Identification of the angiogenesis signaling domain in pleiotrophin defines a mechanism of the angiogenic switch

Neoplasms progress through genetic and epigenetic mutations that deregulate pathways in the malignant cell that stimulate more aggressive growth of the malignant cell itself and/or remodel the tumor microenvironment to support the developing tumor mass. The appearance of new blood vessels in malignant tumors is known as the "angiogenic switch." The angiogenic switch triggers a stage of rapid tumor growth supported by extensive tumor angiogenesis and a more aggressive tumor phenotype and its onset is a poor prognostic indicator for host survival. Identification of the factors that stimulate the angiogenic switch thus is of high importance. Pleiotrophin (PTN the protein, Ptn the gene) is an angiogenic factor and the Ptn gene has been found to be constitutively expressed in many human tumors of different cell types. These studies use a nude mouse model to test if Ptn constitutively expressed in premalignant cells is sufficient to trigger an angiogenic switch in vivo. We introduced an ectopic Ptn gene into "premalignant" SW-13 cells and analyzed the phenotype of SW-13 Ptn cell tumor implants in the flanks of nude mice. SW-13 Ptn cell subcutaneous tumor implants grew very rapidly and had a striking increase in the density of new blood vessels compared to the SW-13 cell tumor implants, suggesting that constitutive PTN signaling in the premalignant SW-13 cell implants in the nude mouse recapitulates fully the angiogenic switch. It was found also that ectopic expression of the C-terminal domain of PTN in SW-13 cell implants was equally effective in initiating an angiogenic switch as the full-length PTN whereas implants of SW-13 cells in nude mice that express the N-terminal domain of PTN grew rapidly but failed to develop tumor angiogenesis. The data suggest the possibility that mutations that activate Ptn in premalignant cells are sufficient to stimulate an angiogenic switch in vivo and, since these mutations are frequently found in human malignancies, that constitutive PTN signaling may be an important contributor to progression of human tumors. The data also suggest that the C-terminal and the N-terminal domains of PTN equally initiate switches in premalignant cells to cells of a more aggressive tumor phenotype but the separate domains of PTN signal different mechanisms and perhaps signal through activation of a separate receptor-like protein.     

Pleiotrophin: a cytokine with diverse functions and a novel signaling pathway.

Pleiotrophin (PTN the protein, Ptn the gene) is a 136 amino acid secreted heparin-binding cytokine that signals diverse functions, including lineage-specific differentiation of glial progenitor cells, neurite outgrowth, and angiogenesis. Pleiotrophin gene expression is found in cells in early differentiation during different development periods and upregulated in cells with an early differentiation phenotype in wound repair. The Ptn gene is a protooncogene. It is strongly expressed in different human tumor cells and expression of the Ptn gene in tumor cells in vivo accelerates growth and stimulates tumor angiogenesis. Separate independent domains have been identified in PTN to signal transformation and tumor angiogenesis. Pleiotrophin is the first ligand of any of the known transmembrane tyrosine phosphatases. Pleiotrophin inactivates the receptor protein tyrosine phosphatase (RPTP) beta/zeta. The interaction of PTN and RPTP beta/zeta increases steady-state tyrosine phosphorylation of beta-catenin. Pleiotrophin thus regulates both normal cell functions and different pathological conditions at many levels. It signals these functions through a transmembrane tyrosine phosphatase.     

多效生长因子（Pleiotrophin）基因沉默后的基因表达谱初步分析

多效生长因子（pleiotrophin,PTN指蛋白,Ptn指基因）是一种可同肝素结合的分泌性的生长/分化因子,具有刺激细胞粘附、迁移、存活、生长和分化等功能.我们前期研究发现,Ptn稳定沉默可以显著降低细胞的生长及成瘤能力.为进一步了解Ptn表达沉默后小鼠基因转录谱的变化,用小鼠表达谱芯片比较了对照及Ptn沉默细胞的基因表达差异.在检测的24 000个基因中,Ptn沉默后上调2倍以上的基因有240个,下调2倍以上的基因有129个.值得引起注意的是,在Ptn沉默的MEFs细胞中,同DDK综合症相关的基因家族,schlafen（Slfn）家族的Slfn 2、Slfn 3、Slfn 4以及基质金属蛋白酶（matrix metalloproteinase,MMP）家族的Mmp 3、Mmp 10、Mmp 13表达均显著上调;而可促进内皮细胞运动,参与血管发生的基因angiomotin(Amot)表达显著下调.通过研究,获得了一系列Ptn沉默后表达变化的基因信息.     

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

Pleiotrophin (PTN, Ptn) is an 18kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway in PTN-stimulated cells, not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTPbeta/zeta signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the ;;dotted" pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTPbeta/zeta signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.     

