positive
charge, suggesting they may interact with the receptor or an associated
second "low affinity" receptor through electrostatic forces but are
unlikely to signal active site-mediated receptor functions. Recently,
N-syndecan has been implicated as a PTN binding protein (13), however, the binding of N-syndecan to PTN is not specific to PTN because basic
fibroblast growth factor (bFGF) competes for the PTN binding sites, and
the glycosaminoglycan chains alone in N-syndecan bind both PTN and bFGF
(13). N-syndecan functions as a low affinity receptor and appears to
regulate binding of bFGF to its high affinity receptor (14, 15). It
seems possible that our results are consistent with a similar model in
which the N and C termini of PTN facilitate the binding of PTN residues
41-64 to sites on a high affinity receptor.
A surprising result of these experiments is the difference of NIH 3T3
cells expressing PTN 1-64 in focus and colony formation compared with
tumor formation. The ability of PTN 1-64 to strongly induce focus and
colony formation suggests a role of the C-terminal domain in loss of
contact inhibition and anchorage independence. PTN residues 65-136 may
contain a domain favoring tumor growth in vivo, such as
tumor angiogenesis.
The Ptn gene is highly expressed in breast cancers and
melanomas and constitutively expressed in cell lines derived from these tumors. Ptn gene expression is not detectable in melanocytes
and normal breast cells (7, 11), suggesting that PTN signally has an
important role in neoplastic growth. This view was strongly supported
when introduction of a dominant negative PTN effector reversed the
malignant phenotype of a human breast cancer cell line that
constitutively expresses the Ptn gene. Our findings provide
a structural basis for further studies on the functions of PTN in
transformation in breast cancer and other human tumors. Our findings
also provide a molecular model system to dissect the functional
responses in tumors constitutively expressing PTN.
We conclude that the potential of PTN to induce transformation is
mediated by several functional domains. Residues 41-64 of PTN
constitute an essential domain necessary for PTN-mediated transformation, whereas the C- and N-terminal lysine-rich domains function nearly equally to support residues 41-64 in
PTN-dependent transformation, perhaps through a
"docking" function to appropriately position PTN with its receptor.
Two internal duplicates of PTN negatively regulate PTN transformation.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL14147, CA66029, and CA49712.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Beth Israel Deaconess
Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA
02215. Fax: 617-667-1276.
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ABBREVIATIONS |
The abbreviations used are:
PTN, pleiotrophin;
WT, wild type;
bFGF, basic fibroblast growth factor.
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