COMMUNICATION
Domain Structure of Pleiotrophin Required for Transformation*

Nan Zhang, Rong Zhong, and Thomas F. DeuelDagger

From the Division of Growth Regulation, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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."

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta 123-136), PTN 1-64 (Delta 65-136), and PTN 1-40 (Delta 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 Delta 65-68 and PTN Delta 42-45/Delta 65-68 were constructed to ablate one or both of the two internal repeats. PTN Lys-91 right-arrow Asn/Arg-92 right-arrow Gln mutations were introduced into WT Ptn by site-directed mutagenesis. PTN 40-136 (Delta 1-40), PTN 69-136 (Delta 1-68), and PTN 101-136 (Delta 1-100) were constructed as N-terminal deletions of human PTN. PTN 13-122 (Delta 1-12, Delta 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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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 Delta 65-68, PTN Delta 42-45/Delta 65-68, PTN Lys-91 right-arrow Asn/Arg-92 right-arrow 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 Delta 42-45, PTN Delta 65-68, PTN Delta 42-45/Delta 65-68, and PTN Lys-9 right-arrow Asn/Arg-92 right-arrow 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 Delta 65-Delta 68, PTN Lys-91 right-arrow Asn/Arg-92 right-arrow 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 Delta 42-45/Delta 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 Delta 65-68 (F), PTN Delta 42-45/Delta 65-68 (G), PTN Lys-91 right-arrow Asn/Arg-92 right-arrow 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 Delta 65-68, PTN Delta 42-45/Delta 65-68, PTN Lys-91 right-arrow Asn/Arg-92 right-arrow 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.

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 Delta 65-68, PTN Delta 42-45/Delta 65-68, PTN Lys-9 right-arrow Asn/Arg-92 right-arrow 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 Delta 65-68 (F), PTN Delta 42-45/Delta 65-68 (G), PTN Lys-91 right-arrow Asn/Arg-92 right-arrow 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).

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 Delta 42-45/Delta 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    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.

Dagger To whom correspondence should be addressed: Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215. Fax: 617-667-1276.

    ABBREVIATIONS

The abbreviations used are: PTN, pleiotrophin; WT, wild type; bFGF, basic fibroblast growth factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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