Dominant negative pleiotrophin induces tetraploidy and aneuploidy in U87MG human glioblastoma cells

Pleiotrophin (PTN, Ptn) is an 18kDa secretory cytokine that is expressed in many human cancers, including glioblastoma. In previous experiments, interruption of the constitutive PTN signaling in human U87MG glioblastoma cells that inappropriately express endogenous Ptn reversed their rapid growth in vitro and their malignant phenotype in vivo. To seek a mechanism for the effect of the dominant-negative PTN, flow cytometry was used to compare the profiles of U87MG cells and four clones of U87MG cells that express the dominant-negative PTN (U87MG/PTN1-40 cells); here, we report that the dominant-negative PTN in U87MG cells induces tetraploidy and aneuploidy and arrests the tetraploid and aneuploid cells in the G1 phase of the cell cycle. The data suggest that PTN signaling may have a critical role in chromosomal segregation and cell cycle progression; the data suggest induction of tetraploidy and aneuploidy in U87MG glioblastoma cells may be an important mechanism that contributes to the loss of the malignant phenotype of U87MG cells.     

Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase beta/zeta-signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin

Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, disrupting the balanced activity of RPTPbeta/zeta and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPbeta/zeta, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPbeta/zeta in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPbeta/zeta, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that beta-catenin and beta-adducin are targets of the PTN/RPTPbeta/zeta-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of beta-catenin and beta-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPbeta/zeta-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of beta-catenin, beta-adducin, and Fyn through the PTN/RPTPbeta/zeta-signaling pathway and that together Fyn, beta-adducin, and beta-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell-cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.     

The Effects of Pleiotrophin in Proliferative Diabetic Retinopathy

Pleiotrophin (PTN), a secreted, multifunctional cytokine, is involved in angiogenic, fibrotic and neurodegenerative diseases. However, little is known about its effects on diabetic retinopathy, a neurovascular disease. To investigate the role of PTN in proliferative diabetic retinopathy (PDR), PTN concentration in the vitreous was evaluated in PDR patients and non-diabetic controls. PTN expression was observed in epiretinal membranes from patients. PTN knockdown was performed using small interfering (si)RNA, and the effects on retinal pigment epithelium (RPE) cells and human umbilical vascular endothelia cells (HUVECs) were observed in vitro under hyperglycemic and hypoxic conditions. Cell attachment, proliferation, migration, tube formation, cell cycle, apoptosis, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation, and VEGF levels were studied. The vitreous PTN concentration in PDR patients was higher than that in non-diabetic controls, and PTN was highly expressed in the fibrovascular membranes of PDR patients. Under hyperglycemic and hypoxic conditions, PTN knockdown reduced cell attachment, proliferation, migration, and tube formation and induced cell cycle arrest and apoptosis in vitro. Mechanically, PTN depletion decreased ERK 1/2 phosphorylation. Recombinant PTN up regulated the concentration of VEGF in vitro, which can be attenuated by the ERK 1/2 inhibitor. Taken together, our results implied that elevated PTN in PDR patients might participate in the critical processes of the development of PDR, most likely playing roles in angiogenesis and proliferation, possibly by activating the ERK 1/2 pathway and regulating VEGF secretion. These findings provide new insight into the roles of PTN in PDR and suggest that PTN may become a new target for therapeutic intervention in PDR.     

Pleiotrophin stimulates tyrosine phosphorylation of beta-adducin through inactivation of the transmembrane receptor protein tyrosine phosphatase beta/zeta

Pleiotrophin (PTN the protein, Ptn the gene) signals through a unique mechanism; it inactivates the tyrosine phosphatase activity of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, and increases tyrosine phosphorylation of the substrates of RPTPbeta/zeta through the continued activity of a yet to be described protein tyrosine kinase(s) in PTN-stimulated cells. We have now found that the cytoskeletal protein beta-adducin interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system, that beta-adducin is a substrate of RPTPbeta/zeta, that beta-adducin is phosphorylated in tyrosine in cells not stimulated by PTN, and that tyrosine phosphorylation of beta-adducin is sharply increased in PTN-stimulated cells, suggesting that beta-adducin is a downstream target of and regulated by the PTN/RPTPbeta/zeta signaling pathway. beta-Catenin was the first downstream target of the PTN/RPTPbeta/zeta signaling pathway to be identified; these data thus also suggest that PTN coordinately regulates steady state levels of tyrosine phosphorylation of the important cytoskeletal proteins beta-adducin and beta-catenin and, through PTN-stimulated tyrosine phosphorylation, beta-adducin may contribute to the disruption of cytoskeletal structure, increased plasticity, and loss of homophilic cell-cell adhesion that are the consequences of PTN stimulation of cells and a characteristic feature of different malignant cells with mutations that activate constitutive expression of the endogenous Ptn gene.     

Domain structure of pleiotrophin required for transformation

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.     

Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways

Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.     

