Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Animal studies demonstrated that PTN can serve as a ratelimiting angiogenic factor during tumor growth, invasion, and metastasis (8 -12). Clinical studies showed elevated serum levels and tumor expression of PTN in samples from patients with colon, stomach, pancreatic, and breast cancer (5, 13). Furthermore, PTN has been implicated in neonatal brain development as well as in neurodegenerative disorders (reviewed in Ref. 14).Obviously, understanding of PTN-mediated signal transduction as well as identification of a receptor for PTN would enhance studies on the biology and pathology of this growth factor family. Our previous studies have shown that the activation of mitogen-activated protein kinase and PI 3-kinase pathways is required for mitogenic activity of PTN, and we had found that the adaptor molecule Shc participated in signal transduction (15). Based on the work of different laboratories in various cell types, it was hypothesized that proteins of 170 -220 kDa that are tyrosine-phosphorylated in response to PTN could be part of the receptor complex (15-17). More recently, several cell membrane-located proteins were shown to bind PTN at low affinity and serve as potential coreceptors or modulators of signal transduction (18 -21), but none of these molecules carried the hallmarks of a signal transducing receptor predicted from the earlier work.To identify a receptor for PTN, we rationalized that panning of a phage display cDNA library against immobilized PTN as a bait would allow us to isolate phage containing a ligand binding fragment of the receptor on their surface. Because of the high levels of expression of PTN during the perinatal development of the nervous system, we hypothesized that fetal brain would most likely also express a PTN receptor. We therefore panned a human fetal brain cDNA phage display library over several rounds against purified PTN that had been tested for biological activity (15). From this we isolated a phage insert homologous to an amino acid sequence stretch in the ECD of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK), a recently described orphan receptor with an apparent molecular mass of

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.The common structural features of a receptor tyrosine kinase (RTK) 1 include an extracellular ligand binding region, a hydrophobic membrane-spanning segment, and a cytoplasmic domain that carries the catalytic function. Following ligand binding, the RTK dimerizes and autophosphorylates (1). The activated RTK initiates signal transduction cascades through binding of SH2 domain-containing proteins to specific receptor phosphotyrosine residues (2). RTKs can regulate a wide variety of cellular processes involved in cell division, differentiation, survival, and motility. A number of RTKs play essential roles during the development of the nervous system by contributing to neuronal differentiation, survival, and function (reviewed in Ref.3). Most of these receptors have specific or shared ligands called neurotrophic factors that have been identified (reviewed in Ref. 3). However, for some of them, named orphan receptors, their ligands are still unknown (4 -6).Anaplastic lymphoma kinase (ALK), a novel orphan neuronal receptor, was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused in the t(2;5) chromosomal rearrangement associated with the non-Hodgkin lymphoma (7). This translocation produces a fusion gene that encodes a soluble chimeric transforming protein comprised of the N-terminal portion of the phosphoprotein nucleophosmin (NPM), a highly conserved RNA-binding nucleolar protein, linked to the cytoplasmic portion of ALK (7). The NPM-ALK fusion protein was localized within both the cytoplasm and the nucleoplasm and also within the nucleoli of t(2;5)-translocation-positive lymphoma cells (8). However, whereas the NPM sequence is essential for the transforming activity (9), the nuclear localization, occurring via the shuttling activity of NPM (10...

Elevated serum pleiotrophin levels can indicate the presence of tumors expressing this factor. Monitoring serum levels of pleiotrophin may prove useful in determining the pharmacologic efficacy of cytotoxic or anti-pleiotrophin therapy.