Use of adenovirus vectors for functional gene analysis in the chicken chorioallantoic membrane

The chicken chorioallantoic membrane (CAM) assay represents one of the most widely used in vivo screening assay for genes with angiogenic (blood vessel-inducing) or angiostatic (inhibition of vessel formation or their destruction) activities. Here we show that adenovirus gene transfer vectors infect cells in the CAM and lead to expression of the viral transgene. Furthermore, infection with an adenovirus vector containing the human vascular endothelial growth factor gene induced the formation of new blood vessels. This improved method saves a considerable amount of time in the identification of genes that can influence blood vessel formation because the expensive and time-consuming production and purification of recombinant protein can be omitted.     

Pleiotrophin,A Multifunctional Tumor Promoter Through Induction of Tumor Angiogenesis,Remodeling of the Tumor Microenvironment,and Activation of Stromal Fibroblasts

Pleiotrophin (PTN, Ptn) is a widely expressed, developmentally regulated 136 amino acid secreted heparin-binding cytokine. It signals through a unique signaling pathway; the PTN receptor is the transmembrane receptor protein tyrosine phosphatase (RPTP)β/ζ. RPTPβ/ζ is inactivated by PTN, which leads to increased tyrosine phosphorylation of the downstream targets of the PTN/RPTPβ/ζ signaling pathway. Pleiotrophin gene expression is found in cells in early differentiation during different developmental periods. It is upregulated in cells with an early differentiation phenotype in wound repair. The Ptn gene also is a proto-oncogene; PTN is expressed in human tumor cells, and, in cell lines derived from human tumors that express Ptn, Ptn expression is constitutive and thus "inappropriate". Importantly, properties of different cells induced by PTN in PTN-stimulated cells are strikingly similar to properties of highly malignant cells. Furthermore, transformed cells into which Ptn is introduced undergo "switches" to malignant cells of higher malignancy with properties that are strikingly similar to properties of PTN-stimulated cells. These unique features of PTN support the conclusion that constitutive PTN signaling in malignant cells that inappropriately express Ptn functions as a potent tumor promoter. Recently, in confirmation, Ptn targeted by the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model was found to promote breast cancers to a more aggressive breast cancer cell phenotype that morphologically closely resembles scirrhous carcinoma in human; in addition, it promoted a striking increase in tumor angiogenesis and a remarkable degree of remodeling of the micro-environment. Pleiotrophin thus regulates both different normal and pathological functions; collectively, the different studies have uncovered the unique ability of a single cytokine PTN, which signals through the unique PTN/RPTPβ/ζ signaling pathway, to induce the many properties associated with tumor promotion in the malignant cells that constitutively express Ptn and in their microenvironment.     

The receptor protein tyrosine phosphatase (RPTP)beta/zeta is expressed in different subtypes of human breast cancer

Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP)beta/zeta, leading to increased tyrosine phosphorylation of different substrate proteins of RPTPbeta/zeta, including beta-catenin, beta-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTPbeta/zeta signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusion protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTPbeta/zeta is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTPbeta/zeta furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTPbeta/zeta changes as the breast cancer become more malignant. The data suggest that the PTN/RPTPbeta/zeta signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.     

Pleiotrophin Gene Therapy for Peripheral Ischemia: Evaluation of Full-Length and Truncated Gene Variants

Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.     

Pleiotrophin induces nitric oxide dependent migration of endothelial progenitor cells

Pleiotrophin (PTN) is produced under ischemic conditions and has been shown to induce angiogenesis in vivo. We studied whether or not PTN exerts chemotaxis of pro-angiogenic early endothelial progenitor cells (EPCs), a population of circulating cells that have been reported to participate in and stimulate angiogenesis. Chemotaxis of EPCs, isolated from blood of healthy humans (n = 5), was measured in transwell assays. PTN at 10-500 ng/ml elicited dose-dependent chemotaxis of both EPCs and human umbilical vein endothelial cells (HUVECs), but not of human coronary artery smooth muscle cells (CASMCs) and T98G glioblastoma cells that lack PTN receptors. The degree of chemotaxis was comparable to that induced by the angiogenic factors VEGF and SDF-1alpha. Chemotaxis to PTN was blocked by the NOS inhibitors L-NNA and L-NMMA, the NO scavenger PTIO, the phosphoinositide-3 kinase inhibitor wortmannin, and the guanylyl cyclase inhibitor ODQ, suggesting dependence of EPC chemotaxis on these pathways. PTN induced NOS-dependent production of NO to a similar degree as did VEGF, as indicated by the NO indicator DAF-2. PTN increased proliferation in EPCs and HUVECs to a similar extent as VEGF, but did not induce proliferation of CASMCs. While L-NNA abolished PTN-induced migration in EPCs and HUVECs, it did not inhibit PTN- and VEGF-enhanced proliferation and also caused proliferation by itself. These data suggest that PTN may mediate its pro-angiogenic effects by increasing the local number of not only endothelial cells but also early EPCs at angiogenic sites.